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Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis JCB: ARTICLE Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis 1 2 1 2 1 1 Daniela A. Sahlender, Rhys C. Roberts, Susan D. Arden, Giulietta Spudich, Marcus J. Taylor, J. Paul Luzio, 2 1 John Kendrick-Jones, and Folma Buss Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England, UK yosin VI plays a role in the maintenance of G-protein to the plasma membrane is dramatically reduced. Golgi morphology and in exocytosis. In a Two further binding partners for optineurin have been yeast 2-hybrid screen we identified optineurin identified: huntingtin and Rab8. We show that myosin VI as a binding partner for myosin VI at the Golgi complex and Rab8 colocalize around the Golgi complex and in and confirmed this interaction in a range of protein inter- vesicles at the plasma membrane and overexpression of action studies. Both proteins colocalize at the Golgi constitutively active Rab8-Q67L recruits myosin VI onto complex and in vesicles at the plasma membrane. When Rab8-positive structures. These results show that optineurin optineurin is depleted from cells using RNA interference, links myosin VI to the Golgi complex and plays a central myosin VI is lost from the Golgi complex, the Golgi is role in Golgi ribbon formation and exocytosis. fragmented and exocytosis of vesicular stomatitis virus Introduction In membrane trafficking pathways, motor proteins moving membrane ruffles (Buss et al., 1998), the Golgi complex, and along cytoskeletal tracks play a major role in transporting secretory vesicles (Buss et al., 1998; Warner et al., 2003). vesicles between donor and acceptor compartments and may Unlike all the other myosins that have been studied so far that also be involved in processes such as cargo sorting, vesicle move toward the plus end of actin filaments, myosin VI moves formation, and steady-state localization of organelles. Short- toward the minus end of actin (Wells et al., 1999). Functional range movement of cargo or vesicles along actin filaments, studies have indicated that myosin VI plays a major role in around internal organelles, or within the cortical regions of the endocytic and secretory membrane traffic pathways (Buss et cell is powered by members of the myosin superfamily, which al., 2001b; Warner et al., 2003) and it has been postulated that is comprised of at least 18 different classes (Hodge and Cope, the diverse functions of myosin VI are mediated by interaction 2000; Berg et al., 2001). Although in recent years the localization with a number of different binding partners (Buss et al., 2004). and functions of a few of these myosins have been identified, Recently, three binding partners of myosin VI were identified, there is still limited information regarding the molecular mech- Dab2, GIPC, and SAP97, all of which target myosin VI to anism linking myosin function and cargo attachment. For vesicular compartments (Bunn et al., 1999; Morris et al., 2002; example, how does a myosin recognize its cargo; how is the Wu et al., 2002). So far, the best-characterized myosin VI– interaction regulated and what influence does cargo binding binding partner is Dab2; its interaction with myosin VI has have on motor activity? been shown to form a dynamic link between cell surface receptors, Myosin VI is a multifunctional motor protein found in a clathrin-mediated endocytosis, and the actin cytoskeleton (Mor- number of different intracellular compartments including en- ris and Cooper, 2001; Morris et al., 2002). In contrast, no bind- docytic vesicles (Buss et al., 2001b; Aschenbrenner et al., 2003), ing partners have been identified that targets myosin VI to the Golgi complex and the secretory pathway. In this paper, we have identified and characterized opti- D.A. Sahlender and R.C. Roberts contributed equally to this work. neurin, a novel myosin VI–binding partner, which is found at Correspondence to Folma Buss:fb1@mole.bio.cam.ac.uk the Golgi complex. Optineurin was first discovered as a binding Abbreviations used in this paper: MTOC, microtubule organizing center; NRK, partner of the adenoviral protein E3-14.7K (14.7K-interacting normal rat kidney; SEAP, soluble secreted form of the alkaline phosphatase; protein-2 and therefore named FIP-2) and was shown to protect siRNA, small interfering RNA; VSV-G, vesicular stomatitis virus G-protein. The online version of this article contains supplemental material. infected cells from TNF-–induced cytolysis (Li et al., 1998b). © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 169, No. 2, April 25, 2005 285–295 http://www.jcb.org/cgi/doi/10.1083/jcb.200501162 JCB 285 THE JOURNAL OF CELL BIOLOGY It is a conserved 67-kD protein with multiple leucine zipper do- which is interesting, because Rab8 is a regulator of post-Golgi mains and a putative zinc finger domain at the COOH termi- membrane traffic from the TGN to the plasma membrane. nus. Optineurin shows strong homology (53% identity) with NF-B essential modulator and was therefore also called Results NEMO-related protein (Schwamborn et al., 2000). Mutations Optineurin binds to the COOH-terminal in the human optineurin gene are associated with adult-onset tail of myosin VI open angle glaucoma (hence it was named “optic neuropathy inducing” protein optineurin; Rezaie et al., 2002). The COOH-terminal tail domain of myosin VI is required for Although optineurin was previously localized to the Golgi targeting to intracellular locations such as clathrin-coated vesi- complex (Schwamborn et al., 2000; Stroissnigg et al., 2002) its cles (Buss et al., 2001a) and the Golgi complex (Warner et al., functions at this organelle have not yet been established. How- 2003). To identify binding partners for myosin VI in the Golgi ever, two binding partners for optineurin have been identified complex we used the whole tail of myosin VI as a bait in a which link it to membrane trafficking events. One is huntingtin, yeast two-hybrid screen of a human umbilical vein epithelial the protein mutated in the neurodegenerative disorder Hunting- cell cDNA library (Morris et al., 2002). A 375–amino acid ton’s disease (Faber et al., 1998), and the other is the small COOH-terminal fragment of optineurin (residues 198–577) GTPase Rab8 (Hattula and Peranen, 2000). Although the precise was identified in the screen as a myosin VI–binding partner cellular functions of the wild-type huntingtin protein are not (Fig. 1 A). To confirm this interaction both myosin VI and op- known, its intracellular localization to the Golgi complex and to tineurin were overexpressed in CHO cells and the binding was endocytic and exocytic vesicles (DiFiglia et al., 1995; Velier et measured in a mammalian two-hybrid assay (Fig. 1 B). Dele- al., 1998), as well as the identity of its binding partners (Harjes tion mutants of optineurin revealed that the myosin VI–binding and Wanker, 2003), suggests a role in membrane trafficking site on optineurin is between amino acids 412 and 520 (not de- pathways (Gauthier et al., 2004). The other optineurin binding picted). To determine the site on myosin VI that binds to op- partner Rab8 belongs to a large family of small GTPases that tineurin a combination of deletion mutants followed by site- participate in and regulates intracellular membrane trafficking directed mutagenesis was used (Fig. 1 B). The results indicate pathways (Zerial and McBride, 2001). In their active GTP- that the globular tail (GT; aa 1034–1276) contains the binding bound state different Rab proteins bind to different membrane site, because the NH -terminal helical part of the tail (HT; aa compartments and recruit specific effector proteins, which are 845–1033; Fig. 1 B, columns 1 and 2) did not bind. Further- not only involved in docking and fusion with the target mem- more, myosin VI interaction with optineurin does not require brane but also in the formation of transport vesicles and in bind- the large insert (LI) found in some isoforms (Fig. 1 B, columns ing motor proteins for vesicle transport (Zerial and McBride, 3 and 4); this part of the tail has previously been shown to be 2001; Hammer and Wu, 2002; Seabra and Coudrier, 2004). involved in targeting myosin VI to clathrin-coated vesicles at Several examples of motor protein–Rab complexes are known; the apical domain of polarized cells (Buss et al., 2001b). Be- for example those involving microtubule motors such as the ki- cause deletion of the COOH-terminal 159 aa of the myosin VI nesin-like protein (Rabkinesin-6) and Rab6 as well as actin- tail did not abolish binding (Fig. 1 B, column 5), we used site- based motor complexes such as myosin Va and Rab27a and directed mutagenesis to change three adjacent residues at a myosin Vb and Rab11 (Hammer and Wu, 2002). time to alanine in order to narrow down the binding site in the Rab8 interacts specifically with the NH terminus of op- remainder of the GT region, i.e., between aa 1060 and 1117. tineurin (Hattula and Peranen, 2000). Rab8 has been localized Fig. 1 B shows that changing WKS to AAA, KVY to AAA, to the Golgi region, to TGN-derived transport vesicles and to and REE to AAA did not reduce binding (Fig. 1, columns 6, 7, the plasma membrane (Huber et al., 1993b) and has been and 9), however mutating RRL to AAA abolished binding of shown to regulate biosynthetic trafficking pathways from the the myosin VI tail to optineurin (column 8), clearly showing TGN to the cell surface (Huber et al., 1993b; Ang et al., 2003). that these residues constitute the binding site for optineurin. In our present study, we have identified an RRL se- In vitro phosphorylation studies using recombinant p21– quence in the tail domain of myosin VI that binds to a site in activated kinase (PAK; Buss et al., 1998) and the tail of myosin the COOH-terminal region of optineurin. Both proteins colo- VI expressed in E. coli indicated that threonine 1088 and threo- calize at the Golgi complex and in vesicular structures close nine 1091 (the sequence TINT; Fig. 1 A) are potential phos- to the plasma membrane. The depletion of optineurin using phorylation sites (unpublished data). These threonines are also small interfering RNA (siRNA) techniques drastically alters the phosphorylated in vivo, because when full-length myosin VI morphology of the Golgi complex and reduces significantly was isolated from A431 cells by immunoprecipitation and sub- transport of vesicular stomatitis virus G-protein (VSV-G) to jected to MALDI mass spectrometric analysis a peptide con- the cell surface indicating that optineurin plays an important taining this phosphorylated TINT motif was detected. This role in Golgi ribbon formation and in exocytosis. In addition, peptide was dephosphorylated after alkaline phosphatase treat- depletion of optineurin causes a marked reduction in the ment (unpublished data). We mutated these two threonines to amount of myosin VI associated with the Golgi complex sug- either alanines or glutamates to mimic the nonphosphorylated gesting that optineurin is the anchor for myosin VI at this or- and phosphorylated states respectively in order to test their ef- ganelle. We further show that myosin VI colocalizes with fects on the binding of myosin VI to optineurin in the mamma- Rab8 at the Golgi complex and at the plasma membrane, lian two-hybrid assay. Whereas no effect was seen with the 286 JCB • VOLUME 169 • NUMBER 2 • 2005 alanine-mutated construct AINA (Fig. 1 B, column 10), TINT mutated to EINE abolished optineurin binding to myosin VI (col- umn 11) but did not abolish myosin VI binding to Dab2, which was used as a control. Because the EINE mutation potentially mimics the phosphorylated form, this data suggests that phos- phorylation regulates the binding of optineurin to myosin VI. A GST pull-down assay gave further confirmation that optineurin binds directly to the tail of myosin VI. In vitro– translated [ S]methionine labeled optineurin or Dab2 as a pos- itive control (Fig. 1 C, lanes 2 and 3) were incubated with ei- ther GST or GST myosin VI tail. The autoradiogram in Fig. 1 C shows that optineurin binds to the myosin VI tail (Fig. 1 C, lane 5), but not to GST alone (lane 4) or to luciferase as a nega- tive control protein (lanes 6 and 7). To confirm that myosin VI binds to optineurin in vivo, we immunoprecipitated endoge- nous myosin VI and endogenous optineurin from the cytosol of A431 cells. When myosin VI was immunoprecipitated with myosin VI tail antibodies optineurin is coimmunoprecipitated (Fig. 1 D, lane 4), indicating that optineurin and myosin VI ex- ist in a protein complex in vivo. Mutant myosin VI (RRL) does not rescue secretion in Snell’s waltzer fibroblasts In fibroblastic cell lines derived from Snell’s waltzer mice (sv/ sv) the absence of myosin VI causes a reduction in constitutive secretion. As shown previously this phenotype can be restored by overexpression of a fully functional myosin VI (Warner et al., 2003). To test whether binding of optineurin is essential for myosin VI function at the Golgi complex in vivo, we per- formed similar rescue experiments in sv/sv fibroblasts. Immor- tal fibroblastic sv/sv or wild-type cell lines were stably trans- binds to purified myosin VI tail. A pull down was performed using in vitro– translated optineurin and GST-myosin VI tail. S-labeled in vitro–trans- lated full-length optineurin (lanes 4 and 5), Dab2 as a positive control (lanes 2 and 3) or luciferase as a negative control (lanes 6 and 7) were incubated with either 5 g GST alone (lanes 2, 4, and 6) or with 5 g GST-myosin VI tail (lanes 3, 5, and 7). Lane 1 shows 50% of the input used for the pulldown with [ S]optineurin in lane 5. The band below Dab2 is possibly caused by degradation (lane 3). (D) Co-immunoprecipi- Figure 1. Optineurin is a myosin VI–binding partner. (A) A carton show- tation of endogenous myosin VI and optineurin from the cytosol of A431 ing the major regions in optineurin and myosin VI tail. Optineurin contains cells. Immunoprecipitation was performed under native conditions using an NH -terminal Rab8-binding domain (aa 141–209; Hattula and Peranen, no antibody, only protein A beads as a control (lane 2, blank), an anti- 2000) and a COOH-terminal myosin VI–binding domain (aa 412–520). body to full-length optineurin (lane 3, a-Optn) or a pAb to the tail of Myosin VI tail has a proposed helical tail domain (HT) and a globular tail myosin VI (lane 4, a-MVI). Lane 1 is equivalent to 5% of the input used for (GT), which contains the optineurin-binding site RRL in position 1107– each immunoprecipitation. Immunoprecipitates were run out on a 10% 1109. The putative phosphorylation site TINT is at position 1088–1091. PAGE gel, blotted, and analyzed using an antibody to optineurin. (E) Con- (B) Using the ProQuest yeast two-hybrid screen optineurin was identified stitutive secretion in Snell’s waltzer fibroblasts cannot be rescued by over- as a myosin interacting protein. The interaction was verified using the expression of the mutant myosin VI lacking the optineurin-binding site mammalian two-hybrid system. To narrow down the optineurin-binding (RRL). Rescue experiments were performed by generating Snell’s waltzer site on the myosin VI tail the whole tail (T; column 1), the helical tail cell lines stably expressing only GFP (SV), the mutant myosin VI without the (HT; column 2) or the globular tail (GT) with or without the large insert optineurin-binding site (RRL), or wild-type whole myosin VI (MVI). Levels (GTLI or GT-LI; columns 3 and 4) were fused to the Gal4 DNA-binding of secretion were compared with wild-type cells stably expressing only domain and coexpressed with full-length optineurin fused to the VP16 acti- GFP (WT). To measure constitutive secretion, a construct, which expresses vating domain in CHO cells. Luciferase activity is shown relative to control a SEAP was transfected into the various Snell’s waltzer cell lines and the cells containing only the myosin VI construct with the empty prey vector. wild-type fibroblasts. The amount of alkaline phosphatase secreted into the Deletion of myosin VI tail at residue 1117 (TC; column 5) did not abolish media was measured 24, 48, and 72 h after transfection and plotted as a binding. Mutating three adjacent amino acids each (WKS, KVY, RRL, and percentage of total maximal secretion in wild-type cells after 72 h. Trans- REE to AAA) revealed RRL as the optineurin-binding site (columns 6–9). fection efficiency was normalized by cotransfection of a control plasmid Two threonine residues in positions 1088 and 1091 (TINT) were mutated to expressing a red fluorescent protein. The data from two separate experi- either alanine (AINA) or glutamate (EINE; columns 10 and 11). (C) Optineurin ments run in triplicate are shown. OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 287 fected with GFP, GFP-tagged whole wild-type myosin VI or myosin VI with mutations in the optineurin-binding site (RRL). Constitutive secretion in these stable cell lines was measured using a soluble secreted form of the alkaline phos- phatase (SEAP) assay as previously described (Warner et al., 2003). We observed that only fully functional myosin VI is able to restore wild-type levels of secretion, whereas cells ex- pressing myosin VI with the mutant optineurin-binding site are unable to increase secretion levels back to those of the wild type (Fig. 1 E). Myosin VI and optineurin colocalize at the Golgi complex and in vesicles underneath the plasma membrane To test the interaction between myosin VI and optineurin in vivo, we investigated their intracellular localization in normal rat kidney (NRK) cells. Previously, both proteins had been lo- calized independently to the Golgi complex (Buss et al., 1998; Hattula and Peranen, 2000; Schwamborn et al., 2000; Stroiss- nigg et al., 2002; Warner et al., 2003). Using an affinity-purified pAb to optineurin and the TGN marker, TGN38, we confirm that optineurin is localized at the Golgi complex (Fig. 2, a–c). Double labeling experiments using optineurin antibodies and an mAb to myosin VI (Warner et al., 2003) showed considerable colocalization of both endogenous proteins at the Golgi complex (Fig. 2, d–f). In addition, both proteins were found in vesicular structures in the cell periphery close to the plasma membrane (Fig. 2, g–i and g–i). We transiently overexpressed optineurin Figure 2. Optineurin and myosin VI colocalize in NRK cells. Endogenous optineurin localizes to the Golgi complex using a pAb to optineurin (a) or fragments of optineurin tagged with either GFP, Flag, or myc and an mAb to TGN38 (b). Optineurin and myosin VI are localized at the at their COOH or NH termini in a number of different cell types Golgi complex using a pAb to optineurin (d) and an mAb to myosin VI (e). for colocalization studies with the aim of expressing optineurin Colocalization in vesicular structures close to the plasma membrane was demonstrated with the pAb to optineurin (g) and by transiently overex- mutants that might function as dominant negative. However, pressing myosin VI tagged with GFP (h). White boxes indicate areas en- none of the transiently expressed optineurin constructs gave the larged in the pictures below (g, h, and i). Endogenous myosin VI, visual- same intracellular localization as endogenous optineurin (un- ized using the pAb against the whole tail, is recruited into aggregates containing overexpressed GFP-optineurin (j–k). The merged images are published data); the overexpressed optineurin was not observed shown in c, f, i, i, and l. Bars, 10 m. at the Golgi complex but showed an overall vesicular distribu- tion and formed aggregates of various sizes in the cytoplasm. It would appear that the addition of the GFP, Flag, or myc tags to and HeLa cells (see Materials and methods) and the best results the NH or COOH terminus of optineurin or even to its frag- were achieved after repeated transfection over a period of 4 d. In ments alters the protein conformation in some way and masks these cells optineurin was depleted to 10% of its original level domains important for intracellular targeting to the Golgi com- as shown by Western blotting of equal amounts of mock and plex. Interestingly, endogenous myosin VI was recruited from siRNA-treated cells 48 h after the second transfection (Fig. 3 A, its cytosolic pool into these optineurin aggregates (Fig. 2, j–l), a and B, a). In the same cells expression levels of myosin VI and indicating that the myosin VI–binding site is maintained and ac- actin were unaffected (Fig. 3 A, a and B, a). In the knockdown cessible. NH -terminal optineurin constructs without the myosin cells immunofluorescence microscopy showed that very little VI–binding domain (aa 412–520) were unable to recruit endog- optineurin was present in the Golgi complex (Fig. 3 A, d and B, enous myosin VI into these aggregates (not depicted). These d), although in some cells residual cytosolic labeling was appar- results strongly indicate that myosin VI binds to and forms a ent (Fig. 3 A, d). To find out what happens to the localization of protein complex with optineurin in vivo. myosin VI in optineurin knockdown cells, we double labeled Depletion of optineurin by siRNA causes siRNA and mock-transfected NRK cells with a myosin VI loss of myosin VI from the Golgi complex COOH-terminal globular tail antibody and with antibodies to GM130 and TGN38. In optineurin-depleted cells there was a Does optineurin play a role in linking myosin VI to the Golgi marked reduction in the amount of myosin VI associated with complex? To address this question we used siRNA to reduce the Golgi complex (Fig. 4, c and g) as compared with mock- cellular expression levels of optineurin (Elbashir et al., 2001). transfected cells (Fig. 4, a and e) indicating that optineurin plays For the knockdown experiments two siRNA duplexes, specific a role in anchoring myosin VI to the Golgi complex. for the nucleotide sequence of optineurin, were tested in NRK 288 JCB • VOLUME 169 • NUMBER 2 • 2005 Figure 4. Knockdown of optineurin in NRK cells causes loss of myosin VI from the Golgi complex. Mock-transfected (a, b, e, and f) or siRNA-trans- fected (c, d, g, and h) NRK cells were used for immunofluorescence and were double labeled with antibodies to the globular tail of myosin VI (a, c, e, and g) and antibodies to TGN38 (b and d) or GM130 (f and h). Asterisks mark the position of the nucleus. Bars, 10 m. membranes into a ribbon like structure (Rambourg and Cler- mont, 1990). After depolymerization of microtubules using drugs such as nocodazole, these tubular connections are lost and the Golgi stacks are disconnected and dispersed throughout the cells, indicating that the cytoskeleton plays a major role in maintaining this perinuclear organization of the Golgi complex (Rogalski and Singer, 1984). Figure 3. Optineurin is depleted from NRK and HeLa cells using siRNA. When optineurin was depleted by siRNA transfection in HeLa (A) or NRK (B) cells were either mock transfected with water or trans- NRK cells (Fig. 4, d and h) or especially in HeLa cells (Fig. 5, fected twice at 48-h intervals with siRNA specific to optineurin. After 4 d d–f) there was a dramatic effect on the structure of the Golgi cells were blotted and probed with antibodies to myosin VI, optineurin, or actin (A, a and B, a). In a parallel experiment mock-transfected, siRNA- complex. In the optineurin-depleted HeLa cells the Golgi com- transfected NRK, or HeLa cells were used for immunofluorescence and plex was fragmented into vesicular and sometimes short tubu- double labeled with antibodies to optineurin (A, b and d; B, b and d) lar structures dispersed throughout the whole cytoplasm. These and GM130 (A, c and e; B, c and e). Bars, 10 m. Golgi fragments not only contained the Golgi matrix (GM130) and TGN (TGN46) markers (Fig. 5, d–f) but also markers for Loss of optineurin disrupts the Golgi the cis- and medial-Golgi stacks (not depicted). Although the ribbon structure Golgi matrix and TGN markers were present in a single struc- In mammalian cells the Golgi complex is formed by stacks of ture, they do not completely colocalize, indicating that some flattened membrane cisternae that are interconnected by tubular compartmentalization and organization of the Golgi was still OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 289 preserved in these fragments (Fig. 5, d–f). The Golgi struc- ture was not disrupted due to a breakdown of the microtubular network, because this network was still intact in optineurin- depleted cells (unpublished data). To characterize the phenotype of optineurin-depleted cells at the ultrastructural level, electron microscopy was per- formed on mock and siRNA-treated HeLa cells. In optineurin knockdown cells mini-stacks of Golgi membranes similar to those in control cells were still observed (Fig. 5, g and h). This indicates that although the loss of optineurin caused a break- down of the Golgi ribbon structure, the overall morphology of individual Golgi stacks was maintained and no large-scale ve- siculation was present. Colocalization of myosin VI and Rab8 is optineurin dependent Optineurin has been shown to form a link between huntingtin and Rab8 (Hattula and Peranen, 2000) and optineurin and hun- tingtin can be colocalized at the Golgi complex (Fig. S1, avail- able at http://www.jcb.org/cgi/content/full/jcb.200501162/ DC1). These proteins are both found at the TGN, in transport vesicles throughout the cytoplasm and at the plasma membrane (Huber et al., 1993b; Velier et al., 1998). Optineurin specifi- cally interacts with wild-type Rab8 and with the constitutively active GTP-bound mutant Rab8-Q67L, but not with the domi- nant-negative GDP-bound mutant Rab8-T22N in vitro (Hattula and Peranen, 2000; Fig. S2 A, available at http://www.jcb.org/ cgi/content/full/jcb.200501162/DC1). By immunofluorescence optineurin was shown to colocalize with wild-type Rab8 and the constitutively active Rab8-Q67L at the Golgi complex and Figure 5. The loss of optineurin results in Golgi fragmentation. Mock (a–c) or optineurin siRNA-treated HeLa cells (d–f and d–f) were double labeled in vesicular and tubular structures in the cytoplasm (Fig. S2 B). in immunofluorescence experiments with TGN46 (b, e, and e) and We suggest that optineurin may link myosin VI to Rab8, GM130 (a, d, and d). White boxes indicate areas enlarged in the as it has been suggested that it links huntingtin to Rab8 pictures below (d, e, and f). The Golgi fragments contain both marker proteins as indicated by the yellow color in the merged images (c, f, and f). (Hattula and Peranen, 2000). To check this idea we deter- The arrows highlight the overlap between the marker proteins in (d–f). mined the intracellular distribution of myosin VI and Rab8 in Bars, 10 m. TEM analysis of mock (g) or siRNA-transfected (h) HeLa NRK cells. Both proteins showed good colocalization around cells. Arrows indicate the position of Golgi stacks that can be found in both mock-transfected and siRNA-transfected cells. Bars, 200 nm. the Golgi complex (Fig. 6, a–c), and also in peripheral vesicles Figure 6. Myosin VI colocalizes with Rab8 at the Golgi complex and the plasma membrane. In NRK cells tran- siently overexpressing Rab8 tagged with GFP (b and e) myosin VI was detected with an antibody to the globular tail (a) or to the whole tail (d). Myosin VI and Rab8 are found in the same labeled structures/vesicles at the Golgi complex (c) and at the plasma membrane (f). Arrows indicate vesicles containing both proteins. Bars, 10 m. 290 JCB • VOLUME 169 • NUMBER 2 • 2005 Figure 8. The transport of VSV-G to the cell surface is dramatically reduced in optineurin-depleted HeLa cells. Mock-treated or siRNA-treated HeLa cells were transfected with ts045-VSV-G-GFP to measure the rate of exocy- tosis . VSV-G at the cell surface was detected in indirect immunofluores- cence using an mAb to the luminal domain of VSV-G and total VSV-G expressed was detected using a pAb to GFP. (A) Representative cells for the 40- and 100-min time points are shown to compare the amount of VSV-G on the cell surface in mock- and optineurin-depleted cells. (B) The ratio of cell surface over total VSV-G fluorescence was measured as described in Materials and methods to determine the rate of transport from the Golgi complex to the cell surface. Error bars: SEM. Bars, 10 m. Figure 7. The recruitment of myosin VI to tubular vesicular structures formed after overexpression of the constitutively active form of Rab8 is lost in optineurin-depleted cells. The constitutively active form of Rab8 (GFP- nous myosin VI onto their membranes and led to a dramatic re- Rab8-Q67L) overexpressed in NRK cells localizes with myosin VI at the Golgi complex (a and b) and recruits myosin VI onto tubular and vesicular distribution of myosin VI (Fig. 7, e and e). The recruitment structures emerging from the Golgi complex (e and f). In optineurin-depleted of myosin VI to these Rab8-positive structures requires op- NRK cells, which are overexpressing GFP-Rab8-Q67L myosin VI no longer tineurin, because in optineurin knockdown cells myosin VI is colocalizes with Rab8-Q67L at the Golgi complex (c and d) and is not recruited to tubular and vesicular structures positive for Rab8-Q67L (g and h). no longer present at the fragmenting Golgi complex (Fig. 7, c White boxes highlight the areas of the cells enlarged in the pictures below and d) or in the tubular structures (Fig. 7, g, h, g, and h). (e,f, g, and h). Endogenous myosin VI was detected with either a pAb to the globular tail (a, c, g, and g) or to the whole tail (e and e). Bars, 10 m. Transport of VSV-G from the Golgi complex to the plasma membrane is reduced in optineurin knockdown cells close to the plasma membrane (Fig. 6, d–f). Overexpression of the constitutively active form of Rab8-Q67L colocalized with Myosin VI has been shown to be important for constitutive se- myosin VI at the Golgi complex (Fig. 7, a and b) but also re- cretion in fibroblasts (Warner et al., 2003). To test whether op- sulted in the formation of long tubular structures emerging tineurin as a myosin VI–binding partner present at the Golgi from the Golgi complex (Fig. 7, f and f). These long tubular complex, plays a role in post-Golgi membrane traffic to the cell carriers containing Rab8-Q67L were able to recruit endoge- surface, we used a thermoreversible folding mutant of the OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 291 VSV-G fused to GFP at its cytoplasmic tail (ts045 VSV-G- myosin VI not only in the motor domain (Buss et al., 1998) but GFP) as a reporter molecule for membrane transport in the also in the globular tail domain at threonine 1088 and 1091, secretory pathway (Wehland et al., 1982; Kreis and Lodish, thus coordinating the regulation of motor function with cargo 1986; Balch et al., 1994). This membrane protein misfolds and binding in the tail domain. is retained in the ER at 39.5 C, but upon a temperature shift to In the absence of optineurin the Golgi complex is frag- 32 C, it can fold and move to the Golgi complex and subse- mented. The ribbon structure of interconnected stacks of mem- quently to the plasma membrane. We measured the amount brane cisternae is broken up and the disconnected Golgi stacks of VSV-G at the cell surface relative to the total amount of are dispersed throughout the cytoplasm. The overall appearance VSV-G expressed in the cell using an antibody specific to the of the fragmented Golgi stacks, however, is similar to the ap- luminal domain of this protein (Pepperkok et al., 1993; See- pearance of the Golgi in control cells and no gross vesiculation mann et al., 2000). was observed at the EM level. Golgi resident proteins such as The loss of optineurin has a dramatic effect on transport TGN46 and GM130 were still present in the Golgi fragments. of VSV-G from the Golgi complex to the cell surface. After 40 These morphological changes are very similar to those ob- min in mock-transfected cells VSV-G could be detected at the served when microtubules are disrupted by drugs such as no- cell surface (Fig. 8 A, b), whereas in optineurin knockdown codazole (Rogalski and Singer, 1984), suggesting that op- cells it is still found in the fragmented Golgi (Fig. 8 A, c) and tineurin might be involved in linking Golgi membranes directly not at the cell surface (Fig. 8 A, d). After 100 min very little or indirectly to microtubules around the microtubule organizing VSV-G still remained in the Golgi complex in the mock-trans- center (MTOC). One of the optineurin-binding partners is hun- fected cells and most was on the cell surface (Fig. 8 A, e and f), tingtin, known to interact with HAP1 (Huntingtin-associated however, in the optineurin-depleted cells VSV-G was still re- protein; Li et al., 1995), which binds directly to the dynactin glued tained in the Golgi complex with only a small proportion of it subunit p150 (Engelender et al., 1997; Li et al., 1998a). The present on the cell surface (Fig. 8 A, g and h). To quantify these dynactin complex is involved in linking the minus end directed observations 30 cells per time point were measured in two in- microtubule motor protein dynein to membrane vesicles (Gill et dependent experiments. At every time point less VSV-G was al., 1991). It has been proposed that huntingtin and HAP1 act as detected at the plasma membrane of siRNA-treated cells com- scaffolding proteins regulating the interaction between the dy- pared with mock-transfected cells (Fig. 8 B). Thus, the trans- nein–dynactin complex and cargo such as Golgi membranes port of VSV-G from the Golgi complex to the cell surface was (Harjes and Wanker, 2003). The absence or inactivation of cy- dramatically delayed in the optineurin-depleted cells and only toplasmic dynein leads to fragmentation and spreading of the at later time points did it reach 50% of the control levels. These Golgi complex into the cytoplasm (a phenotype similar to that results clearly indicate that optineurin plays an important role observed after the loss of optineurin; Harada et al., 1998); thus in post-Golgi membrane trafficking of VSV-G from the Golgi it is possible that optineurin via the huntingtin–HAP1 complex complex to the cell surface. may be involved in targeting dynein to Golgi membranes (Fig. 9). Recent results (Gauthier et al., 2004) indicate a further role for the huntingtin–HAP1 complex in the axonal transport of Discussion vesicles containing brain-derived neurotrophic factor along mi- We have previously shown that myosin VI is present at the crotubules and highlight the importance of huntingtin-binding Golgi complex (Buss et al., 1998) and is involved not only in the partners in the progression of the disease. steady-state organization of the Golgi complex but also in post- The loss of optineurin not only disrupts the structure of Golgi membrane traffic (Warner et al., 2003). To investigate the the Golgi complex, but also dramatically reduces secretion of precise roles that myosin VI plays in these processes we identi- VSV-G to the plasma membrane. Reduced transport is not due fied optineurin, a peripheral Golgi protein as a binding partner to Golgi fragmentation because Golgi stacks dispersed by de- for myosin VI. This interaction found in a yeast two-hybrid polymerization of microtubules remain fully functional with screen was confirmed using a range of different protein–protein only a slight reduction in protein secretion (Van De Moortele et interaction studies. Myosin VI and optineurin colocalize at the al., 1993). Reduced exocytosis of VSV-G in the optineurin Golgi complex and in vesicular structures close to the plasma knockdown cells is a very similar phenotype to that observed in membrane. The loss of optineurin not only leads to Golgi frag- the myosin VI knockout mouse, where secretion of alkaline mentation and a reduction in exocytosis, but we also show that phosphatase is also significantly reduced (Warner et al., 2003). optineurin is essential for targeting myosin VI to the Golgi com- Secretion in these cells lacking myosin VI is not restored by plex. Most interestingly optineurin plays a role in colocalizing mutant myosin VI, which is unable to bind to optineurin, indi- myosin VI and Rab8, a member of the Rab family of G-proteins, cating that a functional complex between myosin VI and op- which are important regulators of membrane traffic events. tineurin is required for constitutive exocytosis. Optineurin binds directly to the sequence RRL (residues Rab8 binds to optineurin and plays an important role in 1107–1109) in the globular tail of myosin VI. This binding site polarized membrane transport both from the Golgi complex to is distinct from the Dab2-binding site and is independent of the the basolateral membrane in polarized epithelial cells and to large insert. The binding of optineurin to myosin VI is regulated dendrites in neurons (Huber et al., 1993a,b). Rab8 is present in by phosphorylation at the TINT (1088–1091) site in the globu- the same intracellular compartments as myosin VI and in this lar tail region. Our results suggest that a PAK phosphorylates paper we show that myosin VI and Rab8 colocalize in the peri- 292 JCB • VOLUME 169 • NUMBER 2 • 2005 VI in the Golgi complex (Fig. 9): First, optineurin together with huntingtin links via the minus end directed motor dynein to the microtubule network and also by directly interacting with myosin VI, is linked to the actin cytoskeleton, thus it may play a role in coordinating microtubule-based and actin-based motor activity around the Golgi complex. Support for such a role is provided by our previous observation that in Snell’s waltzer mice the absence of myosin VI causes a reduction in the size of the Golgi complex (Warner et al., 2003), which would be due to the dynein motor complex pulling the Golgi membranes in toward the MTOC resulting in a smaller more compact Golgi complex. Therefore, in the absence of myosin Figure 9. A cartoon illustrating the possible interaction of optineurin with VI the balance of motor proteins is disturbed and dynein wins motor protein complexes at the Golgi complex. Optineurin may play a the “tug of war”. Second, optineurin might link myosin VI to central role in coordinating actin-based and microtubule-based motor func- tion for maintaining Golgi morphology. The optineurin-binding partner Rab8, a regulatory protein known to be involved in sorting huntingtin has been shown to interact with HAP1, which in turn was reported molecules in the exocytic pathway at the TGN and in mem- glued to form a complex with the dynactin subunit p150 and modulate/ brane fusion at the plasma membrane. The linker function of regulate the dynein–dynactin complex. Loss of minus end–directed dynein motor activity causes in fragmentation of the Golgi ribbon structure. On optineurin may be temporally regulated, for example by phos- the other hand loss of myosin VI results in a reduction in the size of the phorylation of individual proteins, including myosin VI and Golgi complex, because now the dynein complex is still active and able to optineurin (Schwamborn et al., 2000). retract the Golgi complex toward the MTOC. However, if optineurin is depleted, both motor complexes are nonfunctional and the Golgi complex In addition, because optineurin is obviously a dimer with is fragmented. multiple leucine zippers it may induce monomeric myosin VI to dimerize for processive movement of vesicles from the TGN to the plasma membrane. nuclear region around the Golgi complex and both are present Our work provides new information on the intracellular on the same vesicles underneath the plasma membrane. In ad- functions of optineurin, which will help elucidate how muta- dition Rab8 has been localized to the recycling endosome, a tions in the optineurin gene lead to primary open angle glau- compartment shown to be involved in post-Golgi membrane coma (Rezaie et al., 2002). Previous reports, that reduced secre- trafficking to the plasma membrane (Ang et al., 2003, 2004). tion of the glycoprotein myocilin leads to primary open angle However, at present we don’t know whether some myosin VI is glaucoma (Joe et al., 2003), support our results that optineurin also associated with the fraction of Rab8 present in the recy- plays a major role in Golgi morphology and exocytosis. cling endosome. On the other hand because Rab8 may interact via optineurin with myosin VI it suggests a possible pathway Materials and methods whereby TGN-derived vesicles are transported short distances Antibodies along actin filaments by myosin VI, before a kinesin motor The following antibodies were used: affinity-purified rabbit pAb to hu- picks up the cargo for long distance transport along microtu- man full-length optineurin (-Optn; GenBank/EMBLDDBJ accession no. bules. Similarly, close to the plasma membrane the Rab8– AF061034); monoclonal -VSV-G to the luminal domain (a gift from R. Pepperkok and J. Simpson, EMBL, Heidelberg, Germany); polyclonal -opti- optineurin–myosin VI complex might be involved in presenting neurin (Cayman); monoclonal -GM130 (BD Transduction Laboratories); the secretory vesicle to the docking site, thus tethering the vesi- monoclonal -TGN38 (Luzio et al., 1990); monoclonal -GFP (Qbiogene); cle close to the plasma membrane before fusion. Overexpres- polyclonal -GFP (Molecular Probes); monoclonal, polyclonal -TGN46 (Serotec); and monoclonal and pAbs to the whole tail (-MVI) and globu- sion of Rab8-Q67L leads to the formation of long tubular ex- lar tail of myosin VI (-MVI-GT; Buss et al., 1998; Warner et al., 2003). tensions from the Golgi complex and it recruits endogenous Yeast two-hybrid screen myosin VI onto these tubular structures. These results indicate Myosin VI interacting proteins were identified in a ProQuest Two-Hybrid that Rab8 serves as a membrane receptor for myosin VI, re- System (Invitrogen) using the tail domain of chicken intestinal brush border cruiting this myosin motor onto a specific intracellular mem- myosin VI (amino acids 846–1277) containing the large insert to screen a human umbilical vein epithelial cell cDNA library (provided by M. McCor- brane compartment via optineurin. Similarly, it was shown re- mack and T. Rabbits, MRC-LMB, Cambridge, UK) as described previously cently that Rab27a recruits myosin Va onto melanosomes via (Morris et al., 2002). 100 mM 3-amino-1,2,4-TRIzol was used to overcome the linker molecule melanophillin (Seabra and Coudrier, 2004). the observed self-activation by the myosin VI tail domain. The potential myo- sin VI tail domain interacting proteins were identified in the yeast two- Optineurin is the first myosin VI–binding partner identi- hybrid screen by selecting transformants that were able to grow on plates fied at the Golgi complex and may play an adaptor or linker lacking leucine, tryptophan, and histidine in the presence of 100 mM role. The recent demonstration that myosin VI can exist as a 3-amino-1,2,4-TRIzol. Colonies containing candidate cDNAs for interacting proteins were isolated and replated on selection agar to test for activation nonprocessive monomer (Lister et al., 2004) and also possibly of the three reporter genes HIS3, URA3, and lacZ. Colonies positive for all as a processive dimer (Rock et al., 2001; Nishikawa et al., three reporter genes were confirmed in the mammalian two-hybrid assay. 2002) may provide a mechanism for the distinct dual roles for Cloning of optineurin and construction of pEGFP-Rab8-Q67L myosin VI in structural maintenance and vesicle transport ac- The full-length version of human optineurin cDNA was generated by PCR tion away from the Golgi complex. We therefore suggest that from Caco-2 cDNA and subcloned into pEGFP (Clontech) or pRSET (Invitro- optineurin may serve two major “linker” functions for myosin gen) for pAb production. pEGFP-Rab8 WT, containing the human wild-type OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 293 Rab8 cDNA sequence (GenBank/EMBL/DDBJ accession no. 498943) IGEPAL CA-630 (Sigma-Aldrich) and protease inhibitor cocktail (Com- was a gift from P. Row (University of Liverpool, Liverpool, UK) and H. plete; Roche) and processed for immunoprecipitation followed by SDS Davidson (University of Colorado, Denver, CO). The constitutively active PAGE and immunoblotting as described previously (Buss et al., 1998). form of Rab8, Rab8-Q67L, was generated by QuikChange Site-Directed Mutagenesis (Stratagene) according to the manufacturer’s instructions. GST pull-down assay The chicken brush border myosin VI tail residues 840–1277 tagged with Mammalian two-hybrid assay GST was expressed in E. coli BL21 (DE3) cells and purified as described Chicken myosin VI tail fragments HT (aa 845–1033) and TC (aa 845– previously (Buss et al., 1998). Full-length optineurin and Dab2 were 1117) were generated by PCR and inserted into the pM “bait” vector cloned into pcDNA3 (Invitrogen) and in vitro translated using the TNT-cou- S]methionine in vitro– (CLONTECH Laboratories, Inc.). The following alanine mutations were pled Reticulocyte Lysate System (Promega). The [ generated using QuikChange Site-Directed Mutagenesis (Stratagene): translated protein was incubated for 2 h at 4 C with 5 g of GST or GST WKS, KVY, RRL, and REE to AAA at aa positions 1115–1117, 1110– myosin VI tail coupled to glutathione beads in 10 mM Hepes, pH 7.4, 1112, 1107–1109, and 1102–1104, respectively, (named TWKS, etc.) 150 mM NaCl, 1% Triton X-100. After extensive washing the protein com- along with T1088A/T1091A and T1088E/T1091E double mutants. T plexes bound to glutathione beads were separated by SDS PAGE and an- (chicken Myosin VI whole tail with the large insert (LI)), CC (aa 846– alyzed by autoradiography. 1034), GTLI (aa 1035–1277), and GT-LI (aa 1061–1277) were con- structed previously (Morris et al., 2002). The mammalian two-hybrid as- Transmission electron microscopy say was performed as previously described (Morris et al., 2002). Cells were grown in 6-cm tissue cultures dishes and fixed with 2.5% glu- taraldehyde/2% PFA in 0.1 M Na cacodylate buffer, pH 7.2, at 37 C. Cell culture and transfection After washing with 0.1 M Na cacodylate buffer, pH 7.2, cells were fixed NRK cells, CHO cells, and HeLa cells were cultured as described before after in 1% osmium tetroxide in 0.1 M Na cacodylate buffer, pH 7.2, for (Buss et al., 1998). For transient transfection experiments cells were trans- 1 h and washed with 0.05 M Na maleate buffer, pH 5.2. The cells were fected either with 2 g of pEGFP-optineurin, pEGFP-MyosinVI, pEGFP- then en bloc stained with 0.5% uranyl acetate in 0.05 M Na maleate Rab8-WT, or pEGFP-Rab8-Q67L for 16–18 h using FuGENE (Roche Diag- buffer, pH 5.2, dehydrated in ethanol, exchanged into 1,2-epoxy pro- nostics) according to the manufacturer’s instructions. Snell’s waltzer mouse pane, and embedded in Araldite CY212 epoxy resin (Agar Scientific). immortal cell lines (Warner et al., 2003) were transfected using the MEF-1 Ultrathin sections were collected on EM grids and stained with uranyl ac- Nucleofector kit (Amaxa Biosystems) according to the manufacturer’s in- etate and Reynolds lead citrate. The sections were observed in a transmis- structions with chicken full-length myosin VI, the mutant myosin VI (RRL), or sion electron microscope (model CM100; Philips Electron Optics) at an only GFP cloned into pMEP (Buss et al., 2001a). The cells were grown operating voltage of 80 kV. and selection performed as previously described (Warner et al., 2003). Online supplemental material SEAP expression and assay Fig. S1 shows colocalization of optineurin with wild-type huntingtin at the Snell’s waltzer fibroblasts (SV), wild-type fibroblasts (WT; Warner et al., Golgi complex in HeLa cells. Fig. S2 A demonstrates that optineurin binds 2003) and Snell’s waltzer expressing either full-length wild-type myosin VI- to wild-type Rab8 and the Rab8 Q67L mutant but not the Rab8 T22N mu- GFP (MVI) or the mutant myosin VI (RRL) were transiently transfected with tant in vitro. Fig. S2 B shows colocalization of endogenous optineuin with the pSEAP2-control mammalian expression plasmid (CLONTECH Laborato- wild-type GFP-Rab8 and the GFP-Rab8 Q67L mutant at the Golgi com- ries, Inc.) containing SEAP cDNA using the MEF-1 Nucleofector kit (Amaxa plex and in vesicular structures throughout the cytosol. Online supplemen- Biosystems) according to the manufacturer’s instructions. The SEAP assay tal material is available at http://www.jcb.org/cgi/content/full/jcb. was performed as previously described (Warner et al., 2003). 200501162/DC1. We thank Drs R. Duden, R. Pepperkok, J. Simpson, and H. Davidson for gen- Knockdown of optineurin by siRNA erous gifts of antibodies and reagents; and Drs. M. Chibalina and P. Pryor for HeLa and NRK cells were transfected with siRNA duplex (20 M; Dhar- help and advice. macon Research) using OligofectAMINE (Invitrogen) according to the This work was funded by a Wellcome Trust Senior Fellowship (F. Buss), manufacturer’s manual. For efficient knockdown of optineurin cells were a Wellcome Trust studentship (D.A. Sahlender) and a Royal Society USA post- transfected twice with siRNA duplex on day 1 and day 3. On day 5 cells doctoral fellowship (G. Spudich) and supported by the Medical Research were processed for indirect immunofluoresence and the efficiency of Council. Cambridge Institute for Medical Research is in receipt of a strategic knockdown was assayed by Western blotting. In the mock experiment award from the Wellcome Trust. O. Human and rat optineurin the siRNA duplex was substituted with H were targeted with two independent siRNAs: 5-CCACCAGCTGAAA- Submitted: 31 January 2005 GAAGCC-3 and 5-CTGCAGCTCAAGCTGAACT-3. Both siRNAs gave Accepted: 8 March 2005 the same level of knockdown and phenotypes in optineurin-depleted cells. Indirect immunofluorescence microscopy Indirect immunofluorescence staining was performed as described in Buss et References al. (1998). To visualize optineurin at the Golgi complex, cells were pre-per- meabilized with 0.05% saponin in cytosol buffer (Morris and Cooper, 2001) Ang, A.L., H. Folsch, U.M. Koivisto, M. Pypaert, and I. Mellman. 2003. The for 30 s. Cells were analyzed and photographed using 63 or 100 objec- Rab8 GTPase selectively regulates AP-1B–dependent basolateral tives of an Axiophot microscope (Carl Zeiss MicroImaging, Inc.) equipped transport in polarized Madin-Darby canine kidney cells. J. Cell Biol. with a CCD camera. Images were further processed in Adobe Photoshop. 163:339–350. Ang, A.L., T. Taguchi, S. Francis, H. Folsch, L.J. Murrells, M. Pypaert, G. War- VSV-G-GFP transport assay ren, and I. Mellman. 2004. Recycling endosomes can serve as interme- diates during transport from the Golgi to the plasma membrane of 4-d mock- or siRNA-treated HeLa cells were transfected with ts045G VSV- MDCK cells. J. Cell Biol. 167:531–543. G-GFP (a gift from R. Duden, University of London, London, UK) using Fu- GENE as described above. Cells were first incubated for 2 h at 37 C and Aschenbrenner, L., T. Lee, and T. Hasson. 2003. Myo6 facilitates the translocation of endocytic vesicles from cell peripheries. Mol. Biol. Cell. 14:2728–2743. then for 15 h at 39.5 C. Before a temperature shift from 39.5 C to 32 C, 100 g/ml cycloheximide (Sigma-Aldrich) was added to each dish and Balch, W.E., J.M. McCaffery, H. Plutner, and M.G. Farquhar. 1994. Vesicular cells were incubated at 4 C for 30 min. After 20, 40, 60, and 100 min at stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell. 76:841–852. 32 C cells were fixed with 4% PFA and VSV-G present on the cell surface detected with an mAb to the luminal domain of VSV-G. GFP fluorescence Berg, J.S., B.C. Powell, and R.E. Cheney. 2001. A millennial myosin census. gave the total amount of VSV-G expressed in the cell. The ratio of cell sur- Mol. Biol. Cell. 12:780–794. face over total fluorescence was calculated as described previously (Pep- Bunn, R.C., M.A. Jensen, and B.C. Reed. 1999. Protein interactions with the perkok et al., 1993; Seemann et al., 2000) using IP lab software. glucose transporter binding protein GLUT1CBP that provide a link be- tween GLUT1 and the cytoskeleton. Mol. Biol. Cell. 10:819–832. Immunoprecipitation and immunoblotting Buss, F., J. Kendrick-Jones, C. Lionne, A.E. Knight, G.P. Cote, and J. Paul For immunoprecipitation A431 cells were lysed and sonicated in extrac- Luzio. 1998. The localization of myosin VI at the Golgi complex and , 0.5% tion buffer containing PBS, 1 mM EDTA, 5 mM ATP, 5 mM MgCl leading edge of fibroblasts and its phosphorylation and recruitment into 294 JCB • VOLUME 169 • NUMBER 2 • 2005 membrane ruffles of A431 cells after growth factor stimulation. J. Cell Traffic. 3:331–341. Biol. 143:1535–1545. Nishikawa, S., K. Homma, Y. Komori, M. Iwaki, T. Wazawa, A. Hikikoshi Buss, F., S.D. Arden, M. Lindsay, J.P. Luzio, and J. Kendrick-Jones. 2001a. Iwane, J. Saito, R. Ikebe, E. Katayama, T. Yanagida, and M. Ikebe. Myosin VI isoform localized to clathrin-coated vesicles with a role in 2002. Class VI myosin moves processively along actin filaments back- clathrin-mediated endocytosis. EMBO J. 20:3676–3684. ward with large steps. Biochem. Biophys. Res. Commun. 290:311–317. Buss, F., J.P. Luzio, and J. Kendrick-Jones. 2001b. Myosin VI, a new force in Pepperkok, R., J. Scheel, H. Horstmann, H.P. Hauri, G. Griffiths, and T.E. Kreis. clathrin mediated endocytosis. FEBS Lett. 508:295–299. 1993. Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell. 74:71–82. Buss, F., G. Spudich, and J. Kendrick-Jones. 2004. Myosin VI: cellular fuctions and motor properties. Annu. Rev. Cell Dev. Biol. 20:649–676. Rambourg, A., and Y. Clermont. 1990. Three-dimensional electron microscopy: structure of the Golgi apparatus. Eur. J. Cell Biol. 51:189–200. DiFiglia, M., E. Sapp, K. Chase, C. Schwarz, A. Meloni, C. Young, E. Martin, J.P. Vonsattel, R. Carraway, S.A. Reeves, et al. 1995. Huntingtin is a cy- Rezaie, T., A. Child, R. Hitchings, G. Brice, L. Miller, M. Coca-Prados, E. toplasmic protein associated with vesicles in human and rat brain neu- Heon, T. Krupin, R. Ritch, D. Kreutzer, et al. 2002. Adult-onset pri- rons. Neuron. 14:1075–1081. mary open-angle glaucoma caused by mutations in optineurin. Science. 295:1077–1079. Elbashir, S.M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in Rock, R.S., S.E. Rice, A.L. Wells, T.J. Purcell, J.A. Spudich, and H.L. cultured mammalian cells. Nature. 411:494–498. Sweeney. 2001. Myosin VI is a processive motor with a large step size. Proc. Natl. Acad. Sci. USA. 98:13655–13659. Engelender, S., A.H. Sharp, V. Colomer, M.K. Tokito, A. Lanahan, P. Worley, E.L. Holzbaur, and C.A. Ross. 1997. Huntingtin-associated protein 1 Rogalski, A.A., and S.J. Singer. 1984. Associations of elements of the Golgi ap- (HAP1) interacts with the p150Glued subunit of dynactin. Hum. Mol. paratus with microtubules. J. Cell Biol. 99:1092–1100. Genet. 6:2205–2212. Schwamborn, K., R. Weil, G. Courtois, S.T. Whiteside, and A. Israel. 2000. Faber, P.W., G.T. Barnes, J. Srinidhi, J. Chen, J.F. Gusella, and M.E. Mac- Phorbol esters and cytokines regulate the expression of the NEMO- Donald. 1998. Huntingtin interacts with a family of WW domain pro- related protein, a molecule involved in a NF-kappa B-independent path- teins. Hum. Mol. Genet. 7:1463–1474. way. J. Biol. Chem. 275:22780–22789. Gauthier, L.R., B.C. Charrin, M. Borrell-Pages, J.P. Dompierre, H. Rangone, Seabra, M.C., and E. Coudrier. 2004. Rab GTPases and myosin motors in or- F.P. Cordelieres, J. De Mey, M.E. MacDonald, V. Lessmann, S. Hum- ganelle motility. Traffic. 5:393–399. bert, and F. Saudou. 2004. Huntingtin controls neurotrophic support and Seemann, J., E.J. Jokitalo, and G. Warren. 2000. The role of the tethering pro- survival of neurons by enhancing BDNF vesicular transport along micro- teins p115 and GM130 in transport through the Golgi apparatus in vivo. tubules. Cell. 118:127–138. Mol. Biol. Cell. 11:635–645. Gill, S.R., T.A. Schroer, I. Szilak, E.R. Steuer, M.P. Sheetz, and D.W. Cleve- Stroissnigg, H., M. Repitz, A. Miloloza, I. Linhartova, H. Beug, G. Wiche, and land. 1991. Dynactin, a conserved, ubiquitously expressed component of F. Propst. 2002. FIP-2, an IkappaB-kinase-gamma-related protein, is as- an activator of vesicle motility mediated by cytoplasmic dynein. J. Cell sociated with the Golgi apparatus and translocates to the marginal band Biol. 115:1639–1650. during chicken erythroblast differentiation. Exp. Cell Res. 278:133–145. Hammer, J.A., III, and X.S. Wu. 2002. Rabs grab motors: defining the connec- Van De Moortele, S., R. Picart, A. Tixier-Vidal, and C. Tougard. 1993. Nocoda- tions between Rab GTPases and motor proteins. Curr. Opin. Cell Biol. zole and taxol affect subcellular compartments but not secretory activity 14:69–75. of GH3B6 prolactin cells. Eur. J. Cell Biol. 60:217–227. Harada, A., Y. Takei, Y. Kanai, Y. Tanaka, S. Nonaka, and N. Hirokawa. 1998. Velier, J., M. Kim, C. Schwarz, T.W. Kim, E. Sapp, K. Chase, N. Aronin, and M. Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic DiFiglia. 1998. Wild-type and mutant huntingtins function in vesicle traf- dynein. J. Cell Biol. 141:51–59. ficking in the secretory and endocytic pathways. Exp. Neurol. 152:34–40. Harjes, P., and E.E. Wanker. 2003. The hunt for huntingtin function: interaction Warner, C.L., A. Stewart, J.P. Luzio, K.P. Steel, R.T. Libby, J. Kendrick-Jones, partners tell many different stories. Trends Biochem. Sci. 28:425–433. and F. Buss. 2003. Loss of myosin VI reduces secretion and the size of Hattula, K., and J. Peranen. 2000. FIP-2, a coiled-coil protein, links Huntingtin to the Golgi in fibroblasts from Snell’s waltzer mice. EMBO J. 22:569–579. Rab8 and modulates cellular morphogenesis. Curr. Biol. 10:1603–1606. Wehland, J., M.C. Willingham, M.G. Gallo, and I. Pastan. 1982. The morpho- Hodge, T., and M.J. Cope. 2000. A myosin family tree. J. Cell Sci. 113:3353– logic pathway of exocytosis of the vesicular stomatitis virus G protein in 3354. cultured fibroblasts. Cell. 28:831–841. Huber, L.A., M.J. de Hoop, P. Dupree, M. Zerial, K. Simons, and C. Dotti. Wells, A.L., A.W. Lin, L.Q. Chen, D. Safer, S.M. Cain, T. Hasson, B.O. Car- 1993a. Protein transport to the dendritic plasma membrane of cultured ragher, R.A. Milligan, and H.L. Sweeney. 1999. Myosin VI is an actin- neurons is regulated by rab8p. J. Cell Biol. 123:47–55. based motor that moves backwards. Nature. 401:505–508. Huber, L.A., S. Pimplikar, R.G. Parton, H. Virta, M. Zerial, and K. Simons. Wu, H., J.E. Nash, P. Zamorano, and C.C. Garner. 2002. Interaction of SAP97 1993b. Rab8, a small GTPase involved in vesicular traffic between the with minus-end-directed actin motor myosin VI. Implications for AMPA TGN and the basolateral plasma membrane. J. Cell Biol. 123:35–45. receptor trafficking. J. Biol. Chem. 277:30928–30934. Joe, M.K., S. Sohn, W. Hur, Y. Moon, Y.R. Choi, and C. Kee. 2003. Accumula- Zerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nat. tion of mutant myocilins in ER leads to ER stress and potential cytotoxic- Rev. Mol. Cell Biol. 2:107–117. ity in human trabecular meshwork cells. Biochem. Biophys. Res. Commun. 312:592–600. Kreis, T.E., and H.F. Lodish. 1986. Oligomerization is essential for transport of ve- sicular stomatitis viral glycoprotein to the cell surface. Cell. 46:929–937. Li, S.H., C.A. Gutekunst, S.M. Hersch, and X.J. Li. 1998a. Interaction of huntingtin-associated protein with dynactin P150Glued. J. Neurosci. 18:1261–1269. Li, X.J., S.H. Li, A.H. Sharp, F.C. Nucifora Jr., G. Schilling, A. Lanahan, P. Wor- ley, S.H. Snyder, and C.A. Ross. 1995. A huntingtin-associated protein enriched in brain with implications for pathology. Nature. 378:398–402. Li, Y., J. Kang, and M.S. Horwitz. 1998b. Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-induc- ible cellular protein containing leucine zipper domains. Mol. Cell. Biol. 18:1601–1610. Lister, I., S. Schmitz, M. Walker, J. Trinick, F. Buss, C. Veigel, and J. Ken- drick-Jones. 2004. A monomeric myosin VI with a large working stroke. EMBO J. 23:1729–1738. Luzio, J.P., B. Brake, G. Banting, K.E. Howell, P. Braghetta, and K.K. Stanley. 1990. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38). Biochem. J. 270:97–102. Morris, S.M., and J.A. Cooper. 2001. Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2. Traffic. 2:111–123. Morris, S.M., S.D. Arden, R.C. Roberts, J. Kendrick-Jones, J.A. Cooper, J.P. Luzio, and F. Buss. 2002. Myosin VI binds to and localises with Dab2, po- tentially linking receptor-mediated endocytosis and the actin cytoskeleton. OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 295 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

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Copyright © 2005, The Rockefeller University Press
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JCB: ARTICLE Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis 1 2 1 2 1 1 Daniela A. Sahlender, Rhys C. Roberts, Susan D. Arden, Giulietta Spudich, Marcus J. Taylor, J. Paul Luzio, 2 1 John Kendrick-Jones, and Folma Buss Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, England, UK MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, England, UK yosin VI plays a role in the maintenance of G-protein to the plasma membrane is dramatically reduced. Golgi morphology and in exocytosis. In a Two further binding partners for optineurin have been yeast 2-hybrid screen we identified optineurin identified: huntingtin and Rab8. We show that myosin VI as a binding partner for myosin VI at the Golgi complex and Rab8 colocalize around the Golgi complex and in and confirmed this interaction in a range of protein inter- vesicles at the plasma membrane and overexpression of action studies. Both proteins colocalize at the Golgi constitutively active Rab8-Q67L recruits myosin VI onto complex and in vesicles at the plasma membrane. When Rab8-positive structures. These results show that optineurin optineurin is depleted from cells using RNA interference, links myosin VI to the Golgi complex and plays a central myosin VI is lost from the Golgi complex, the Golgi is role in Golgi ribbon formation and exocytosis. fragmented and exocytosis of vesicular stomatitis virus Introduction In membrane trafficking pathways, motor proteins moving membrane ruffles (Buss et al., 1998), the Golgi complex, and along cytoskeletal tracks play a major role in transporting secretory vesicles (Buss et al., 1998; Warner et al., 2003). vesicles between donor and acceptor compartments and may Unlike all the other myosins that have been studied so far that also be involved in processes such as cargo sorting, vesicle move toward the plus end of actin filaments, myosin VI moves formation, and steady-state localization of organelles. Short- toward the minus end of actin (Wells et al., 1999). Functional range movement of cargo or vesicles along actin filaments, studies have indicated that myosin VI plays a major role in around internal organelles, or within the cortical regions of the endocytic and secretory membrane traffic pathways (Buss et cell is powered by members of the myosin superfamily, which al., 2001b; Warner et al., 2003) and it has been postulated that is comprised of at least 18 different classes (Hodge and Cope, the diverse functions of myosin VI are mediated by interaction 2000; Berg et al., 2001). Although in recent years the localization with a number of different binding partners (Buss et al., 2004). and functions of a few of these myosins have been identified, Recently, three binding partners of myosin VI were identified, there is still limited information regarding the molecular mech- Dab2, GIPC, and SAP97, all of which target myosin VI to anism linking myosin function and cargo attachment. For vesicular compartments (Bunn et al., 1999; Morris et al., 2002; example, how does a myosin recognize its cargo; how is the Wu et al., 2002). So far, the best-characterized myosin VI– interaction regulated and what influence does cargo binding binding partner is Dab2; its interaction with myosin VI has have on motor activity? been shown to form a dynamic link between cell surface receptors, Myosin VI is a multifunctional motor protein found in a clathrin-mediated endocytosis, and the actin cytoskeleton (Mor- number of different intracellular compartments including en- ris and Cooper, 2001; Morris et al., 2002). In contrast, no bind- docytic vesicles (Buss et al., 2001b; Aschenbrenner et al., 2003), ing partners have been identified that targets myosin VI to the Golgi complex and the secretory pathway. In this paper, we have identified and characterized opti- D.A. Sahlender and R.C. Roberts contributed equally to this work. neurin, a novel myosin VI–binding partner, which is found at Correspondence to Folma Buss:fb1@mole.bio.cam.ac.uk the Golgi complex. Optineurin was first discovered as a binding Abbreviations used in this paper: MTOC, microtubule organizing center; NRK, partner of the adenoviral protein E3-14.7K (14.7K-interacting normal rat kidney; SEAP, soluble secreted form of the alkaline phosphatase; protein-2 and therefore named FIP-2) and was shown to protect siRNA, small interfering RNA; VSV-G, vesicular stomatitis virus G-protein. The online version of this article contains supplemental material. infected cells from TNF-–induced cytolysis (Li et al., 1998b). © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 169, No. 2, April 25, 2005 285–295 http://www.jcb.org/cgi/doi/10.1083/jcb.200501162 JCB 285 THE JOURNAL OF CELL BIOLOGY It is a conserved 67-kD protein with multiple leucine zipper do- which is interesting, because Rab8 is a regulator of post-Golgi mains and a putative zinc finger domain at the COOH termi- membrane traffic from the TGN to the plasma membrane. nus. Optineurin shows strong homology (53% identity) with NF-B essential modulator and was therefore also called Results NEMO-related protein (Schwamborn et al., 2000). Mutations Optineurin binds to the COOH-terminal in the human optineurin gene are associated with adult-onset tail of myosin VI open angle glaucoma (hence it was named “optic neuropathy inducing” protein optineurin; Rezaie et al., 2002). The COOH-terminal tail domain of myosin VI is required for Although optineurin was previously localized to the Golgi targeting to intracellular locations such as clathrin-coated vesi- complex (Schwamborn et al., 2000; Stroissnigg et al., 2002) its cles (Buss et al., 2001a) and the Golgi complex (Warner et al., functions at this organelle have not yet been established. How- 2003). To identify binding partners for myosin VI in the Golgi ever, two binding partners for optineurin have been identified complex we used the whole tail of myosin VI as a bait in a which link it to membrane trafficking events. One is huntingtin, yeast two-hybrid screen of a human umbilical vein epithelial the protein mutated in the neurodegenerative disorder Hunting- cell cDNA library (Morris et al., 2002). A 375–amino acid ton’s disease (Faber et al., 1998), and the other is the small COOH-terminal fragment of optineurin (residues 198–577) GTPase Rab8 (Hattula and Peranen, 2000). Although the precise was identified in the screen as a myosin VI–binding partner cellular functions of the wild-type huntingtin protein are not (Fig. 1 A). To confirm this interaction both myosin VI and op- known, its intracellular localization to the Golgi complex and to tineurin were overexpressed in CHO cells and the binding was endocytic and exocytic vesicles (DiFiglia et al., 1995; Velier et measured in a mammalian two-hybrid assay (Fig. 1 B). Dele- al., 1998), as well as the identity of its binding partners (Harjes tion mutants of optineurin revealed that the myosin VI–binding and Wanker, 2003), suggests a role in membrane trafficking site on optineurin is between amino acids 412 and 520 (not de- pathways (Gauthier et al., 2004). The other optineurin binding picted). To determine the site on myosin VI that binds to op- partner Rab8 belongs to a large family of small GTPases that tineurin a combination of deletion mutants followed by site- participate in and regulates intracellular membrane trafficking directed mutagenesis was used (Fig. 1 B). The results indicate pathways (Zerial and McBride, 2001). In their active GTP- that the globular tail (GT; aa 1034–1276) contains the binding bound state different Rab proteins bind to different membrane site, because the NH -terminal helical part of the tail (HT; aa compartments and recruit specific effector proteins, which are 845–1033; Fig. 1 B, columns 1 and 2) did not bind. Further- not only involved in docking and fusion with the target mem- more, myosin VI interaction with optineurin does not require brane but also in the formation of transport vesicles and in bind- the large insert (LI) found in some isoforms (Fig. 1 B, columns ing motor proteins for vesicle transport (Zerial and McBride, 3 and 4); this part of the tail has previously been shown to be 2001; Hammer and Wu, 2002; Seabra and Coudrier, 2004). involved in targeting myosin VI to clathrin-coated vesicles at Several examples of motor protein–Rab complexes are known; the apical domain of polarized cells (Buss et al., 2001b). Be- for example those involving microtubule motors such as the ki- cause deletion of the COOH-terminal 159 aa of the myosin VI nesin-like protein (Rabkinesin-6) and Rab6 as well as actin- tail did not abolish binding (Fig. 1 B, column 5), we used site- based motor complexes such as myosin Va and Rab27a and directed mutagenesis to change three adjacent residues at a myosin Vb and Rab11 (Hammer and Wu, 2002). time to alanine in order to narrow down the binding site in the Rab8 interacts specifically with the NH terminus of op- remainder of the GT region, i.e., between aa 1060 and 1117. tineurin (Hattula and Peranen, 2000). Rab8 has been localized Fig. 1 B shows that changing WKS to AAA, KVY to AAA, to the Golgi region, to TGN-derived transport vesicles and to and REE to AAA did not reduce binding (Fig. 1, columns 6, 7, the plasma membrane (Huber et al., 1993b) and has been and 9), however mutating RRL to AAA abolished binding of shown to regulate biosynthetic trafficking pathways from the the myosin VI tail to optineurin (column 8), clearly showing TGN to the cell surface (Huber et al., 1993b; Ang et al., 2003). that these residues constitute the binding site for optineurin. In our present study, we have identified an RRL se- In vitro phosphorylation studies using recombinant p21– quence in the tail domain of myosin VI that binds to a site in activated kinase (PAK; Buss et al., 1998) and the tail of myosin the COOH-terminal region of optineurin. Both proteins colo- VI expressed in E. coli indicated that threonine 1088 and threo- calize at the Golgi complex and in vesicular structures close nine 1091 (the sequence TINT; Fig. 1 A) are potential phos- to the plasma membrane. The depletion of optineurin using phorylation sites (unpublished data). These threonines are also small interfering RNA (siRNA) techniques drastically alters the phosphorylated in vivo, because when full-length myosin VI morphology of the Golgi complex and reduces significantly was isolated from A431 cells by immunoprecipitation and sub- transport of vesicular stomatitis virus G-protein (VSV-G) to jected to MALDI mass spectrometric analysis a peptide con- the cell surface indicating that optineurin plays an important taining this phosphorylated TINT motif was detected. This role in Golgi ribbon formation and in exocytosis. In addition, peptide was dephosphorylated after alkaline phosphatase treat- depletion of optineurin causes a marked reduction in the ment (unpublished data). We mutated these two threonines to amount of myosin VI associated with the Golgi complex sug- either alanines or glutamates to mimic the nonphosphorylated gesting that optineurin is the anchor for myosin VI at this or- and phosphorylated states respectively in order to test their ef- ganelle. We further show that myosin VI colocalizes with fects on the binding of myosin VI to optineurin in the mamma- Rab8 at the Golgi complex and at the plasma membrane, lian two-hybrid assay. Whereas no effect was seen with the 286 JCB • VOLUME 169 • NUMBER 2 • 2005 alanine-mutated construct AINA (Fig. 1 B, column 10), TINT mutated to EINE abolished optineurin binding to myosin VI (col- umn 11) but did not abolish myosin VI binding to Dab2, which was used as a control. Because the EINE mutation potentially mimics the phosphorylated form, this data suggests that phos- phorylation regulates the binding of optineurin to myosin VI. A GST pull-down assay gave further confirmation that optineurin binds directly to the tail of myosin VI. In vitro– translated [ S]methionine labeled optineurin or Dab2 as a pos- itive control (Fig. 1 C, lanes 2 and 3) were incubated with ei- ther GST or GST myosin VI tail. The autoradiogram in Fig. 1 C shows that optineurin binds to the myosin VI tail (Fig. 1 C, lane 5), but not to GST alone (lane 4) or to luciferase as a nega- tive control protein (lanes 6 and 7). To confirm that myosin VI binds to optineurin in vivo, we immunoprecipitated endoge- nous myosin VI and endogenous optineurin from the cytosol of A431 cells. When myosin VI was immunoprecipitated with myosin VI tail antibodies optineurin is coimmunoprecipitated (Fig. 1 D, lane 4), indicating that optineurin and myosin VI ex- ist in a protein complex in vivo. Mutant myosin VI (RRL) does not rescue secretion in Snell’s waltzer fibroblasts In fibroblastic cell lines derived from Snell’s waltzer mice (sv/ sv) the absence of myosin VI causes a reduction in constitutive secretion. As shown previously this phenotype can be restored by overexpression of a fully functional myosin VI (Warner et al., 2003). To test whether binding of optineurin is essential for myosin VI function at the Golgi complex in vivo, we per- formed similar rescue experiments in sv/sv fibroblasts. Immor- tal fibroblastic sv/sv or wild-type cell lines were stably trans- binds to purified myosin VI tail. A pull down was performed using in vitro– translated optineurin and GST-myosin VI tail. S-labeled in vitro–trans- lated full-length optineurin (lanes 4 and 5), Dab2 as a positive control (lanes 2 and 3) or luciferase as a negative control (lanes 6 and 7) were incubated with either 5 g GST alone (lanes 2, 4, and 6) or with 5 g GST-myosin VI tail (lanes 3, 5, and 7). Lane 1 shows 50% of the input used for the pulldown with [ S]optineurin in lane 5. The band below Dab2 is possibly caused by degradation (lane 3). (D) Co-immunoprecipi- Figure 1. Optineurin is a myosin VI–binding partner. (A) A carton show- tation of endogenous myosin VI and optineurin from the cytosol of A431 ing the major regions in optineurin and myosin VI tail. Optineurin contains cells. Immunoprecipitation was performed under native conditions using an NH -terminal Rab8-binding domain (aa 141–209; Hattula and Peranen, no antibody, only protein A beads as a control (lane 2, blank), an anti- 2000) and a COOH-terminal myosin VI–binding domain (aa 412–520). body to full-length optineurin (lane 3, a-Optn) or a pAb to the tail of Myosin VI tail has a proposed helical tail domain (HT) and a globular tail myosin VI (lane 4, a-MVI). Lane 1 is equivalent to 5% of the input used for (GT), which contains the optineurin-binding site RRL in position 1107– each immunoprecipitation. Immunoprecipitates were run out on a 10% 1109. The putative phosphorylation site TINT is at position 1088–1091. PAGE gel, blotted, and analyzed using an antibody to optineurin. (E) Con- (B) Using the ProQuest yeast two-hybrid screen optineurin was identified stitutive secretion in Snell’s waltzer fibroblasts cannot be rescued by over- as a myosin interacting protein. The interaction was verified using the expression of the mutant myosin VI lacking the optineurin-binding site mammalian two-hybrid system. To narrow down the optineurin-binding (RRL). Rescue experiments were performed by generating Snell’s waltzer site on the myosin VI tail the whole tail (T; column 1), the helical tail cell lines stably expressing only GFP (SV), the mutant myosin VI without the (HT; column 2) or the globular tail (GT) with or without the large insert optineurin-binding site (RRL), or wild-type whole myosin VI (MVI). Levels (GTLI or GT-LI; columns 3 and 4) were fused to the Gal4 DNA-binding of secretion were compared with wild-type cells stably expressing only domain and coexpressed with full-length optineurin fused to the VP16 acti- GFP (WT). To measure constitutive secretion, a construct, which expresses vating domain in CHO cells. Luciferase activity is shown relative to control a SEAP was transfected into the various Snell’s waltzer cell lines and the cells containing only the myosin VI construct with the empty prey vector. wild-type fibroblasts. The amount of alkaline phosphatase secreted into the Deletion of myosin VI tail at residue 1117 (TC; column 5) did not abolish media was measured 24, 48, and 72 h after transfection and plotted as a binding. Mutating three adjacent amino acids each (WKS, KVY, RRL, and percentage of total maximal secretion in wild-type cells after 72 h. Trans- REE to AAA) revealed RRL as the optineurin-binding site (columns 6–9). fection efficiency was normalized by cotransfection of a control plasmid Two threonine residues in positions 1088 and 1091 (TINT) were mutated to expressing a red fluorescent protein. The data from two separate experi- either alanine (AINA) or glutamate (EINE; columns 10 and 11). (C) Optineurin ments run in triplicate are shown. OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 287 fected with GFP, GFP-tagged whole wild-type myosin VI or myosin VI with mutations in the optineurin-binding site (RRL). Constitutive secretion in these stable cell lines was measured using a soluble secreted form of the alkaline phos- phatase (SEAP) assay as previously described (Warner et al., 2003). We observed that only fully functional myosin VI is able to restore wild-type levels of secretion, whereas cells ex- pressing myosin VI with the mutant optineurin-binding site are unable to increase secretion levels back to those of the wild type (Fig. 1 E). Myosin VI and optineurin colocalize at the Golgi complex and in vesicles underneath the plasma membrane To test the interaction between myosin VI and optineurin in vivo, we investigated their intracellular localization in normal rat kidney (NRK) cells. Previously, both proteins had been lo- calized independently to the Golgi complex (Buss et al., 1998; Hattula and Peranen, 2000; Schwamborn et al., 2000; Stroiss- nigg et al., 2002; Warner et al., 2003). Using an affinity-purified pAb to optineurin and the TGN marker, TGN38, we confirm that optineurin is localized at the Golgi complex (Fig. 2, a–c). Double labeling experiments using optineurin antibodies and an mAb to myosin VI (Warner et al., 2003) showed considerable colocalization of both endogenous proteins at the Golgi complex (Fig. 2, d–f). In addition, both proteins were found in vesicular structures in the cell periphery close to the plasma membrane (Fig. 2, g–i and g–i). We transiently overexpressed optineurin Figure 2. Optineurin and myosin VI colocalize in NRK cells. Endogenous optineurin localizes to the Golgi complex using a pAb to optineurin (a) or fragments of optineurin tagged with either GFP, Flag, or myc and an mAb to TGN38 (b). Optineurin and myosin VI are localized at the at their COOH or NH termini in a number of different cell types Golgi complex using a pAb to optineurin (d) and an mAb to myosin VI (e). for colocalization studies with the aim of expressing optineurin Colocalization in vesicular structures close to the plasma membrane was demonstrated with the pAb to optineurin (g) and by transiently overex- mutants that might function as dominant negative. However, pressing myosin VI tagged with GFP (h). White boxes indicate areas en- none of the transiently expressed optineurin constructs gave the larged in the pictures below (g, h, and i). Endogenous myosin VI, visual- same intracellular localization as endogenous optineurin (un- ized using the pAb against the whole tail, is recruited into aggregates containing overexpressed GFP-optineurin (j–k). The merged images are published data); the overexpressed optineurin was not observed shown in c, f, i, i, and l. Bars, 10 m. at the Golgi complex but showed an overall vesicular distribu- tion and formed aggregates of various sizes in the cytoplasm. It would appear that the addition of the GFP, Flag, or myc tags to and HeLa cells (see Materials and methods) and the best results the NH or COOH terminus of optineurin or even to its frag- were achieved after repeated transfection over a period of 4 d. In ments alters the protein conformation in some way and masks these cells optineurin was depleted to 10% of its original level domains important for intracellular targeting to the Golgi com- as shown by Western blotting of equal amounts of mock and plex. Interestingly, endogenous myosin VI was recruited from siRNA-treated cells 48 h after the second transfection (Fig. 3 A, its cytosolic pool into these optineurin aggregates (Fig. 2, j–l), a and B, a). In the same cells expression levels of myosin VI and indicating that the myosin VI–binding site is maintained and ac- actin were unaffected (Fig. 3 A, a and B, a). In the knockdown cessible. NH -terminal optineurin constructs without the myosin cells immunofluorescence microscopy showed that very little VI–binding domain (aa 412–520) were unable to recruit endog- optineurin was present in the Golgi complex (Fig. 3 A, d and B, enous myosin VI into these aggregates (not depicted). These d), although in some cells residual cytosolic labeling was appar- results strongly indicate that myosin VI binds to and forms a ent (Fig. 3 A, d). To find out what happens to the localization of protein complex with optineurin in vivo. myosin VI in optineurin knockdown cells, we double labeled Depletion of optineurin by siRNA causes siRNA and mock-transfected NRK cells with a myosin VI loss of myosin VI from the Golgi complex COOH-terminal globular tail antibody and with antibodies to GM130 and TGN38. In optineurin-depleted cells there was a Does optineurin play a role in linking myosin VI to the Golgi marked reduction in the amount of myosin VI associated with complex? To address this question we used siRNA to reduce the Golgi complex (Fig. 4, c and g) as compared with mock- cellular expression levels of optineurin (Elbashir et al., 2001). transfected cells (Fig. 4, a and e) indicating that optineurin plays For the knockdown experiments two siRNA duplexes, specific a role in anchoring myosin VI to the Golgi complex. for the nucleotide sequence of optineurin, were tested in NRK 288 JCB • VOLUME 169 • NUMBER 2 • 2005 Figure 4. Knockdown of optineurin in NRK cells causes loss of myosin VI from the Golgi complex. Mock-transfected (a, b, e, and f) or siRNA-trans- fected (c, d, g, and h) NRK cells were used for immunofluorescence and were double labeled with antibodies to the globular tail of myosin VI (a, c, e, and g) and antibodies to TGN38 (b and d) or GM130 (f and h). Asterisks mark the position of the nucleus. Bars, 10 m. membranes into a ribbon like structure (Rambourg and Cler- mont, 1990). After depolymerization of microtubules using drugs such as nocodazole, these tubular connections are lost and the Golgi stacks are disconnected and dispersed throughout the cells, indicating that the cytoskeleton plays a major role in maintaining this perinuclear organization of the Golgi complex (Rogalski and Singer, 1984). Figure 3. Optineurin is depleted from NRK and HeLa cells using siRNA. When optineurin was depleted by siRNA transfection in HeLa (A) or NRK (B) cells were either mock transfected with water or trans- NRK cells (Fig. 4, d and h) or especially in HeLa cells (Fig. 5, fected twice at 48-h intervals with siRNA specific to optineurin. After 4 d d–f) there was a dramatic effect on the structure of the Golgi cells were blotted and probed with antibodies to myosin VI, optineurin, or actin (A, a and B, a). In a parallel experiment mock-transfected, siRNA- complex. In the optineurin-depleted HeLa cells the Golgi com- transfected NRK, or HeLa cells were used for immunofluorescence and plex was fragmented into vesicular and sometimes short tubu- double labeled with antibodies to optineurin (A, b and d; B, b and d) lar structures dispersed throughout the whole cytoplasm. These and GM130 (A, c and e; B, c and e). Bars, 10 m. Golgi fragments not only contained the Golgi matrix (GM130) and TGN (TGN46) markers (Fig. 5, d–f) but also markers for Loss of optineurin disrupts the Golgi the cis- and medial-Golgi stacks (not depicted). Although the ribbon structure Golgi matrix and TGN markers were present in a single struc- In mammalian cells the Golgi complex is formed by stacks of ture, they do not completely colocalize, indicating that some flattened membrane cisternae that are interconnected by tubular compartmentalization and organization of the Golgi was still OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 289 preserved in these fragments (Fig. 5, d–f). The Golgi struc- ture was not disrupted due to a breakdown of the microtubular network, because this network was still intact in optineurin- depleted cells (unpublished data). To characterize the phenotype of optineurin-depleted cells at the ultrastructural level, electron microscopy was per- formed on mock and siRNA-treated HeLa cells. In optineurin knockdown cells mini-stacks of Golgi membranes similar to those in control cells were still observed (Fig. 5, g and h). This indicates that although the loss of optineurin caused a break- down of the Golgi ribbon structure, the overall morphology of individual Golgi stacks was maintained and no large-scale ve- siculation was present. Colocalization of myosin VI and Rab8 is optineurin dependent Optineurin has been shown to form a link between huntingtin and Rab8 (Hattula and Peranen, 2000) and optineurin and hun- tingtin can be colocalized at the Golgi complex (Fig. S1, avail- able at http://www.jcb.org/cgi/content/full/jcb.200501162/ DC1). These proteins are both found at the TGN, in transport vesicles throughout the cytoplasm and at the plasma membrane (Huber et al., 1993b; Velier et al., 1998). Optineurin specifi- cally interacts with wild-type Rab8 and with the constitutively active GTP-bound mutant Rab8-Q67L, but not with the domi- nant-negative GDP-bound mutant Rab8-T22N in vitro (Hattula and Peranen, 2000; Fig. S2 A, available at http://www.jcb.org/ cgi/content/full/jcb.200501162/DC1). By immunofluorescence optineurin was shown to colocalize with wild-type Rab8 and the constitutively active Rab8-Q67L at the Golgi complex and Figure 5. The loss of optineurin results in Golgi fragmentation. Mock (a–c) or optineurin siRNA-treated HeLa cells (d–f and d–f) were double labeled in vesicular and tubular structures in the cytoplasm (Fig. S2 B). in immunofluorescence experiments with TGN46 (b, e, and e) and We suggest that optineurin may link myosin VI to Rab8, GM130 (a, d, and d). White boxes indicate areas enlarged in the as it has been suggested that it links huntingtin to Rab8 pictures below (d, e, and f). The Golgi fragments contain both marker proteins as indicated by the yellow color in the merged images (c, f, and f). (Hattula and Peranen, 2000). To check this idea we deter- The arrows highlight the overlap between the marker proteins in (d–f). mined the intracellular distribution of myosin VI and Rab8 in Bars, 10 m. TEM analysis of mock (g) or siRNA-transfected (h) HeLa NRK cells. Both proteins showed good colocalization around cells. Arrows indicate the position of Golgi stacks that can be found in both mock-transfected and siRNA-transfected cells. Bars, 200 nm. the Golgi complex (Fig. 6, a–c), and also in peripheral vesicles Figure 6. Myosin VI colocalizes with Rab8 at the Golgi complex and the plasma membrane. In NRK cells tran- siently overexpressing Rab8 tagged with GFP (b and e) myosin VI was detected with an antibody to the globular tail (a) or to the whole tail (d). Myosin VI and Rab8 are found in the same labeled structures/vesicles at the Golgi complex (c) and at the plasma membrane (f). Arrows indicate vesicles containing both proteins. Bars, 10 m. 290 JCB • VOLUME 169 • NUMBER 2 • 2005 Figure 8. The transport of VSV-G to the cell surface is dramatically reduced in optineurin-depleted HeLa cells. Mock-treated or siRNA-treated HeLa cells were transfected with ts045-VSV-G-GFP to measure the rate of exocy- tosis . VSV-G at the cell surface was detected in indirect immunofluores- cence using an mAb to the luminal domain of VSV-G and total VSV-G expressed was detected using a pAb to GFP. (A) Representative cells for the 40- and 100-min time points are shown to compare the amount of VSV-G on the cell surface in mock- and optineurin-depleted cells. (B) The ratio of cell surface over total VSV-G fluorescence was measured as described in Materials and methods to determine the rate of transport from the Golgi complex to the cell surface. Error bars: SEM. Bars, 10 m. Figure 7. The recruitment of myosin VI to tubular vesicular structures formed after overexpression of the constitutively active form of Rab8 is lost in optineurin-depleted cells. The constitutively active form of Rab8 (GFP- nous myosin VI onto their membranes and led to a dramatic re- Rab8-Q67L) overexpressed in NRK cells localizes with myosin VI at the Golgi complex (a and b) and recruits myosin VI onto tubular and vesicular distribution of myosin VI (Fig. 7, e and e). The recruitment structures emerging from the Golgi complex (e and f). In optineurin-depleted of myosin VI to these Rab8-positive structures requires op- NRK cells, which are overexpressing GFP-Rab8-Q67L myosin VI no longer tineurin, because in optineurin knockdown cells myosin VI is colocalizes with Rab8-Q67L at the Golgi complex (c and d) and is not recruited to tubular and vesicular structures positive for Rab8-Q67L (g and h). no longer present at the fragmenting Golgi complex (Fig. 7, c White boxes highlight the areas of the cells enlarged in the pictures below and d) or in the tubular structures (Fig. 7, g, h, g, and h). (e,f, g, and h). Endogenous myosin VI was detected with either a pAb to the globular tail (a, c, g, and g) or to the whole tail (e and e). Bars, 10 m. Transport of VSV-G from the Golgi complex to the plasma membrane is reduced in optineurin knockdown cells close to the plasma membrane (Fig. 6, d–f). Overexpression of the constitutively active form of Rab8-Q67L colocalized with Myosin VI has been shown to be important for constitutive se- myosin VI at the Golgi complex (Fig. 7, a and b) but also re- cretion in fibroblasts (Warner et al., 2003). To test whether op- sulted in the formation of long tubular structures emerging tineurin as a myosin VI–binding partner present at the Golgi from the Golgi complex (Fig. 7, f and f). These long tubular complex, plays a role in post-Golgi membrane traffic to the cell carriers containing Rab8-Q67L were able to recruit endoge- surface, we used a thermoreversible folding mutant of the OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 291 VSV-G fused to GFP at its cytoplasmic tail (ts045 VSV-G- myosin VI not only in the motor domain (Buss et al., 1998) but GFP) as a reporter molecule for membrane transport in the also in the globular tail domain at threonine 1088 and 1091, secretory pathway (Wehland et al., 1982; Kreis and Lodish, thus coordinating the regulation of motor function with cargo 1986; Balch et al., 1994). This membrane protein misfolds and binding in the tail domain. is retained in the ER at 39.5 C, but upon a temperature shift to In the absence of optineurin the Golgi complex is frag- 32 C, it can fold and move to the Golgi complex and subse- mented. The ribbon structure of interconnected stacks of mem- quently to the plasma membrane. We measured the amount brane cisternae is broken up and the disconnected Golgi stacks of VSV-G at the cell surface relative to the total amount of are dispersed throughout the cytoplasm. The overall appearance VSV-G expressed in the cell using an antibody specific to the of the fragmented Golgi stacks, however, is similar to the ap- luminal domain of this protein (Pepperkok et al., 1993; See- pearance of the Golgi in control cells and no gross vesiculation mann et al., 2000). was observed at the EM level. Golgi resident proteins such as The loss of optineurin has a dramatic effect on transport TGN46 and GM130 were still present in the Golgi fragments. of VSV-G from the Golgi complex to the cell surface. After 40 These morphological changes are very similar to those ob- min in mock-transfected cells VSV-G could be detected at the served when microtubules are disrupted by drugs such as no- cell surface (Fig. 8 A, b), whereas in optineurin knockdown codazole (Rogalski and Singer, 1984), suggesting that op- cells it is still found in the fragmented Golgi (Fig. 8 A, c) and tineurin might be involved in linking Golgi membranes directly not at the cell surface (Fig. 8 A, d). After 100 min very little or indirectly to microtubules around the microtubule organizing VSV-G still remained in the Golgi complex in the mock-trans- center (MTOC). One of the optineurin-binding partners is hun- fected cells and most was on the cell surface (Fig. 8 A, e and f), tingtin, known to interact with HAP1 (Huntingtin-associated however, in the optineurin-depleted cells VSV-G was still re- protein; Li et al., 1995), which binds directly to the dynactin glued tained in the Golgi complex with only a small proportion of it subunit p150 (Engelender et al., 1997; Li et al., 1998a). The present on the cell surface (Fig. 8 A, g and h). To quantify these dynactin complex is involved in linking the minus end directed observations 30 cells per time point were measured in two in- microtubule motor protein dynein to membrane vesicles (Gill et dependent experiments. At every time point less VSV-G was al., 1991). It has been proposed that huntingtin and HAP1 act as detected at the plasma membrane of siRNA-treated cells com- scaffolding proteins regulating the interaction between the dy- pared with mock-transfected cells (Fig. 8 B). Thus, the trans- nein–dynactin complex and cargo such as Golgi membranes port of VSV-G from the Golgi complex to the cell surface was (Harjes and Wanker, 2003). The absence or inactivation of cy- dramatically delayed in the optineurin-depleted cells and only toplasmic dynein leads to fragmentation and spreading of the at later time points did it reach 50% of the control levels. These Golgi complex into the cytoplasm (a phenotype similar to that results clearly indicate that optineurin plays an important role observed after the loss of optineurin; Harada et al., 1998); thus in post-Golgi membrane trafficking of VSV-G from the Golgi it is possible that optineurin via the huntingtin–HAP1 complex complex to the cell surface. may be involved in targeting dynein to Golgi membranes (Fig. 9). Recent results (Gauthier et al., 2004) indicate a further role for the huntingtin–HAP1 complex in the axonal transport of Discussion vesicles containing brain-derived neurotrophic factor along mi- We have previously shown that myosin VI is present at the crotubules and highlight the importance of huntingtin-binding Golgi complex (Buss et al., 1998) and is involved not only in the partners in the progression of the disease. steady-state organization of the Golgi complex but also in post- The loss of optineurin not only disrupts the structure of Golgi membrane traffic (Warner et al., 2003). To investigate the the Golgi complex, but also dramatically reduces secretion of precise roles that myosin VI plays in these processes we identi- VSV-G to the plasma membrane. Reduced transport is not due fied optineurin, a peripheral Golgi protein as a binding partner to Golgi fragmentation because Golgi stacks dispersed by de- for myosin VI. This interaction found in a yeast two-hybrid polymerization of microtubules remain fully functional with screen was confirmed using a range of different protein–protein only a slight reduction in protein secretion (Van De Moortele et interaction studies. Myosin VI and optineurin colocalize at the al., 1993). Reduced exocytosis of VSV-G in the optineurin Golgi complex and in vesicular structures close to the plasma knockdown cells is a very similar phenotype to that observed in membrane. The loss of optineurin not only leads to Golgi frag- the myosin VI knockout mouse, where secretion of alkaline mentation and a reduction in exocytosis, but we also show that phosphatase is also significantly reduced (Warner et al., 2003). optineurin is essential for targeting myosin VI to the Golgi com- Secretion in these cells lacking myosin VI is not restored by plex. Most interestingly optineurin plays a role in colocalizing mutant myosin VI, which is unable to bind to optineurin, indi- myosin VI and Rab8, a member of the Rab family of G-proteins, cating that a functional complex between myosin VI and op- which are important regulators of membrane traffic events. tineurin is required for constitutive exocytosis. Optineurin binds directly to the sequence RRL (residues Rab8 binds to optineurin and plays an important role in 1107–1109) in the globular tail of myosin VI. This binding site polarized membrane transport both from the Golgi complex to is distinct from the Dab2-binding site and is independent of the the basolateral membrane in polarized epithelial cells and to large insert. The binding of optineurin to myosin VI is regulated dendrites in neurons (Huber et al., 1993a,b). Rab8 is present in by phosphorylation at the TINT (1088–1091) site in the globu- the same intracellular compartments as myosin VI and in this lar tail region. Our results suggest that a PAK phosphorylates paper we show that myosin VI and Rab8 colocalize in the peri- 292 JCB • VOLUME 169 • NUMBER 2 • 2005 VI in the Golgi complex (Fig. 9): First, optineurin together with huntingtin links via the minus end directed motor dynein to the microtubule network and also by directly interacting with myosin VI, is linked to the actin cytoskeleton, thus it may play a role in coordinating microtubule-based and actin-based motor activity around the Golgi complex. Support for such a role is provided by our previous observation that in Snell’s waltzer mice the absence of myosin VI causes a reduction in the size of the Golgi complex (Warner et al., 2003), which would be due to the dynein motor complex pulling the Golgi membranes in toward the MTOC resulting in a smaller more compact Golgi complex. Therefore, in the absence of myosin Figure 9. A cartoon illustrating the possible interaction of optineurin with VI the balance of motor proteins is disturbed and dynein wins motor protein complexes at the Golgi complex. Optineurin may play a the “tug of war”. Second, optineurin might link myosin VI to central role in coordinating actin-based and microtubule-based motor func- tion for maintaining Golgi morphology. The optineurin-binding partner Rab8, a regulatory protein known to be involved in sorting huntingtin has been shown to interact with HAP1, which in turn was reported molecules in the exocytic pathway at the TGN and in mem- glued to form a complex with the dynactin subunit p150 and modulate/ brane fusion at the plasma membrane. The linker function of regulate the dynein–dynactin complex. Loss of minus end–directed dynein motor activity causes in fragmentation of the Golgi ribbon structure. On optineurin may be temporally regulated, for example by phos- the other hand loss of myosin VI results in a reduction in the size of the phorylation of individual proteins, including myosin VI and Golgi complex, because now the dynein complex is still active and able to optineurin (Schwamborn et al., 2000). retract the Golgi complex toward the MTOC. However, if optineurin is depleted, both motor complexes are nonfunctional and the Golgi complex In addition, because optineurin is obviously a dimer with is fragmented. multiple leucine zippers it may induce monomeric myosin VI to dimerize for processive movement of vesicles from the TGN to the plasma membrane. nuclear region around the Golgi complex and both are present Our work provides new information on the intracellular on the same vesicles underneath the plasma membrane. In ad- functions of optineurin, which will help elucidate how muta- dition Rab8 has been localized to the recycling endosome, a tions in the optineurin gene lead to primary open angle glau- compartment shown to be involved in post-Golgi membrane coma (Rezaie et al., 2002). Previous reports, that reduced secre- trafficking to the plasma membrane (Ang et al., 2003, 2004). tion of the glycoprotein myocilin leads to primary open angle However, at present we don’t know whether some myosin VI is glaucoma (Joe et al., 2003), support our results that optineurin also associated with the fraction of Rab8 present in the recy- plays a major role in Golgi morphology and exocytosis. cling endosome. On the other hand because Rab8 may interact via optineurin with myosin VI it suggests a possible pathway Materials and methods whereby TGN-derived vesicles are transported short distances Antibodies along actin filaments by myosin VI, before a kinesin motor The following antibodies were used: affinity-purified rabbit pAb to hu- picks up the cargo for long distance transport along microtu- man full-length optineurin (-Optn; GenBank/EMBLDDBJ accession no. bules. Similarly, close to the plasma membrane the Rab8– AF061034); monoclonal -VSV-G to the luminal domain (a gift from R. Pepperkok and J. Simpson, EMBL, Heidelberg, Germany); polyclonal -opti- optineurin–myosin VI complex might be involved in presenting neurin (Cayman); monoclonal -GM130 (BD Transduction Laboratories); the secretory vesicle to the docking site, thus tethering the vesi- monoclonal -TGN38 (Luzio et al., 1990); monoclonal -GFP (Qbiogene); cle close to the plasma membrane before fusion. Overexpres- polyclonal -GFP (Molecular Probes); monoclonal, polyclonal -TGN46 (Serotec); and monoclonal and pAbs to the whole tail (-MVI) and globu- sion of Rab8-Q67L leads to the formation of long tubular ex- lar tail of myosin VI (-MVI-GT; Buss et al., 1998; Warner et al., 2003). tensions from the Golgi complex and it recruits endogenous Yeast two-hybrid screen myosin VI onto these tubular structures. These results indicate Myosin VI interacting proteins were identified in a ProQuest Two-Hybrid that Rab8 serves as a membrane receptor for myosin VI, re- System (Invitrogen) using the tail domain of chicken intestinal brush border cruiting this myosin motor onto a specific intracellular mem- myosin VI (amino acids 846–1277) containing the large insert to screen a human umbilical vein epithelial cell cDNA library (provided by M. McCor- brane compartment via optineurin. Similarly, it was shown re- mack and T. Rabbits, MRC-LMB, Cambridge, UK) as described previously cently that Rab27a recruits myosin Va onto melanosomes via (Morris et al., 2002). 100 mM 3-amino-1,2,4-TRIzol was used to overcome the linker molecule melanophillin (Seabra and Coudrier, 2004). the observed self-activation by the myosin VI tail domain. The potential myo- sin VI tail domain interacting proteins were identified in the yeast two- Optineurin is the first myosin VI–binding partner identi- hybrid screen by selecting transformants that were able to grow on plates fied at the Golgi complex and may play an adaptor or linker lacking leucine, tryptophan, and histidine in the presence of 100 mM role. The recent demonstration that myosin VI can exist as a 3-amino-1,2,4-TRIzol. Colonies containing candidate cDNAs for interacting proteins were isolated and replated on selection agar to test for activation nonprocessive monomer (Lister et al., 2004) and also possibly of the three reporter genes HIS3, URA3, and lacZ. Colonies positive for all as a processive dimer (Rock et al., 2001; Nishikawa et al., three reporter genes were confirmed in the mammalian two-hybrid assay. 2002) may provide a mechanism for the distinct dual roles for Cloning of optineurin and construction of pEGFP-Rab8-Q67L myosin VI in structural maintenance and vesicle transport ac- The full-length version of human optineurin cDNA was generated by PCR tion away from the Golgi complex. We therefore suggest that from Caco-2 cDNA and subcloned into pEGFP (Clontech) or pRSET (Invitro- optineurin may serve two major “linker” functions for myosin gen) for pAb production. pEGFP-Rab8 WT, containing the human wild-type OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 293 Rab8 cDNA sequence (GenBank/EMBL/DDBJ accession no. 498943) IGEPAL CA-630 (Sigma-Aldrich) and protease inhibitor cocktail (Com- was a gift from P. Row (University of Liverpool, Liverpool, UK) and H. plete; Roche) and processed for immunoprecipitation followed by SDS Davidson (University of Colorado, Denver, CO). The constitutively active PAGE and immunoblotting as described previously (Buss et al., 1998). form of Rab8, Rab8-Q67L, was generated by QuikChange Site-Directed Mutagenesis (Stratagene) according to the manufacturer’s instructions. GST pull-down assay The chicken brush border myosin VI tail residues 840–1277 tagged with Mammalian two-hybrid assay GST was expressed in E. coli BL21 (DE3) cells and purified as described Chicken myosin VI tail fragments HT (aa 845–1033) and TC (aa 845– previously (Buss et al., 1998). Full-length optineurin and Dab2 were 1117) were generated by PCR and inserted into the pM “bait” vector cloned into pcDNA3 (Invitrogen) and in vitro translated using the TNT-cou- S]methionine in vitro– (CLONTECH Laboratories, Inc.). The following alanine mutations were pled Reticulocyte Lysate System (Promega). The [ generated using QuikChange Site-Directed Mutagenesis (Stratagene): translated protein was incubated for 2 h at 4 C with 5 g of GST or GST WKS, KVY, RRL, and REE to AAA at aa positions 1115–1117, 1110– myosin VI tail coupled to glutathione beads in 10 mM Hepes, pH 7.4, 1112, 1107–1109, and 1102–1104, respectively, (named TWKS, etc.) 150 mM NaCl, 1% Triton X-100. After extensive washing the protein com- along with T1088A/T1091A and T1088E/T1091E double mutants. T plexes bound to glutathione beads were separated by SDS PAGE and an- (chicken Myosin VI whole tail with the large insert (LI)), CC (aa 846– alyzed by autoradiography. 1034), GTLI (aa 1035–1277), and GT-LI (aa 1061–1277) were con- structed previously (Morris et al., 2002). The mammalian two-hybrid as- Transmission electron microscopy say was performed as previously described (Morris et al., 2002). Cells were grown in 6-cm tissue cultures dishes and fixed with 2.5% glu- taraldehyde/2% PFA in 0.1 M Na cacodylate buffer, pH 7.2, at 37 C. Cell culture and transfection After washing with 0.1 M Na cacodylate buffer, pH 7.2, cells were fixed NRK cells, CHO cells, and HeLa cells were cultured as described before after in 1% osmium tetroxide in 0.1 M Na cacodylate buffer, pH 7.2, for (Buss et al., 1998). For transient transfection experiments cells were trans- 1 h and washed with 0.05 M Na maleate buffer, pH 5.2. The cells were fected either with 2 g of pEGFP-optineurin, pEGFP-MyosinVI, pEGFP- then en bloc stained with 0.5% uranyl acetate in 0.05 M Na maleate Rab8-WT, or pEGFP-Rab8-Q67L for 16–18 h using FuGENE (Roche Diag- buffer, pH 5.2, dehydrated in ethanol, exchanged into 1,2-epoxy pro- nostics) according to the manufacturer’s instructions. Snell’s waltzer mouse pane, and embedded in Araldite CY212 epoxy resin (Agar Scientific). immortal cell lines (Warner et al., 2003) were transfected using the MEF-1 Ultrathin sections were collected on EM grids and stained with uranyl ac- Nucleofector kit (Amaxa Biosystems) according to the manufacturer’s in- etate and Reynolds lead citrate. The sections were observed in a transmis- structions with chicken full-length myosin VI, the mutant myosin VI (RRL), or sion electron microscope (model CM100; Philips Electron Optics) at an only GFP cloned into pMEP (Buss et al., 2001a). The cells were grown operating voltage of 80 kV. and selection performed as previously described (Warner et al., 2003). Online supplemental material SEAP expression and assay Fig. S1 shows colocalization of optineurin with wild-type huntingtin at the Snell’s waltzer fibroblasts (SV), wild-type fibroblasts (WT; Warner et al., Golgi complex in HeLa cells. Fig. S2 A demonstrates that optineurin binds 2003) and Snell’s waltzer expressing either full-length wild-type myosin VI- to wild-type Rab8 and the Rab8 Q67L mutant but not the Rab8 T22N mu- GFP (MVI) or the mutant myosin VI (RRL) were transiently transfected with tant in vitro. Fig. S2 B shows colocalization of endogenous optineuin with the pSEAP2-control mammalian expression plasmid (CLONTECH Laborato- wild-type GFP-Rab8 and the GFP-Rab8 Q67L mutant at the Golgi com- ries, Inc.) containing SEAP cDNA using the MEF-1 Nucleofector kit (Amaxa plex and in vesicular structures throughout the cytosol. Online supplemen- Biosystems) according to the manufacturer’s instructions. The SEAP assay tal material is available at http://www.jcb.org/cgi/content/full/jcb. was performed as previously described (Warner et al., 2003). 200501162/DC1. We thank Drs R. Duden, R. Pepperkok, J. Simpson, and H. Davidson for gen- Knockdown of optineurin by siRNA erous gifts of antibodies and reagents; and Drs. M. Chibalina and P. Pryor for HeLa and NRK cells were transfected with siRNA duplex (20 M; Dhar- help and advice. macon Research) using OligofectAMINE (Invitrogen) according to the This work was funded by a Wellcome Trust Senior Fellowship (F. Buss), manufacturer’s manual. For efficient knockdown of optineurin cells were a Wellcome Trust studentship (D.A. Sahlender) and a Royal Society USA post- transfected twice with siRNA duplex on day 1 and day 3. On day 5 cells doctoral fellowship (G. Spudich) and supported by the Medical Research were processed for indirect immunofluoresence and the efficiency of Council. Cambridge Institute for Medical Research is in receipt of a strategic knockdown was assayed by Western blotting. In the mock experiment award from the Wellcome Trust. O. Human and rat optineurin the siRNA duplex was substituted with H were targeted with two independent siRNAs: 5-CCACCAGCTGAAA- Submitted: 31 January 2005 GAAGCC-3 and 5-CTGCAGCTCAAGCTGAACT-3. Both siRNAs gave Accepted: 8 March 2005 the same level of knockdown and phenotypes in optineurin-depleted cells. Indirect immunofluorescence microscopy Indirect immunofluorescence staining was performed as described in Buss et References al. (1998). To visualize optineurin at the Golgi complex, cells were pre-per- meabilized with 0.05% saponin in cytosol buffer (Morris and Cooper, 2001) Ang, A.L., H. Folsch, U.M. Koivisto, M. Pypaert, and I. Mellman. 2003. The for 30 s. Cells were analyzed and photographed using 63 or 100 objec- Rab8 GTPase selectively regulates AP-1B–dependent basolateral tives of an Axiophot microscope (Carl Zeiss MicroImaging, Inc.) equipped transport in polarized Madin-Darby canine kidney cells. J. Cell Biol. with a CCD camera. Images were further processed in Adobe Photoshop. 163:339–350. Ang, A.L., T. Taguchi, S. Francis, H. Folsch, L.J. Murrells, M. Pypaert, G. War- VSV-G-GFP transport assay ren, and I. Mellman. 2004. Recycling endosomes can serve as interme- diates during transport from the Golgi to the plasma membrane of 4-d mock- or siRNA-treated HeLa cells were transfected with ts045G VSV- MDCK cells. J. Cell Biol. 167:531–543. G-GFP (a gift from R. Duden, University of London, London, UK) using Fu- GENE as described above. Cells were first incubated for 2 h at 37 C and Aschenbrenner, L., T. Lee, and T. Hasson. 2003. Myo6 facilitates the translocation of endocytic vesicles from cell peripheries. Mol. Biol. Cell. 14:2728–2743. then for 15 h at 39.5 C. Before a temperature shift from 39.5 C to 32 C, 100 g/ml cycloheximide (Sigma-Aldrich) was added to each dish and Balch, W.E., J.M. McCaffery, H. Plutner, and M.G. Farquhar. 1994. Vesicular cells were incubated at 4 C for 30 min. After 20, 40, 60, and 100 min at stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell. 76:841–852. 32 C cells were fixed with 4% PFA and VSV-G present on the cell surface detected with an mAb to the luminal domain of VSV-G. GFP fluorescence Berg, J.S., B.C. Powell, and R.E. Cheney. 2001. A millennial myosin census. gave the total amount of VSV-G expressed in the cell. The ratio of cell sur- Mol. Biol. Cell. 12:780–794. face over total fluorescence was calculated as described previously (Pep- Bunn, R.C., M.A. Jensen, and B.C. Reed. 1999. Protein interactions with the perkok et al., 1993; Seemann et al., 2000) using IP lab software. glucose transporter binding protein GLUT1CBP that provide a link be- tween GLUT1 and the cytoskeleton. Mol. Biol. Cell. 10:819–832. Immunoprecipitation and immunoblotting Buss, F., J. Kendrick-Jones, C. Lionne, A.E. Knight, G.P. Cote, and J. Paul For immunoprecipitation A431 cells were lysed and sonicated in extrac- Luzio. 1998. The localization of myosin VI at the Golgi complex and , 0.5% tion buffer containing PBS, 1 mM EDTA, 5 mM ATP, 5 mM MgCl leading edge of fibroblasts and its phosphorylation and recruitment into 294 JCB • VOLUME 169 • NUMBER 2 • 2005 membrane ruffles of A431 cells after growth factor stimulation. J. Cell Traffic. 3:331–341. Biol. 143:1535–1545. Nishikawa, S., K. Homma, Y. Komori, M. Iwaki, T. Wazawa, A. Hikikoshi Buss, F., S.D. Arden, M. Lindsay, J.P. Luzio, and J. Kendrick-Jones. 2001a. Iwane, J. Saito, R. Ikebe, E. Katayama, T. Yanagida, and M. Ikebe. Myosin VI isoform localized to clathrin-coated vesicles with a role in 2002. Class VI myosin moves processively along actin filaments back- clathrin-mediated endocytosis. EMBO J. 20:3676–3684. ward with large steps. Biochem. Biophys. Res. Commun. 290:311–317. Buss, F., J.P. Luzio, and J. Kendrick-Jones. 2001b. Myosin VI, a new force in Pepperkok, R., J. Scheel, H. Horstmann, H.P. Hauri, G. Griffiths, and T.E. Kreis. clathrin mediated endocytosis. FEBS Lett. 508:295–299. 1993. Beta-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell. 74:71–82. Buss, F., G. Spudich, and J. Kendrick-Jones. 2004. Myosin VI: cellular fuctions and motor properties. Annu. Rev. Cell Dev. Biol. 20:649–676. Rambourg, A., and Y. Clermont. 1990. Three-dimensional electron microscopy: structure of the Golgi apparatus. Eur. J. Cell Biol. 51:189–200. DiFiglia, M., E. Sapp, K. Chase, C. Schwarz, A. Meloni, C. Young, E. Martin, J.P. Vonsattel, R. Carraway, S.A. Reeves, et al. 1995. Huntingtin is a cy- Rezaie, T., A. Child, R. Hitchings, G. Brice, L. Miller, M. Coca-Prados, E. toplasmic protein associated with vesicles in human and rat brain neu- Heon, T. Krupin, R. Ritch, D. Kreutzer, et al. 2002. Adult-onset pri- rons. Neuron. 14:1075–1081. mary open-angle glaucoma caused by mutations in optineurin. Science. 295:1077–1079. Elbashir, S.M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in Rock, R.S., S.E. Rice, A.L. Wells, T.J. Purcell, J.A. Spudich, and H.L. cultured mammalian cells. Nature. 411:494–498. Sweeney. 2001. Myosin VI is a processive motor with a large step size. Proc. Natl. Acad. Sci. USA. 98:13655–13659. Engelender, S., A.H. Sharp, V. Colomer, M.K. Tokito, A. Lanahan, P. Worley, E.L. Holzbaur, and C.A. Ross. 1997. Huntingtin-associated protein 1 Rogalski, A.A., and S.J. Singer. 1984. Associations of elements of the Golgi ap- (HAP1) interacts with the p150Glued subunit of dynactin. Hum. Mol. paratus with microtubules. J. Cell Biol. 99:1092–1100. Genet. 6:2205–2212. Schwamborn, K., R. Weil, G. Courtois, S.T. Whiteside, and A. Israel. 2000. Faber, P.W., G.T. Barnes, J. Srinidhi, J. Chen, J.F. Gusella, and M.E. Mac- Phorbol esters and cytokines regulate the expression of the NEMO- Donald. 1998. Huntingtin interacts with a family of WW domain pro- related protein, a molecule involved in a NF-kappa B-independent path- teins. Hum. Mol. Genet. 7:1463–1474. way. J. Biol. Chem. 275:22780–22789. Gauthier, L.R., B.C. Charrin, M. Borrell-Pages, J.P. Dompierre, H. Rangone, Seabra, M.C., and E. Coudrier. 2004. Rab GTPases and myosin motors in or- F.P. Cordelieres, J. De Mey, M.E. MacDonald, V. Lessmann, S. Hum- ganelle motility. Traffic. 5:393–399. bert, and F. Saudou. 2004. Huntingtin controls neurotrophic support and Seemann, J., E.J. Jokitalo, and G. Warren. 2000. The role of the tethering pro- survival of neurons by enhancing BDNF vesicular transport along micro- teins p115 and GM130 in transport through the Golgi apparatus in vivo. tubules. Cell. 118:127–138. Mol. Biol. Cell. 11:635–645. Gill, S.R., T.A. Schroer, I. Szilak, E.R. Steuer, M.P. Sheetz, and D.W. Cleve- Stroissnigg, H., M. Repitz, A. Miloloza, I. Linhartova, H. Beug, G. Wiche, and land. 1991. Dynactin, a conserved, ubiquitously expressed component of F. Propst. 2002. FIP-2, an IkappaB-kinase-gamma-related protein, is as- an activator of vesicle motility mediated by cytoplasmic dynein. J. Cell sociated with the Golgi apparatus and translocates to the marginal band Biol. 115:1639–1650. during chicken erythroblast differentiation. Exp. Cell Res. 278:133–145. Hammer, J.A., III, and X.S. Wu. 2002. Rabs grab motors: defining the connec- Van De Moortele, S., R. Picart, A. Tixier-Vidal, and C. Tougard. 1993. Nocoda- tions between Rab GTPases and motor proteins. Curr. Opin. Cell Biol. zole and taxol affect subcellular compartments but not secretory activity 14:69–75. of GH3B6 prolactin cells. Eur. J. Cell Biol. 60:217–227. Harada, A., Y. Takei, Y. Kanai, Y. Tanaka, S. Nonaka, and N. Hirokawa. 1998. Velier, J., M. Kim, C. Schwarz, T.W. Kim, E. Sapp, K. Chase, N. Aronin, and M. Golgi vesiculation and lysosome dispersion in cells lacking cytoplasmic DiFiglia. 1998. Wild-type and mutant huntingtins function in vesicle traf- dynein. J. Cell Biol. 141:51–59. ficking in the secretory and endocytic pathways. Exp. Neurol. 152:34–40. Harjes, P., and E.E. Wanker. 2003. The hunt for huntingtin function: interaction Warner, C.L., A. Stewart, J.P. Luzio, K.P. Steel, R.T. Libby, J. Kendrick-Jones, partners tell many different stories. Trends Biochem. Sci. 28:425–433. and F. Buss. 2003. Loss of myosin VI reduces secretion and the size of Hattula, K., and J. Peranen. 2000. FIP-2, a coiled-coil protein, links Huntingtin to the Golgi in fibroblasts from Snell’s waltzer mice. EMBO J. 22:569–579. Rab8 and modulates cellular morphogenesis. Curr. Biol. 10:1603–1606. Wehland, J., M.C. Willingham, M.G. Gallo, and I. Pastan. 1982. The morpho- Hodge, T., and M.J. Cope. 2000. A myosin family tree. J. Cell Sci. 113:3353– logic pathway of exocytosis of the vesicular stomatitis virus G protein in 3354. cultured fibroblasts. Cell. 28:831–841. Huber, L.A., M.J. de Hoop, P. Dupree, M. Zerial, K. Simons, and C. Dotti. Wells, A.L., A.W. Lin, L.Q. Chen, D. Safer, S.M. Cain, T. Hasson, B.O. Car- 1993a. Protein transport to the dendritic plasma membrane of cultured ragher, R.A. Milligan, and H.L. Sweeney. 1999. Myosin VI is an actin- neurons is regulated by rab8p. J. Cell Biol. 123:47–55. based motor that moves backwards. Nature. 401:505–508. Huber, L.A., S. Pimplikar, R.G. Parton, H. Virta, M. Zerial, and K. Simons. Wu, H., J.E. Nash, P. Zamorano, and C.C. Garner. 2002. Interaction of SAP97 1993b. Rab8, a small GTPase involved in vesicular traffic between the with minus-end-directed actin motor myosin VI. Implications for AMPA TGN and the basolateral plasma membrane. J. Cell Biol. 123:35–45. receptor trafficking. J. Biol. Chem. 277:30928–30934. Joe, M.K., S. Sohn, W. Hur, Y. Moon, Y.R. Choi, and C. Kee. 2003. Accumula- Zerial, M., and H. McBride. 2001. Rab proteins as membrane organizers. Nat. tion of mutant myocilins in ER leads to ER stress and potential cytotoxic- Rev. Mol. Cell Biol. 2:107–117. ity in human trabecular meshwork cells. Biochem. Biophys. Res. Commun. 312:592–600. Kreis, T.E., and H.F. Lodish. 1986. Oligomerization is essential for transport of ve- sicular stomatitis viral glycoprotein to the cell surface. Cell. 46:929–937. Li, S.H., C.A. Gutekunst, S.M. Hersch, and X.J. Li. 1998a. Interaction of huntingtin-associated protein with dynactin P150Glued. J. Neurosci. 18:1261–1269. Li, X.J., S.H. Li, A.H. Sharp, F.C. Nucifora Jr., G. Schilling, A. Lanahan, P. Wor- ley, S.H. Snyder, and C.A. Ross. 1995. A huntingtin-associated protein enriched in brain with implications for pathology. Nature. 378:398–402. Li, Y., J. Kang, and M.S. Horwitz. 1998b. Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-induc- ible cellular protein containing leucine zipper domains. Mol. Cell. Biol. 18:1601–1610. Lister, I., S. Schmitz, M. Walker, J. Trinick, F. Buss, C. Veigel, and J. Ken- drick-Jones. 2004. A monomeric myosin VI with a large working stroke. EMBO J. 23:1729–1738. Luzio, J.P., B. Brake, G. Banting, K.E. Howell, P. Braghetta, and K.K. Stanley. 1990. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38). Biochem. J. 270:97–102. Morris, S.M., and J.A. Cooper. 2001. Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2. Traffic. 2:111–123. Morris, S.M., S.D. Arden, R.C. Roberts, J. Kendrick-Jones, J.A. Cooper, J.P. Luzio, and F. Buss. 2002. Myosin VI binds to and localises with Dab2, po- tentially linking receptor-mediated endocytosis and the actin cytoskeleton. OPTINEURIN LINKS MYOSIN VI TO THE GOLGI COMPLEX • SAHLENDER ET AL. 295

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Published: Apr 25, 2005

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