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Huntingtin–HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease

Huntingtin–HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is... JCB: ARTICLE <doi>10.1083/jcb.200509091</doi><aid>200509091</aid>Huntingtin–HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington’s disease Arun Pal, Fedor Severin, Barbara Lommer, Anna Shevchenko, and Marino Zerial Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany he molecular mechanisms underlying the targeting Remarkably, endogenous HAP40 was up- regulated in fi - of Huntingtin (Htt) to endosomes and its multifaceted broblasts and brain tissue from human patients affected T role in endocytosis are poorly understood. In this by Huntington’s disease (HD) as well as in STHdhQ study, we have identifi ed Htt-associated protein 40 (HAP40) striatal cells established from a HD mouse model. These as a novel effector of the small guanosine triphosphatase cells consistently displayed altered endosome motility and Rab5, a key regulator of endocytosis. HAP40 mediates endocytic activity, which was restored by the ablation of the recruitment of Htt by Rab5 onto early endosomes. HAP40. In revealing an unexpected link between Rab5, HAP40 overexpression caused a drastic reduction of early HAP40, and Htt, we uncovered a new mechanism regu- endosomal motility through their displacement from mi- lating cytoskeleton-dependent endosome dynamics and crotubules and preferential association with actin fi laments. its dysfunction under pathological conditions. Introduction Huntington’s disease (HD) is a neurodegenerative disorder expansion confers the adjacent proline-rich sequence in Htt caused by expansion of the CAG repeat in the gene encoding alterations in binding affi nity to HIPs/HAPs. Thus, release Huntingtin (Htt), which confers to the protein an expanded from or sequestration of these molecules by mutant Htt has NH -terminal polyglutamine (polyQ) stretch of >35 residues been implicated in the pathogenetic mechanisms. (for review see Harjes and Wanker, 2003). The function of Htt For example, the tighter binding of HAP1 to mutant Htt is is largely unclear. It has been shown to interact with microtu- thought to impair the correct dynactin–dynein motor complex bules (Hoffner et al., 2002) and to display anti-apoptotic activ- assembly and cause a traffi cking defect, leading to neuronal de- ity (Rigamonti et al., 2000, 2001). Insights into its function generation (Block-Galarza et al., 1997; Engelender et al., 1997; came from studies of Htt-interacting proteins (HIPs) and Htt- Gauthier et al., 2004). Consistently, mutant Htt was recently associated proteins (HAPs). For example, interactions with shown to release dynein from microtubules and reduce the mo- HIP1, HIP1R, PACSIN1, SH3GL3, and HIP14 have implicated tility of EGFP –brain-derived neurotrophic factor–containing Htt in clathrin-mediated endocytosis. Studies of HAP1 have vesicles in vivo (Gauthier et al., 2004). However, the upstream suggested a role for Htt in axonal transport in neurons by link- events that target Htt and its partners to their various sites of ing vesicles to the dynein–dynactin motor complex (Block- function and the mechanisms whereby they regulate intracellu- Galarza et al., 1997; Engelender et al., 1997). The polyQ lar traffi cking remain elusive. In this study, we report an unexpected link between Htt and the small GTPase Rab5 via the adaptor protein HAP40. Correspondence to Marino Zerial: zerial@mpi-cbg.de Correspondence to Marino Zerial: zerial@mpi-cbg.de Rab5 is a key regulator of endocytosis that orchestrates the F F.. Severin’ Severin’s present address is Biotechnology Centre, University of T s present address is Biotechnology Centre, University of Technology echnology recruitment of multiple effector proteins on the early endo- Dresden, Cellular Machines, 01307 Dresden, Ger Dresden, Cellular Machines, 01307 Dresden, Germany many.. some membrane to regulate organelle tethering, fusion, and Abbreviations used in this paper: EEA1, early endosome antigen 1; F-actin, fi Abbreviations used in this paper: EEA1, early endosome antigen 1; F-actin, fi la- la- microtubule-dependent motility (Zerial and McBride, 2001). mentous actin; GDI, GDP dissociation inhibitor; GDP mentous actin; GDI, GDP dissociation inhibitor; GDP, guanosine diphosphate; , guanosine diphosphate; HAP HAP, Htt-associated protein; HD, Huntington’ , Htt-associated protein; HD, Huntington’s disease; HIP; Htt-interacting pro- s disease; HIP; Htt-interacting pro- Our data extend the analysis of the Rab5 effector machinery by tein; Htt, Huntingtin; LAMP tein; Htt, Huntingtin; LAMP, lysosome-associated membrane protein; polyQ, , lysosome-associated membrane protein; polyQ, functionally implicating the interaction between Rab5 and Htt polyglutamine; RNAi, RNA inter polyglutamine; RNAi, RNA interference; siRNA, shor ference; siRNA, short inter t interfering RNA; Tfr fering RNA; Tfr, trans- , trans- ferrin receptor ferrin receptor.. in the regulation of the differential association of early endo- The online version of this ar The online version of this article contains supplemental material. ticle contains supplemental material. somes with the actin and tubulin cytoskeleton. © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 172, No. 4, February 13, 2006 605–618 http://www.jcb.org/cgi/doi/10.1083/jcb.200509091 JCB 605 THE JOURNAL OF CELL BIOLOGY eluted together with HAP40 from GTPγS- but not GDP-bound GST-Rab5 (Fig. 2 D, lanes 7 and 8). None of the other Htt frag- ments displayed such binding (Fig. 2 D, lanes 1–6), which is consistent with the reported interaction map between HAP40 and Htt (Peters and Ross, 2001). Neither the Htt fragments nor HAP40 displayed binding to Rab4, Rab6, Rab7, or Rab11 (Fig. 2 D), suggesting the interaction with Rab5 is specifi c. Thus, we conclude that HAP40 binds to the COOH-terminal part of Htt and links the complex to active Rab5. By applying a 10-fold excess of the COOH-terminal fragment of Htt onto the Rab5 column to reduce binding of free HAP40 to Rab5 (Fig. 2 E), we estimated the stoichiometry of the Rab5/HAP40/Htt interaction in this assay to be 1:1:1 (see Rab5 affi nity…cloning). Htt is recruited onto early endosome in a HAP40- and Rab5-dependent manner Figure 1. Htt and HAP40 elute from immobilized Rab5. (A) SDS-PAGE of proteins eluted from immobilized GST-Rab5 that was loaded with GTPγS. We began testing the functional relevance of this interaction The indicated bands were found to correspond to Htt, HAP40, Rab5-GST, in vivo by immunofl uorescence microscopy analysis of HeLa and EEA1 by mass spectrometry analysis. (B) Western blot analysis of cells. First, we verifi ed that the anti-HAP40 antibody resulted chromatographic eluates from GST-Rab5 (lanes 1 and 2) and -Rab4 (lanes 3 and 4) affi nity columns preloaded with GDP (D; lanes 1 and 3) or GTPγS in specifi c staining above background levels for detection of (T; lanes 2 and 4). The bovine brain cytosol (BBC; lane 5) was used as a the endogenous antigen (Fig. 3 A). Endogenous HAP40 dis- source of proteins. Blots were probed for Htt and HAP40 as indicated. played a diffuse staining in the cytoplasm and accumulated in the nucleus, whereas endogenous Htt localized to discrete cytoplasmic structures (Fig. 3 B) as reported previously (Peters Results and Ross, 2001; Tao and Tartakoff, 2001). Colocalization of The Htt–HAP40 complex is a novel endogenous HAP40 and Htt was hardly detectable (Fig. 3 B). Rab5 effector Early endosomes labeled with EGFP-Rab5 displayed little co- Affi nity chromatography revealed several downstream effectors localization with endogenous Htt (7 ± 5% overlap; n = 10; of the small GTPase Rab5 (Christoforidis et al. 1999). Surpris- Fig. 3 C). However, the association of HAP40 with early en- ingly, among the proteins specifi cally eluted from the GST- dosomes dramatically increased upon overexpression. In cells Rab5–GTPγS but not from the Rab5–guanosine diphosphate overexpressing HAP40 (see Fig. 7 D) but not EGFP-Rab5 (GDP) nor GST-Rab4 af nity column, we identifi fi ed Htt and alone, HAP40 signifi cantly colocalized with endogenous Htt on HAP40 (Peters and Ross, 2001) by mass spectrometry and im- EGFP-Rab5–positive early endosomes (Fig. 3, compare B with munoblotting (Fig. 1, A and B). Therefore, we investigated the D; endogenous Htt and EGFP-Rab5: 43 ± 6% overlap, n = 10; function of HAP40 and Htt with respect to Rab5. HAP40 and EGFP-Rab5: 31 ± 7% overlap, n = 10; Fig. 3, We fi rst tested whether HAP40, Htt, or both interact compare C with D). We experimentally verifi ed that the HAP40 directly and specifi cally with Rab5. To this end, full-length fl uorescence signals (Fig. 3 D) were not caused by bleed- HAP40 was cloned from a rat brain cDNA library and in vitro through of the Htt signals (because of extended AlexaFluor568 translated. Because of its large molecular mass (348 kD; Fig. emission in the Cy5 channel) and that swapping these fl uores- 2 A), fragments of wild-type Htt were translated to facilitate cent dyes on the secondary antibodies resulted in similar distri- the analysis. In vitro translation yielded major products of pre- bution patterns of Htt and HAP40 (see Cell culture procedures). dicted size as well as lower molecular mass bands presumably The endosomal colocalization of endogenous HAP40 and as a result of internal initiation (Fig. 2 B). Immobilized Rab5- Htt was even more striking upon expression of the activated GST fusion protein preloaded with either GDP or GTPγS were EGFP-Rab5Q79L mutant (Htt: 52 ± 7% overlap, n = 8), incubated with the translation products and washed, and bound which caused the characteristic swelling of early endosomes proteins were eluted with glutathione analyzed by SDS-PAGE (Fig. 4 A; Stenmark et al., 1994). and autoradiography (Christoforidis et al., 1999). Similar to Given the dif culties detecting endogenous HAP40 and fi early endosome antigen 1 (EEA1), which served as a positive Htt on early endosomes in untreated cells (Fig. 3, B and C) we control, HAP40 displayed specifi c binding to GTP γS- versus sought to verify the changes in the localization of both proteins GDP-bound Rab5 (Fig. 2 C, compare lanes 1 and 11 with lanes upon the overexpression of HAP40 biochemically. To this end, 2 and 12). In contrast, none of the Htt fragments exhibited sig- we prepared early endosomes from HeLa cells for Western blot nifi cant binding to Rab5 (Fig. 2 C, lanes 3–10). Because Htt analysis. Indeed, we found that the levels of HAP40 and Htt in- was purifi ed on the GST-Rab5 af nity column, we ne fi xt tested creased on early endosomes from the HAP40 overexpressor whether binding of Htt to Rab5 occurs indirectly and requires compared with untreated cells (Fig. 3 E). Early endosome HAP40 as a bridge. HAP40 and Htt fragments were cotranslated (EEA1 and transferrin receptor [Tfr]) as well as lysosomal in vitro (Fig. 2 B, lanes 6–9) and applied on the Rab5 columns. (lysosome-associated membrane protein (LAMP); Eskelinen Indeed, the COOH-terminal part of Htt (Fig. 2 A, Htt4) was et al., 2003) and Golgi (GM130; Nakamura et al., 1995) markers 606 JCB • VOLUME 172 • NUMBER 4 • 2006 remained unchanged, confi rming equal loading and the speci- ficity of changes through elevated HAP40 on early endosomes. To confi rm the requirement of HAP40 for the recruitment of Htt onto early endosomes, we transfected HeLa cells with short interfering RNA (siRNA) duplexes against HAP40 and un- related siRNA (against GFP) as control. The HAP40 siRNA spe- cifi cally and ef ciently reduced the protein le fi vels by 90% (Fig. 4 C), whereas the level of EEA1 remained unchanged. When cells were cotransfected with the expression vector for EGFP- Rab5Q79L and HAP40 siRNA, Htt was no longer detectable on the enlarged endosomes (8 ± 6% overlap, n = 9; Fig. 4 B), con- fi rming HAP40 as a prerequisite to bridge Htt to active Rab5. Collectively, these data suggest that active Rab5 and HAP40 are rate limiting for the recruitment of Htt onto early endosomes. The Htt–HAP40 complex inhibits the binding and motility of early endosomes on microtubules Htt has previously been shown to bind microtubules and reg- ulate microtubule–motor interactions (Block-Galarza et al., 1997; Engelender et al., 1997; Hoffner et al., 2002; Gauthier et al., 2004). Because Rab5 regulates endosome motility along microtubules (Nielsen et al., 1999; Hoepfner et al., 2005), we explored the role of the Htt–HAP40 complex in this process. First, by using a cell- and cytosol-free assay that recapitulates the Rab5-dependent movement of early endosomes along mi- crotubules (Hoepfner et al., 2005), we found that Htt–HAP40 inhibited microtubule-dependent early endosome motility. Addition of the GTPγS- (containing Htt and HAP40; Fig. 1 B) but not the GDP-loaded Rab5 column eluate reduced the motility compared with control conditions (Fig. 5 A). This inhibition was Figure 2. Htt–HAP40 complex is a novel Rab5 effector. (A) Schematic 23 glutamine residues (23Q), the adjacent proline-rich sequence (P), the caspase-3 cleavage site, and the four recombinant fragments (Htt1–4) cloned in pcDNA3.1 using the indicated restriction sites. (B) Autoradio- graph of in vitro–translated Htt fragments (Htt1–4) and HAP40. Full-length HAP40 cDNA and Htt fragments were in vitro translated in the presence of [ S]methionine, separated by SDS-PAGE, and autoradiographed. Given their large size, the bands of strongest intensity (arrows) and correspond- ing to the predicted masses for HAP40 (40 kD, lane 1), Htt1 (65 kD, lane 2), Htt2 (97 kD, lane 3), Htt3 (84 kD, lane 4), and Htt4 (102 kD, lane 5) were accompanied by multiple products because of either initiation of translation at internal sites or premature termination. Lanes 6–9: each Htt fragment was cotranslated with HAP40. (C) Autoradiograph of the in vitro–translated proteins in B eluted from immobilized GST-Rab5 preloaded with GTPγS (T) or GDP (D). (D) Autoradiograph of Htt fragments cotrans- lated in vitro with HAP40 and eluted from various immobilized Rab pro- teins as indicated. The experiment was performed as described in C, but Htt fragments and HAP40 were cotranslated (see B, lanes 6–9) and ap- plied onto immobilized Rab proteins. Besides HAP40, the COOH-terminal Htt fragment (Htt4) was eluted from GST-Rab5 beads (compare lanes 7 with 8), whereas Htt1 (lanes 1 and 2), Htt2 (lanes 3 and 4), and Htt3 (lanes 5 and 6) did not show signifi cant specifi c association with Rab5. Moreover, none of the Htt fragments or HAP40 displayed specifi c interac- tions with GST-Rab4, 6, 7, or 11. Positive controls (POS, lanes 9 and 10) for each Rab protein were EEA1 (170 kD) for Rab5, Rabenosyn-5 (89 kD) for Rab4 (de Renzis et al., 2002), VPS39 (100 kD) for Rab7 (Rink et al., 2005), and GapCenA (115 kD) for Rab6 and 11 (Cuif et al., 1999 and unpublished data). (E) Htt and HAP40 elute in equimolar amounts from the Rab5 column. A mixture of Htt4 and HAP40 was applied onto Rab5 columns and eluted as in D but with a 10-fold excess of Htt4 to prevent HAP40 from binding freely to Rab5. (C–E) Arrows point to correct transla- tion products as in B. THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 607 Figure 3. Overexpressed HAP40 recruits Htt onto early endosomes. Immunofl uorescence microscopy analysis of HeLa cells expressing EGFP-Rab5 and/or HAP40 and immuno- stained for HAP40 and Htt as indicated. (A) The anti-HAP40 antibody specifi cally recognizes its antigen (green). Cells were stained with the antibody alone (anti-HAP40) or pre- mixed with HAP40-GST fusion (anti-HAP40 & GST-HAP40) or GST protein (anti-HAP40 & GST). The image after specifi c de- pletion of the antibody (anti-HAP40 & GST-HAP40) was ob- tained at sixfold prolonged exposure time compared with the others. (B) Endogenous Htt (red) and endogenous HAP40 (green); <1% overlapping (n = 10). (C) Endogenous Htt (red) and EGFP-Rab5 (green); 7% overlapping (SD ± 5%, n = 10). (D) Overexpressed HAP40 (red) recruits endogenous Htt (red) onto endosomes labeled with EGFP-Rab5WT (green); 43% overlapping of endogenous Htt with EGFP-Rab5 (merge Rab5 & Htt; SD ± 6%, n = 10) and 31% overlapping of overex- pressed HAP40 with EGFP-Rab5 (merge Rab5 & HAP40; SD ± 7%, n = 10). Cells were cotransfected with HAP40 and GFP-Rab5WT expression constructs. Arrowheads in insets (magnifi ed images of boxed areas) highlight colocalization. Bar, 10 μm. (E) Western blot analysis of early endosomes prepared from untreated (WT) or HeLa cells overexpressing HAP40 (HAP40). Blots were probed for HAP40, Htt, EEA1, transferrin receptor (Tfr), LAMP1, and GM130 as indicated. specifi cally rescued with anti-Htt but not unrelated antibodies. assay as described previously (Nielsen et al., 1999) with The addition of 1 μM GST-HAP40 fusion protein completely some modifi cations to improve the quantitative assessment. blocked the in vitro motility (Fig. 5, A and B). Second, we An early endosome–enriched fraction was prepared from performed a biochemical early endosome–microtubule- binding HeLa cells pulsed with rhodamine–transferrin, incubated with 608 JCB • VOLUME 172 • NUMBER 4 • 2006 Figure 4. Htt is recruited onto early endo- somes in a Rab5- and HAP40-dependent fashion. Immunofl uorescence microscopy anal- ysis of HeLa cells transfected with expression vectors for EGFP-Rab5Q79L alone or cotrans- fected with siRNA duplexes against HAP40 as indicated. (A) EGFP-Rab5Q79L (green) recruits endogenous Htt (red) onto endosomes; 52% overlapping (SD ± 7%, n = 8). Cells were transfected with EGFP-Rab5Q79L expression construct alone. (B) RNAi against HAP40 (siHAP40) leads to the loss of endogenous Htt (red) from endosomes; 8% overlapping (SD ± 6%, n = 9). Cells were cotransfected with HAP40 siRNA and EGFP-Rab5Q79L (green) expression constructs. Bar, 10 μm. (C) Knockdown of HAP40 protein by RNAi as shown by Western blot analysis. EEA1 and HAP40 expression levels in untransfected cells (control) and cells transfected with unrelated (siGFP) or siRNA against HAP40 (siHAP40). taxol- stabilized microtubules, ATP, and factors to be tested, compare lane 5 with lanes 4 and 2). To rule out the idea that the and centrifuged through a sucrose cushion. The resulting pellet observed differences result from the bundling of microtubules of microtubule-associated material was analyzed by immuno- causing unspecifi c cosedimentation of any membranous struc- blotting (Fig. 6 A) and fl uorimetrically for the rhodamine– ture, we probed the pellets for nonendosomal contaminants in transferrin content (Fig. 6 D). In a dilution series for calibration, the fraction. Both the lysosomal (LAMP1) and Golgi marker we verifi ed that the amount of endosomes and fl uorescence in- (GM130) pelleted with similar effi ciency in all samples. Collec- tensity correlated linearly (see Microtubule and actin spin-down tively, these data indicate that Htt–HAP40 specifi cally lowers assays). Western blotting revealed that the β-tubulin content was the binding of early endosomes to microtubules. similar between samples, ruling out secondary effects on micro- Having validated the assay, we next quantifi ed the amount tubule stability (Fig. 6 A, compare lanes 2–5 with lane 1). The of endosomes bound to microtubules fl uorimetrically. Omission addition of 1 μM GST-HAP40 protein decreased the amount of either microtubules or early endosomes reduced the fl uores- of early endosomes in the pellet as revealed by EEA1 and Tfr cence signal to background levels (Fig. 6 D). As reported previ- (Fig. 6 A, compare lane 3 with lane 2). The GST-Rab5–GTPγS ously (Nielsen et al., 1999), the association of early endosomes column eluate adding 0.3 μM HAP40 (Fig. 6 C) caused a with microtubules was energy dependent and required active similar inhibition (Fig. 6A, compare lane 4 with lane 2) that was Rab5. Omitting ATP or substituting it with the nonhydrolyzable rescued through the addition of antiserum against Htt (Fig. 6 A, adenylyl-imidodiphosphate analogue resulted in an 50% THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 609 reduction in microtubule binding. The requirement for ATP can be explained by the role of PI3-K in the assembly and mainte- nance of a functional Rab5 domain on endosomes (Zerial and McBride, 2001) and in recruitment of the endosomal kinesin KIF16B (Hoepfner et al., 2005). Extraction of Rab proteins from membranes by the addition of 1 μM of recombinant Rab–GDP dissociation inhibitor (GDI; Ullrich et al., 1994) or treatment with RN-tre, a GTPase-activating protein for Rab5 (Lanzetti et al., 2000), caused an 40% reduction of endosomes in the pellet (Fig. 6 D). As for endosome motility (Fig. 5 A), Htt and HAP40 inhibited endosome–microtubule binding. The GST-Rab5–GTPγS column eluate led to an 25% reduc- tion in binding (Fig. 6 D), but supplementing the reaction with antibodies against Htt restored binding to control levels. The addition of 1 μM HAP40-GST fusion protein decreased the binding by 60%, whereas GST alone did not have any effect. Collectively, these data underpin the ability of HAP40 and Htt to destabilize endosome–microtubule association. We next performed time-lapse video microscopy studies to correlate these biochemical fi ndings in vitro with the regula- tion of early endosome dynamics in vivo. We detected a dras- tic reduction in motility of EGFP-Rab5–positive endosomes in HeLa cells overexpressing HAP40 (Fig. 7 A) compared with cells transfected with EGFP-Rab5 alone (Fig. 7 A and Vid- eos 1 and 2, available at http://www.jcb.org/cgi/content/full/ jcb.200509091/DC1). Whereas some residual motility activ- ity was observed in the cell periphery, early endosomes in the perinuclear region appeared static, with frequent short-range movements almost completely impaired in long-range motility (Nielsen et al., 1999, Rink et al. 2005). Collectively, these data suggest that the Rab5-dependent recruitment of Htt onto endo- somes by HAP40 disrupts early endosome–microtubule inter- actions, thus leading to a reduction in organelle motility. HD cells display increased levels of HAP40 and are impaired in early endosome motility We next asked whether alterations of endosome motility could occur in cells bearing the HD mutation. Primary fi broblasts from fi ve healthy individuals and fi ve unrelated HD patients were transfected with EGFP-Rab5 to compare the motility of early endosomes. The identity of the HD cell lines was con- fi rmed by Western blotting to detect the polyQ-expanded Htt. Because the cells were derived from patients heterozygous for Figure 5. Htt and HAP40 decrease in vitro reconstituted motility of early the HD gene, they express both normal and mutant Htt. endosomes along microtubules. (A) Purifi ed early endosomes labeled by internalization of rhodamine–transferrin were mixed with buffer (control) The latter is known to display a lower mobility in SDS-PAGE alone or with various eluates of bovine brain cytosol proteins that were af- (Trottier et al., 1995), thus causing a doublet on the blot (Fig. 7 D). fi nity purifi ed on GST-Rab5 columns. Eluate obtained from columns loaded Strikingly, we observed a severe reduction in early endosome with GDP was added directly to the sample (+GDP-eluate). Eluate ob- tained from columns loaded with GTPγS was added directly to the sample motility in all HD cell lines compared with fi broblasts from (+GTP-eluate) or after preincubation with antiserum against the cytoplas- healthy individuals (Fig. 7 B and Videos 3 and 4, available mic domain of Tfr (+eluate + anti-Tfr), antiserum against Htt (+eluate at http://www.jcb.org/cgi/content/full/jcb.200509091/DC1). + anti-Htt), or without eluate but with 1 μM of recombinant GST-HAP40 fu- sion protein (+HAP40). In vitro motility of early endosomes along micro- The similarity between this phenotype and the alterations in- tubules recorded using time-lapse fl uorescence video microscopy (see duced upon HAP40 overexpression in HeLa cells (Fig. 7 A and Materials and methods) was quantifi ed by counting motility events per Videos 1 and 2) hinted to a common molecular basis. Interest- video. Error bars show the SD of 10 videos. (B) Videos recorded under control conditions (control) or with 1 μM GST-HAP40 fusion protein ingly, we discovered an 10-fold up-regulation of endogenous (HAP40) used for the analysis in A are displayed as merged stacks of over- HAP40 levels by Western blotting in all HD cell lines com- laid images collected at 2-s intervals over 2 min. When represented in this pared with normal fi broblasts (Fig. 7 D). As a control, the lev- manner, a moving object will generate a trajectory consisting of a linear series of overlapping spots. Bar, 10 μm. els of EEA1 remained unchanged. Moreover, we found that 610 JCB • VOLUME 172 • NUMBER 4 • 2006 HAP40 protein levels were signifi cantly elevated in striatal tissue (caudate, putamen, accumbens, and globus pallidus) from hu- man postmortem brains affected by HD (adult onset grade) compared with control brains (Fig. 8 B). Our data suggest that the motility block in HD cells may be caused by elevated HAP40 levels, as mimicked by overexpression of HAP40 in HeLa cells. Consistently, endogenous Htt localized to EGFP- Rab5–labeled endosomes in fi broblasts from human HD pa- tients as well as striatal STHdhQ cells from a HD mouse model (Trettel et al., 2000) but not in cells lacking the mutant Htt (Fig. 9). This phenotype is also caused by overexpressed HAP40 in HeLa cells (Fig. 3 D). Our data do not exclude the possibility that the observed inhibition of early endosome motility may have other under- lying causes. As a test to our hypothesis, we attempted to rescue the inhibition of early endosome motility by specifi cally ab- lating HAP40 from the HD fi broblasts by RNA interference (RNAi). Transfection of HAP40 siRNA effi ciently reduced the HAP40 protein levels in these cells (Fig. 7 E) as in HeLa cells (Fig. 4 C). Indeed, endosome motility was restored by RNAi against HAP40 (Fig. 7 C and Video 5, available at http://www. jcb.org/cgi/content/full/jcb.200509091/DC1) but not against GFP, suggesting that the up-regulation of HAP40 is indeed the underlying mechanism of the motility defect in HD fi broblasts. Given the ability of HAP40 to reduce endosome– microtubule binding in vitro (Fig. 6, A and D), we investigated whether the observed motility block in HD cell lines was caused by a release of endosomes from microtubules in vivo. Immunofl uorescence analysis on cells transfected with EGFP-Rab5 showed a consid- erable alignment of early endosomes to microtubules in healthy fi broblasts (82 ± 9% overlap, n = 10) but to a much lesser extent in HD cell lines (15 ± 6% overlap, n = 9; Fig. 7, compare F with G). In contrast, early endosomes were strikingly aligned with fi lamentous actin (F-actin) in all fi ve HD cell lines (44 ± 8% overlap, n = 10) but not in healthy fi broblasts (2 ± 1% overlap, n = 10; Fig.7, compare G with F). A similar in samples prepared for the early endosome– microtubule/actin-binding assays. Samples were prepared as described for A and B either with GST- HAP40 fusion (lane 1) or GST-Rab5–GTPγS column eluate (lane 2). 10-μl aliquots of each sample were separated by SDS-PAGE and immunoblotted for HAP40. Bands corresponded to the correct masses of GST-HAP40 or HAP40 as indicated on the right. Nonrelevant lanes on the same blot were sliced out in Adobe Photoshop to juxtapose the lanes shown. (D) Quantifi - cations of early endosomes bound to microtubules. Binding was performed as described for A, but the resulting pellet of microtubule- associated mate- rial was lysed to release the rhodamine–transferrin label of early endo- some. Fluorescence (arbitrary units) served as a direct measure for the Figure 6. Modulation of binding of early endosomes to microtubules and amount of early endosomes bound to microtubules. Error bars represent F-actin by Htt and HAP40. (A) Reduction of binding of early endosomes the SD of samples in triplicate. Binding was performed in the presence (EE) to microtubules in vitro caused by Htt and HAP40. A spin-down assay of 15 μg of early endosomes and 16 μg of microtubules (control), in was performed (see Materials and methods), and the resulting pellet of mi- the absence of microtubules (−MT), in the absence of early endosomes crotubule-associated material was analyzed by immunoblotting with anti- (−EE), in the absence of ATP (−ATP), with all three components omitted bodies against proteins indicated on the right. (B) Stimulation of binding of (buffer), with 2 mM adenylyl-imidodiphosphate (+AMP-PNP), with 1 μM early endosomes to F-actin in vitro caused by Htt and HAP40. A spin-down of recombinant RN-tre (+RN-tre), with 1 μM Rab-GDI (+GDI), with 1 μM assay was performed as described for A but with 10 μg of freshly polymer- HAP40-GST fusion protein (+HAP40), with 1 μM GST (+GST), with 10 μg ized F-actin replacing the microtubule. (A and B) Microtubules (A) or F-actin GST-Rab5–GTPγS column eluate (+eluate), or with eluate preincubated (B) spun alone (−EE, lane 1), with early endosomes (control; +EE, lane 2), with anti-Htt antiserum (+eluate+anti-Htt). (E) Quantifi cations of early en- with early endosomes and 1 μM GST-HAP40 fusion protein (+EE+HAP40, dosomes bound to F-actin. Binding was performed as described for B, and lane 3), with early endosomes and 10 μg GST-Rab5–GTPγS column elu- quantifi cations were made as in D. Binding was performed in the presence ate (+EE+eluate, lane 4), or eluate preincubated with anti-Htt antiserum of 15 μg of early endosomes, 10 μg of F-actin (control), and with the (+EE+eluate+anti-Htt, lane 5). (C) Comparison of HAP40 protein levels addition of various components as in D. THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 611 Figure 7. Elevated protein levels of HAP40 shift early endosomes from microtubules to actin fi laments, causing a severe decrease of motility in vivo. HeLa cells and human primary fi broblasts expressing EGFP-Rab5 were imaged using time-lapse video microscopy. (A–C) Images generated by merging a stack of overlaid images collected at 300-ms intervals over 2 min, as for Fig. 5 B. Videos corresponding to A–C are available as online supplemental materials (Videos 1–5, available at http://www.jcb.org/cgi/content/full/jcb.200509091/DC1). (A) In HeLa cells, coexpression of EGFP-Rab5 and HAP40 (right) led to a drastic reduction in endosome motility compared with EGFP-Rab5 alone (WT, left). (B) In fi broblasts from HD patients (HD), such reduction was also evident from the comparison with fi broblasts from healthy individuals (WT). (C) RNAi against HAP40 in these HD fi broblasts (HAP40 RNAi) restores endo- some motility. HD fi broblast cell lines were cotransfected with EGFP-Rab5 expression vector and siRNA against HAP40. (D) Similar to HeLa cells overex- pressing HAP40 (compare lane 1 with lane 2), endogenous protein levels of HAP40 were elevated in fi ve HD fi broblast cell lines (lanes 8–12) compared with fi broblasts from fi ve healthy individuals (lanes 3–7). Blots were also probed with anti-EEA1 and anti-Htt antiserum to confi rm equal loading and the identity of the HD cell lines (see Results). (E) Knockdown of HAP40 protein levels in fi broblasts from all fi ve HD cell lines by RNAi. Western blot analysis of EEA1 and HAP40 in untransfected cells (lanes 1, 4, 7, 10, and 13), cells transfected with unrelated siRNA against GFP (lanes 2, 5, 8, 11, and 14), or HAP40 siRNA (lanes 3, 6, 9, 12, and 15). (F) EGFP-Rab5–labeled endosomes align primarily with microtubules in healthy fi broblasts. Primary human fi broblasts from healthy individuals were transfected with EGFP-Rab5 (green) and fi xed and immunostained for microtubules and F-actin. The same cell is shown with its β-tubulin staining (red) on the left and for F-actin (red) on the right, as indicated. Arrowheads in the insets (magnifi cations of the boxed areas) point to endosomes aligned with microtubules. Overlap of EGFP-Rab5 with tubulin signals was 82% (SD ± 9%, n = 10) for healthy fi broblasts and 15% (SD ± 6%, n = 9) for HD cell lines (see G). (G) The cell processed as in F shows that EGFP-Rab5–labeled endosomes align strikingly with F-actin in fi bro- blasts from HD patients. Arrowheads in the insets point to endosomes (green) aligned with F-actin (red). Overlap of EGFP-Rab5 with actin was 2% (SD ± 1%, n = 10) for healthy fi broblasts (see F) and 44% (SD ± 8%, n = 10) for HD cell lines. Bar, 10 μm. 612 JCB • VOLUME 172 • NUMBER 4 • 2006 phenotype was obtained by the overexpression of HAP40 in apparently occur in peripheral tissues such as fi broblasts as well HeLa cells (not depicted). as in neuronal systems and, therefore, are of potential relevance for HD. The Htt–HAP40 complex enhances the binding of early endosomes to actin Increased HAP40 levels cause alterations The alignment of early endosomes on F-actin in HD cell lines in endocytic traffi cking could be a secondary effect from the inhibition of endosome– To gain some insights into possible alterations of endocytic microtubule interactions. To directly test the role of HAP40 in transport caused by increased levels of HAP40, we tested the the association between early endosomes and actin fi laments uptake of transferrin in HeLa cells overexpressing HAP40 and (Fig. 6, B and E), we modifi ed the biochemical sedimentation in fi broblasts from healthy and HD patients. Fig. 10 A shows assay used to study endosome–microtubule interactions (Fig. 6, that the uptake of transferrin was reduced by 30% in HeLa cells A and D) by replacing taxol-stabilized microtubules with freshly overexpressing HAP40 compared with mock-transfected or in vitro–polymerized F-actin. Unlike for endosome–microtubule control cells. Consistent with this result, fi broblasts from HD binding, depletion of active Rab5 from endosomal membranes patients displaying higher levels of endogenous HAP40 (Fig. 7 D) by treatment with either Rab-GDI or RN-tre did not decrease displayed a similarly reduced uptake of transferrin compared endosome–actin interactions (Fig. 6 E). Thus, a basal level of with normal fi broblasts (Fig. 10 B). As for the block of endo- endosome–F-actin binding activity was independent of Rab5. some motility, the inhibitory effect on transferrin uptake was However, the addition of HAP40-GST fusion protein stimulated rescued by the depletion of HAP40 by RNAi. These data sug- binding (260%) over control levels, whereas GST alone was gest that the inhibitory effects on endosome motility caused by ineffective. Evidently, endosome binding to microtubules and up-regulation of HAP40 also result in defects in cargo transport F-actin is reciprocally regulated through HAP40 because con- through the endocytic pathway. centrations (1 μM) inhibiting binding to microtubules (Fig. 6 D) clearly stimulated binding to F-actin (Fig. 6 E). This effect Discussion was Rab5 dependent because when Rab-GDI or RN-tre were added together with HAP40, the stimulation was nearly abol- The key fi nding of this study is that the complex between ished (Fig. 6 E). Again, immunoblotting confi rmed that the HAP40 and Htt is a direct downstream effector of Rab5 that amount of actin in the pellets was unaffected by any added pro- regulates the dynamics of early endosomes through a switch tein and that all changes in pelletable fl uorescence corresponded from microtubules to F-actin. These fi ndings provide important consistently to altered band intensities of early endosomal new insights into how the motility of early endosomes is regu- markers (EEA1 and TFr) but not to others (LAMP1 and GM130), lated under physiological and pathological conditions. Htt has indicating specifi c effects of Htt–HAP40 on endosome–actin been implicated in clathrin-mediated endocytosis, regulation of binding (Fig. 6 B). the actin cytoskeleton, and microtubule-dependent transport along the endocytic pathway via interactions with its numerous Alterations in early endosome motility binding partners (Harjes and Wanker, 2003). Such multiplicity in a HD mouse model of striatal cells of roles implies that the activities of Htt in endocytic membrane Next, we investigated the role of HAP40 in Rab5 dynamics in an traffi cking need to be spatially and temporally coordinated. experimental system that is more relevant for HD using immor- In showing for the fi rst time that a complex between Htt and one talized STHdhQ striatal cells (Trettel et al., 2000). These cells of its binding partners, HAP40, can be recruited onto endo- were established from embryonic normal or HD knock-in mice somes by interacting directly with Rab5, our data provide novel 7/7 and either express normal (STHdhQ ) or mutant Htt as a result insights into the mechanisms governing the targeting of Htt to of a CAG expansion inserted into the endogenous Htt gene (het- early endosomes and its regulatory activity on cytoskeleton- 7/111 111/111 erozygous STHdhQ and homozygous STHdhQ ). Thus, dependent dynamics. they refl ect the closest situation to HD patients as normal, and Under normal physiological conditions, early endosomes mutant Htt are expressed at endogenous levels. Remarkably, undergo frequent short-range movements on actin but also we again found endogenous HAP40 protein levels elevated in long-range bidirectional movements along microtubules 7/111 111/111 7/7 STHdhQ and STHdhQ compared with STHdhQ cells (Nielsen et al., 1999; Gasman et al., 2003; Rink et al. 2005). (Fig. 8 B), which is consistent with the data on HD fi broblasts Plus end movement of early endosomes along microtubules is (Fig. 7 D) and brain tissue from HD patients (Fig. 8 B). Live cell propelled by KIF16B (Hoepfner et al. 2005). Overexpression imaging revealed EGFP-Rab5–positive organelles moving bidi- of HAP40, which is rate limiting for the recruitment of Htt rectionally in neuronlike outgrowths as well as in the cell body on the membrane, caused the detachment of early endosomes 7/7 of normal STHdhQ cells (Fig. 8 A and Video 6, available at from microtubules and their preferential association with actin http://www.jcb.org/cgi/content/full/jcb.200509091/DC1). Con- fi laments, thus limiting both their velocity and range of 111/111 7/111 versely, STHdhQ and STHdhQ cells clearly displayed movements. Therefore, the Htt–HAP40–Rab5 complex is a key a drastic reduction of endosome dynamics (Fig. 8 A and Videos regulator of the switch from one type of fi laments to another. 7 and 8), which is consistent with the observations on HD Our data are consistent with previous studies documenting fi broblasts (Fig. 7 B and Videos 3 and 4). Collectively, defects alterations in microtubule-dependent motility in HD model of Rab5 dynamics caused by pathogenic excess of HAP40 systems (Block-Galarza et al., 1997; Engelender et al., 1997; THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 613 Figure 8. Elevated endogenous HAP40 pro- tein levels impair Rab5 dynamics in vivo in 111/111 7/111 STHdhQ and STHdhQ striatal cells from a HD mouse model. (A) Cells expressing 7/7 111/111 normal (STHdhQ ) or mutant (STHdhQ 7/111 and STHdhQ ) Htt were transfected with EGFP-Rab5 plasmid, differentiated, and im- aged using time-lapse video microscopy. Over- laid images were generated as described for Fig. 5 B. Videos corresponding to A are avail- able as online supplemental material (Videos 6–8, available at http://www.jcb.org/cgi/ content/full/jcb.200509091/DC1). Motility of Rab5 compartments was drastically reduced in 111/111 7/111 STHdhQ and STHdhQ compared 7/7 with STHdhQ cells. Insets show magnifi ca- tions of boxed areas. Bar, 10 μm. (B) Endog- enous protein levels of HAP40 are elevated in 111/111 7/111 STHdhQ (lane 1) and STHdhQ (lane 2) 7/7 compared with STHdhQ cells (lane 3) as well as in striatal tissue from fi ve human postmortem brains affected by HD (lanes 4–8) compared with healthy control brains (lanes 9–13). Blots were also probed with anti-EEA1 and anti-Htt antiserum to confi rm equal load- ing and the expression of wild-type and mutant Htt as described for Fig. 7 D. Gauthier et al., 2004). However, these studies have exclusively nucleus (Landles and Bates, 2004). The majority of HAP40 is implicated alterations between mutant Htt and HIP/HAP also nuclear under normal conditions (Fig. 3; Peters and Ross, effectors. For example, wild-type Htt has been shown to interact 2001), although the functional signifi cance of this localization Glued with the dynactin subunit p150 via HAP1 and mutant Htt to is unclear at present. Our data indicate that HAP40 fulfi lls a disrupt the dynein motor complex in axonal transport (Gauthier function in the cytoplasm. It is interesting in this respect to note et al., 2004). In this study, we have uncovered a different mech- that HAP40 adds to the increasing list of proteins implicated anism based on the up-regulation of an Htt adaptor, HAP40. in a dual role in endocytic traffi cking and nuclear signaling Compelling evidence in support of this mechanism was pro- (Miaczynska et al., 2004). The molecular mechanism underly- vided by the rescue of the motility block upon depletion of ing the up-regulation of HAP40 unexpectedly observed in cells HAP40 by RNAi both in HD fi broblasts and in striatal cells. and brain tissue from HD patients remains to be determined. Because the COOH-terminal part of Htt is responsible for the The most likely explanation is that it arises as a consequence of underlying interactions with HAP40 and Rab5, this endosomal alterations in gene expression caused by mutated Htt in the recruitment affects normal as well as mutant Htt. Consistently, nucleus. Messenger RNA microarray studies have revealed wild-type (overexpression of HAP40 in HeLa cells), heterozy- many transcriptional abnormalities in HD (Chan et al., 2002; 7/111 111/111 gous (STHdhQ ), and homozygous (STHdhQ ) HD cells Sipione et al., 2002; Sugars and Rubinsztein, 2003), although display very similar phenotypes. In this way, functional compe- no changes for HAP40 or any other Htt-interacting partners tition with the Rab5-dependent endosomal kinesin KIF16B and have been reported so far. Up-regulation of HAP40 at the pro- disruption of the dynein–dynactin complex (Gauthier et al., 2004) tein level might thus serve as a new diagnostic indicator for the could account for the compromised bidirectional microtubule- disease. However, it is premature to judge to what extent this based motility. Therefore, our data suggest that multiple defects abnormality and the consequent alterations in membrane dy- may contribute to the block of vesicular transport in HD cells, namics contribute to the pathogenetic mechanism of HD as which implicates various HIP/HAPs interacting with different compared with nuclear activities of Htt and HAP40. Although regions of Htt. endosome motility is not perturbed in HD cells to the extent of Previous fi ndings have shown that Htt is also linked to compromising cell viability, we nevertheless uncovered defects Rab8 through FIP-2 (Hattula and Peranen, 2000) and that this in endocytic transport, specifi cally a decrease in transferrin interaction regulates cell polarization and morphogenesis. uptake. These observations may be of particular functional rel- Whereas the mechanism underlying the latter process is unclear, evance in neurons given the importance of long-range transport in light of our observation, we propose that Htt is a multifunc- (Block-Galarza et al., 1997; Engelender et al., 1997; Gauthier tional protein that may be recruited by different Rab GTPases et al., 2004). Therefore, it is possible that alterations in endo- through different adaptors at different intracellular locations to some motility and transport may further compromise the patho- regulate organelle dynamics. logical state induced by mutated Htt on the survival and function The primary pathological cause of HD is attributed to the of neurons or some selected populations of neurons where en- abnormal activity of Htt and its interacting partners in the dosomal transport is particularly rate limiting in vivo. Addressing 614 JCB • VOLUME 172 • NUMBER 4 • 2006 7/111 Figure 9. Htt and HAP40 are recruited onto Rab5 vesicles in primary fi broblasts from human HD patients and STHdhQ striatal cells from a HD mouse model. (A) Image gallery of the same fi broblast cell from a healthy individual transfected with EGFP-Rab5 (green) and fi xed and immunostained for endog- enous HAP40 (red) and Htt (red). Boxed areas are magnifi ed in the top panels. (B) The same analysis of a fi broblast from a HD patient. Yellow (arrowheads) indicates colocalization of EGFP-Rab5 and Htt (merge Rab5 and Htt,) or of EGFP-Rab5 and HAP40 (merge Rab5 and HAP40). Overlapping of EGFP-Rab5 with Htt was 6% (SD ± 3%, n = 10) for fi broblasts from healthy individuals (A) and 46% (SD ± 8%, n = 10) from HD patients (B). Overlapping of EGFP- Rab5 with HAP40 was 4% (SD ± 3%, n = 10) for fi broblasts from healthy individuals (A) and 51% (SD ± 9%, n = 10) from HD patients (B). (C) Image gal- 7/7 lery of the same differentiated STHdhQ cell (homozygous for normal Htt) transfected with EGFP-Rab5 (green) and immunostained for endogenous HAP40 7/111 (red) and Htt (red). (D) The same analysis of a differentiated STHdhQ cell (heterozygous for mutant Htt). Overlapping of EGFP-Rab5 with Htt was 5% 7/7 7/111 (SD ± 4%, n = 10) for STHdhQ cells (C) and 83% (SD ± 12%, n = 10) for STHdhQ cells (D). Overlapping of EGFP-Rab5 with HAP40 was 4% 7/7 7/111 (SD ± 3%, n = 10) for STHdhQ cells (C) and 86% (SD ± 15%, n = 10) for STHdhQ cells (D). The analysis was restricted to cellular outgrowths. 111/111 Essentially the same results were obtained for STHdhQ cells (homozygous for mutant Htt; not depicted). Bar, 10 μm. this hypothesis requires a more thorough investigation of Materials and methods HAP40 and Htt function in the transport of different types of Rab5 affi nity chromatography and HAP40 and Htt cloning cargo along the endocytic/recycling as well as degradative GST-Rab5 affi nity chromatography with bovine brain cytosol or in vitro– pathway in neuronal cells rather than fi broblasts. A deeper anal- translated proteins was performed as described previously (Christoforidis ysis of the role of HAP40 on the molecular level will hopefully et al., 1999). HAP40 was cloned from rat brain cDNA by PCR, Htt frag- ments were cloned by PCR on full-length Htt cDNA in pBlueScript (a gift improve our understanding of both HD pathogenesis and the from M. Sherman, Boston University School of Medicine, Boston, MA), mechanisms coordinating organelle–cytoskeleton interactions and all PCR products were cloned into pcDNA3.1 (Invitrogen) and se- during membrane traffi cking. quenced to confi rm their identity. To estimate the stoichiometry of the THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 615 Cell culture procedures HeLa and human primary fi broblast cells were grown according to stan- dard procedures. Immortalized STHdhQ striatal cell lines from control and HD knock-in mice were cultured and differentiated as described previously (Trettel et al., 2000). For transient expression studies, cells were transfected using LipofectAMINE 2000 (Invitrogen) and used 12 h after transfection for immunoblot analysis or intracellular localization studies according to standard protocols. Fixed cells were analyzed using a 100×/NA 1.40 plan-Apochromat oil immersion lens (Carl Zeiss MicroImaging, Inc.) on a microscope (Axiovert S100TV; Carl Zeiss MicroImaging, Inc.). Illumination was performed with a 100-W mercury lamp with fi lter sets for GFP, Alexa- Fluor568, DAPI, and Cy5 fl uorescence. Images were acquired at 1,300 × 1,000 pixels with a digital camera (SP 1.4.4; Diagnostic Instruments) con- trolled by the MetaVue 6.1 software package (Universal Imaging Corp.). Raw images from various color channels were assembled and colorized in Adobe Photoshop 7.0. Brightness and contrast were adjusted for visual clarity. To validate immunofl uorescence microscopy studies using Alexa- Fluor568- and Cy5-conjugated secondary antibodies, HAP40 was overex- pressed in HeLa cells and cells stained with a mixture of anti-HAP40 and -Htt antisera. Single addition of secondary antibody against mouse IgG (for Htt) conjugated with AlexaFluor568 resulted in no detectable bleed- through into the Cy5 channel at the exposure time and fi lter settings used for HAP40 detection with Cy5. Moreover, signals for HAP40 that were ob- tained with Cy5 looked very similar in the presence or absence of Alexa- Fluor568. Finally, when these dyes were swapped on the secondary antibodies, very similar distribution patterns for HAP40 and Htt were obtained. Collectively, the colocalization between HAP40 and Htt revealed in Figs. 3 and 9 is not caused by bleed-through. The percentage of colocal- ization between immunofl uorescence signals was determined as follows: raw signals from discrete membrane structures were manually counted and inspected for overlapping with signals in other channels to calculate the percentage of colocalization. Only signals above value 70 on the eight-bit tonal scale were considered specifi c and used for the analysis. Typically, Figure 10. Elevated levels of HAP40 reduces transferrin uptake. (A) HeLa 10 cells (n = 10) were counted to calculate the mean colocalization cells transfected with HAP40 expression (HAP40), empty plasmid (mock), and SD. For overlapping studies of EGFP-labeled early endosomes with cy- or untreated (WT) were serum starved, allowed to internalize biotinylated toskeletal fi laments, these organelles were considered colocalized to either transferrin (Tf) for the indicated times, washed, and lysed, and internalized microtubules or F-actin if at least three early endosomes in a row were transferrin was quantifi ed. The mean values of triplicate samples from one clearly aligned to a fi lament. In areas where microtubules and F-actin con- representative experiment out of three with SD (error bars, often omitted by verged (e.g., close to the cell edge), early endosomes could often not be plot symbols) are shown. (B) The same uptake for primary human fi bro- assigned to either type of fi lament. The sum of early endosomes aligned to blasts untreated or transfected with siRNA duplexes against HAP40 microtubules, F-actin, and unassignable early endosomes (100%) was (siHAP40) or unrelated GFP (siGFP). The mean values of different cell lines used to calculate percentages of colocalization. Internalized biotinylated from fi ve healthy persons (5× WT), fi ve HD patients (5× HD), the same but transferrin was detected with ruthenium-labeled antitransferrin antibodies treated with siHAP40 (5× HD siHAP40), or the mean values of triplicate and subsequently quantifi ed by ECL analysis as described previously sample obtained from one HD line treated with unrelated siGFP (1× HD (Hoepfner et al. 2005). siGFP) are shown. Error bars (mostly omitted by plot symbols) represent the SD between cell lines (5× WT, 5× HD, and 5× HD siHAP40) or of tripli- cate samples (1× HD siGFP). Cell lines and tissue samples The primary human fi broblast cell lines GM00023, GM00024, GM00037, GM00038, and GM00041 (Apparently Healthy cell collection) and GM04281, GM04723, GM03621, GM04869, and GM04847 (Hun- bound complex, GST-Rab5 was incubated in the presence of a 10-fold ex- tington Disease cell collection) were obtained from Coriell Cell Repositories. cess of the COOH-terminal fragment of Htt over HAP40. Band intensities Cell lines were established from both genders from healthy, unrelated obtained under these conditions were quantifi ed in ImageJ software individuals by the age of 3–31 yr or from clinically affected onset HD (National Institutes of Health; Htt4/HAP40 = 6:1) and corrected for the patients by the age of 19–32 yr. Immortalized striatal cell lines established number of [ S]methionine residues incorporated (Htt4/HAP40 = 22:4). from mouse embryos (STHdhQ) were gifts from M. MacDonald (Richard B. The resulting ratio was 1:1. Assuming a 1:1 ratio of Rab protein to Simches Research Center, Boston, MA; Trettel et al., 2000). Human effector, the stoichiometry of the Rab5/HAP40/Htt interaction was esti- postmortem brain samples (CAP; Globus Pallidus) from healthy and HD mated at 1:1:1. grades 3 and 4 donors were provided by the Harvard Brain Tissue Resource Center. Antibodies and recombinant proteins Monoclonal mouse antibodies against Htt were obtained from Chemicon Protein identifi cation by mass spectrometry and Y. Trottier (Institut de Génétique et de Biologie Moléculaire et Cellu- Gel-separated Coomassie-stained proteins were excised from the gel slab laire, Université Louis Pasteur, Illkirch, France), and polyclonal rabbit anti- and in-gel digested with trypsin as described previously (Shevchenko et al., body against HAP40 was obtained from Chemicon. Microtubules were 1996). Tryptic peptides were sequenced by nanoelectrospray tandem revealed with mouse monoclonal anti–β-tubulin antibody (BD Biosciences), mass spectrometry on hybrid quadrupole time-of-fl ight mass spectrometers and F-actin was revealed with AlexaFluor568-conjugated phalloidin (Invit- (Q-TOF I; Micromass, Ltd. and QSTAR Pulsar I; MDS Sciex) as described rogen). Secondary anti–mouse and rabbit IgG for immunofl uorescence mi- previously (Shevchenko et al., 1997). Database searching was performed croscopy were conjugated to AlexaFluor568 and Cy5 (Invitrogen). Rabbit by Mascot software (Matrix Science, Ltd.). polyclonal anti-EEA1 antibody was described previously (Christoforidis et al. 1999), mouse monoclonal antibody against the cytoplasmic tail of Tfr was purchased from Invitrogen, mouse monoclonal anti-LAMP1 antibody In vitro motility of early endosomes was obtained from Becton Dickinson, and mouse monoclonal anti-GM130 The in vitro motility assay was essentially performed as described previ- antibody was obtained from Abcam. Rab-GDI and RN-tre were prepared ously (Nielsen et al., 1999) with the following modifi cations (Hoepfner as described previously (Ullrich et al., 1994; Lanzetti et al., 2000). GST et al., 2005). KHMG (110 mM KCl, 50 mM Hepes-KOH, pH 7.4, 2 mM and HAP40-GST fusion proteins were affi nity purifi ed from Escherichia coli MgCl2, and 10% glycerol) was used as an assay buffer. For preparation lysates according to standard procedures. of the antifade solution, BRB80 buffer was substituted with KHMG, and the 616 JCB • VOLUME 172 • NUMBER 4 • 2006 solution was further supplemented with 10% serum. The energy mix con- Center is acknowledged for providing human postmortem brain samples from sisted of 75 mM creatine phosphate, 10 mM ATP, 10 mM GTP, and 20 mM healthy and HD grades 3 and 4 donors. MgCl2 in BRB80. For the assay, taxol-stabilized microtubules were per- This work was supported by the Max Planck Society and grants from fused in a microscopy chamber and allowed to bind to the coverslip. Next, the Human Frontier Science Program (RG-0260/1999-M) and the European 10 μl of 10% nonspecifi c rabbit serum in antifade solution was perfused in Union (HPRN-CT-2000-00081). A. Pal, F. Severin, and B. Lommer are long- the chamber followed by 10 μl of the assay mixture (2 μl of 5 mg/ml fl uo- term Max Planck fellows. rescently labeled early endosome, 1 μl of energy mix, 6 μl of the antifade solution, and 1 μl of saturated hemoglobin solution in KHMG) and was in- Submitted: 15 September 2005 cubated for 5 min at RT. At least three videos per sample were recorded Accepted: 10 January 2006 (60 frames with 2-s intervals) and analyzed as described previously (Nielsen et al., 1999). References In vivo motility of early endosomes Cells were grown on glass coverslips, transfected with EGFP-Rab5 alone Block-Galarza, J., K.O. Chase, E. Sapp, K.T. Vaughn, R.B. Vallee, M. DiFiglia, or together with siRNA against HAP40, and transferred to custom-built and N. Aronin. 1997. Fast transport and retrograde movement of huntingtin aluminum microscope slide chambers (Nielsen et al., 1999) just before and HAP 1 in axons. Neuroreport. 8:2247–2251. observation. 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The equation of the linear regression analysis was y = 4,548.4x − Harjes, P., and E.E. Wanker. 2003. The hunt for huntingtin function: interaction 2,653.1 with R = 0.9879, where y is the emission in arbitrary units and partners tell many different stories. Trends Biochem. Sci. 28:425–433. x is the concentration of early endosomes in protein mass units. For binding Hattula, K., and J. Peranen. 2000. FIP-2, a coiled-coil protein, links Huntingtin to of early endosomes to F-actin, globular-actin (Cytoskeleton, Inc.) was po- Rab8 and modulates cellular morphogenesis. Curr. Biol. 10:1603–1606. lymerized in KHMG buffer at RT for 1 h and centrifuged for 30 min at Hoepfner, S., F. Severin, A. Cabezas, B. Habermann, A. Runge, D. Gillooly, H. 100,000 g. The resulting pellet was washed with KHMG buffer and resus- Stenmark, and M. Zerial. 2005. Modulation of receptor recycling and pended at 10 mg/ml. The assay was then performed with 10 μg of F-actin degradation by the endosomal kinesin KIF16B. Cell. 121:437–450. as described for microtubules. Hoffner, G., P. Kahlem, and P.J. Djian. 2002. Perinuclear localization of hun- tingtin as a consequence of its binding to microtubules through an inter- Online supplemental material action with beta-tubulin: relevance to Huntington’s disease. J. Cell Sci. 115:941–948. All videos show EGFP-Rab5 dynamics, and images were acquired as described in In vivo motility of early endosomes. Video 1 shows a wild- Landles, C., and G.P. Bates. 2004. Huntingtin and the molecular pathogenesis type HeLa cell transfected with plasmid for EGFP-Rab5 expression only. of Huntington’s disease. EMBO Rep. 5:958–963. Video 2 shows a HeLa cell cotransfected with plasmids for EGFP-Rab5 and Lanzetti, L., V. Rybin, M.G. Malabarba, S. Christoforidis, G. Scita, M. Zerial, HAP40. Video 3 shows a primary human fi broblast from a healthy person and P.P. Di Fiore. 2000. The Eps8 protein coordinates EGF receptor sig- transfected with plasmid for EFGP-Rab5 expression only, whereas Video 4 nalling through Rac and traffi cking through Rab5. Nature. 408:374–377. shows the fi broblast from a HD patient. Video 5 shows a primary human Miaczynska, M., L. Pelkmans, and M. Zerial. 2004. Not just a sink: endosomes fi broblast from a HD patient cotransfected with plasmid for EGFP-Rab5 in control of signal transduction. Curr. Opin. Cell Biol. 16:400–406. transfection and siRNA duplexes against HAP40. Video 6 shows a striatal Nakamura, N., C. Rabouille, R. Watson, T. Nilsson, N. Hui, P. Slusarewicz, 7/7 STHdhQ cell transfected with plasmid for EGFP-Rab5 expression only, T.E. Kreis, and G. Warren. 1995. Characterization of a cis-Golgi matrix 7/111 Video 7 shows the same for an STHdhQ cell, and Video 8 shows this protein, GM130. J. Cell Biol. 131:1715–1726. 111/111 for an STHdhQ cell. Online supplemental material is available at Nielsen, E., F. Severin, J.M. Backert, A.A. Hyman, and M. Zerial. 1999. Rab5 http://www.jcb.org/cgi/content/full/jcb.200509091/DC1. regulates motility of early endosomes on microtubules. Nat. Cell Biol. 1:376–382. We thank M. Sherman for full-length Htt cDNA, M. Matyash for the rat brain Peters, M.F., and C.A. Ross. 2001. Isolation of a 40-kDa Huntingtin-associated cDNA library, M. MacDonald for STHdhQ striatal cell lines, and Y. Trottier for protein. J. Biol. Chem. 276:3188–3194. invaluable advice, discussions, and for providing anti-Htt antibodies. We Rigamonti, D., J.H. Bauer, C. De-Fraja, L. Conti, S. Sipione, C. Sciorati, E. thank K. Simons, J. Howard, L. Pelkman, and J. Rink for critical reading of the Clementi, A. Hackam, M.R. Hayden, Y. Li, et al. 2000. Wild-type hun- manuscript. The Coriell Cell Repositories is acknowledged for providing the tingtin protects from apoptosis upstream of caspase-3. J. Neurosci. human primary fi broblast cell lines, and the Harvard Brain Tissue Resource 20:3705–3713. THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 617 Rigamonti, D., S. Sipione, D. Goffredo, C. Zuccato, E. Fossale, and E. Cattaneo. 2001. Huntingtin’s neuroprotective activity occurs via inhibition of pro- caspase-9 processing. J. Biol. Chem. 276:14545–14548. Rink, J., E. Ghigo, Y. Kalaidzidis, and M. Zerial. 2005. Rab conversion as a mech- anism of progression from early to late endosomes. Cell. 122:735–749. Shevchenko, A., M. Wilm, O. Vorm, and M. Mann. 1996. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68:850–858. Shevchenko, A., I. Chernushevich, W. Ens, K.G. Standing, B. Thomson, M. Wilm, and M. Mann. 1997. Rapid ‘de novo’ peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/ time-of-fl ight mass spectrometer. Rapid Commun. Mass Spectrom. 11:1015–1024. Sipione, S., D. Rigamonti, M. Valenza, C. Zuccato, L. Conti, J. Pritchard, C. Kooperberg, J.M. Olson, and E. Cattaneo. 2002. Early transcriptional profi les in huntingtin-inducible striatal cells by microarray analyses. Hum. Mol. Genet. 11:1953–1965. Stenmark, H., R.G. Parton, O. Steele-Mortimer, A. Lutcke, J. Gruenberg, and M. Zerial. 1994. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 13:1287–1296. Sugars, K.L., and D.C. Rubinsztein. 2003. Transcriptional abnormalities in Huntington disease. Trends Genet. 19:233–238. Tao, T., and A.M. Tartakoff. 2001. Nuclear relocation of normal huntingtin. Traf c fi . 2:385–394. Trettel, F., D. Rigamonti, P. Hilditch-Maguire, V.C. Wheeler, A.H. Sharp, F. Persichetti, E. Cattaneo, and M.E. MacDonald. 2000. Dominant phe- Q111 notypes produced by the HD mutation in STHdh striatal cells. Hum. Mol. Genet. 9:2799–2809. Trottier, Y., D. Devys, G. Imbert, F. Saudou, I. An, Y. Lutz, C. Weber, Y. Agid, E.C. Hirsch, and J.L. Mandel. 1995. Cellular localization of the Huntington’s disease protein and discrimination of the normal and mutated form. Nat. Genet. 10:104–110. Ullrich, O., H. Horiuchi, C. Bucci, and M. Zerial. 1994. Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature. 368:157–160. Zerial, M., and H.M. McBride. 2001. Rab proteins as membrane organizers. Nat. Rev. Mol. Cell Biol. 2:107–117. 618 JCB • VOLUME 172 • NUMBER 4 • 2006 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Huntingtin–HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington's disease

The Journal of Cell Biology , Volume 172 (4) – Feb 13, 2006

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Abstract

JCB: ARTICLE <doi>10.1083/jcb.200509091</doi><aid>200509091</aid>Huntingtin–HAP40 complex is a novel Rab5 effector that regulates early endosome motility and is up-regulated in Huntington’s disease Arun Pal, Fedor Severin, Barbara Lommer, Anna Shevchenko, and Marino Zerial Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany he molecular mechanisms underlying the targeting Remarkably, endogenous HAP40 was up- regulated in fi - of Huntingtin (Htt) to endosomes and its multifaceted broblasts and brain tissue from human patients affected T role in endocytosis are poorly understood. In this by Huntington’s disease (HD) as well as in STHdhQ study, we have identifi ed Htt-associated protein 40 (HAP40) striatal cells established from a HD mouse model. These as a novel effector of the small guanosine triphosphatase cells consistently displayed altered endosome motility and Rab5, a key regulator of endocytosis. HAP40 mediates endocytic activity, which was restored by the ablation of the recruitment of Htt by Rab5 onto early endosomes. HAP40. In revealing an unexpected link between Rab5, HAP40 overexpression caused a drastic reduction of early HAP40, and Htt, we uncovered a new mechanism regu- endosomal motility through their displacement from mi- lating cytoskeleton-dependent endosome dynamics and crotubules and preferential association with actin fi laments. its dysfunction under pathological conditions. Introduction Huntington’s disease (HD) is a neurodegenerative disorder expansion confers the adjacent proline-rich sequence in Htt caused by expansion of the CAG repeat in the gene encoding alterations in binding affi nity to HIPs/HAPs. Thus, release Huntingtin (Htt), which confers to the protein an expanded from or sequestration of these molecules by mutant Htt has NH -terminal polyglutamine (polyQ) stretch of >35 residues been implicated in the pathogenetic mechanisms. (for review see Harjes and Wanker, 2003). The function of Htt For example, the tighter binding of HAP1 to mutant Htt is is largely unclear. It has been shown to interact with microtu- thought to impair the correct dynactin–dynein motor complex bules (Hoffner et al., 2002) and to display anti-apoptotic activ- assembly and cause a traffi cking defect, leading to neuronal de- ity (Rigamonti et al., 2000, 2001). Insights into its function generation (Block-Galarza et al., 1997; Engelender et al., 1997; came from studies of Htt-interacting proteins (HIPs) and Htt- Gauthier et al., 2004). Consistently, mutant Htt was recently associated proteins (HAPs). For example, interactions with shown to release dynein from microtubules and reduce the mo- HIP1, HIP1R, PACSIN1, SH3GL3, and HIP14 have implicated tility of EGFP –brain-derived neurotrophic factor–containing Htt in clathrin-mediated endocytosis. Studies of HAP1 have vesicles in vivo (Gauthier et al., 2004). However, the upstream suggested a role for Htt in axonal transport in neurons by link- events that target Htt and its partners to their various sites of ing vesicles to the dynein–dynactin motor complex (Block- function and the mechanisms whereby they regulate intracellu- Galarza et al., 1997; Engelender et al., 1997). The polyQ lar traffi cking remain elusive. In this study, we report an unexpected link between Htt and the small GTPase Rab5 via the adaptor protein HAP40. Correspondence to Marino Zerial: zerial@mpi-cbg.de Correspondence to Marino Zerial: zerial@mpi-cbg.de Rab5 is a key regulator of endocytosis that orchestrates the F F.. Severin’ Severin’s present address is Biotechnology Centre, University of T s present address is Biotechnology Centre, University of Technology echnology recruitment of multiple effector proteins on the early endo- Dresden, Cellular Machines, 01307 Dresden, Ger Dresden, Cellular Machines, 01307 Dresden, Germany many.. some membrane to regulate organelle tethering, fusion, and Abbreviations used in this paper: EEA1, early endosome antigen 1; F-actin, fi Abbreviations used in this paper: EEA1, early endosome antigen 1; F-actin, fi la- la- microtubule-dependent motility (Zerial and McBride, 2001). mentous actin; GDI, GDP dissociation inhibitor; GDP mentous actin; GDI, GDP dissociation inhibitor; GDP, guanosine diphosphate; , guanosine diphosphate; HAP HAP, Htt-associated protein; HD, Huntington’ , Htt-associated protein; HD, Huntington’s disease; HIP; Htt-interacting pro- s disease; HIP; Htt-interacting pro- Our data extend the analysis of the Rab5 effector machinery by tein; Htt, Huntingtin; LAMP tein; Htt, Huntingtin; LAMP, lysosome-associated membrane protein; polyQ, , lysosome-associated membrane protein; polyQ, functionally implicating the interaction between Rab5 and Htt polyglutamine; RNAi, RNA inter polyglutamine; RNAi, RNA interference; siRNA, shor ference; siRNA, short inter t interfering RNA; Tfr fering RNA; Tfr, trans- , trans- ferrin receptor ferrin receptor.. in the regulation of the differential association of early endo- The online version of this ar The online version of this article contains supplemental material. ticle contains supplemental material. somes with the actin and tubulin cytoskeleton. © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 172, No. 4, February 13, 2006 605–618 http://www.jcb.org/cgi/doi/10.1083/jcb.200509091 JCB 605 THE JOURNAL OF CELL BIOLOGY eluted together with HAP40 from GTPγS- but not GDP-bound GST-Rab5 (Fig. 2 D, lanes 7 and 8). None of the other Htt frag- ments displayed such binding (Fig. 2 D, lanes 1–6), which is consistent with the reported interaction map between HAP40 and Htt (Peters and Ross, 2001). Neither the Htt fragments nor HAP40 displayed binding to Rab4, Rab6, Rab7, or Rab11 (Fig. 2 D), suggesting the interaction with Rab5 is specifi c. Thus, we conclude that HAP40 binds to the COOH-terminal part of Htt and links the complex to active Rab5. By applying a 10-fold excess of the COOH-terminal fragment of Htt onto the Rab5 column to reduce binding of free HAP40 to Rab5 (Fig. 2 E), we estimated the stoichiometry of the Rab5/HAP40/Htt interaction in this assay to be 1:1:1 (see Rab5 affi nity…cloning). Htt is recruited onto early endosome in a HAP40- and Rab5-dependent manner Figure 1. Htt and HAP40 elute from immobilized Rab5. (A) SDS-PAGE of proteins eluted from immobilized GST-Rab5 that was loaded with GTPγS. We began testing the functional relevance of this interaction The indicated bands were found to correspond to Htt, HAP40, Rab5-GST, in vivo by immunofl uorescence microscopy analysis of HeLa and EEA1 by mass spectrometry analysis. (B) Western blot analysis of cells. First, we verifi ed that the anti-HAP40 antibody resulted chromatographic eluates from GST-Rab5 (lanes 1 and 2) and -Rab4 (lanes 3 and 4) affi nity columns preloaded with GDP (D; lanes 1 and 3) or GTPγS in specifi c staining above background levels for detection of (T; lanes 2 and 4). The bovine brain cytosol (BBC; lane 5) was used as a the endogenous antigen (Fig. 3 A). Endogenous HAP40 dis- source of proteins. Blots were probed for Htt and HAP40 as indicated. played a diffuse staining in the cytoplasm and accumulated in the nucleus, whereas endogenous Htt localized to discrete cytoplasmic structures (Fig. 3 B) as reported previously (Peters Results and Ross, 2001; Tao and Tartakoff, 2001). Colocalization of The Htt–HAP40 complex is a novel endogenous HAP40 and Htt was hardly detectable (Fig. 3 B). Rab5 effector Early endosomes labeled with EGFP-Rab5 displayed little co- Affi nity chromatography revealed several downstream effectors localization with endogenous Htt (7 ± 5% overlap; n = 10; of the small GTPase Rab5 (Christoforidis et al. 1999). Surpris- Fig. 3 C). However, the association of HAP40 with early en- ingly, among the proteins specifi cally eluted from the GST- dosomes dramatically increased upon overexpression. In cells Rab5–GTPγS but not from the Rab5–guanosine diphosphate overexpressing HAP40 (see Fig. 7 D) but not EGFP-Rab5 (GDP) nor GST-Rab4 af nity column, we identifi fi ed Htt and alone, HAP40 signifi cantly colocalized with endogenous Htt on HAP40 (Peters and Ross, 2001) by mass spectrometry and im- EGFP-Rab5–positive early endosomes (Fig. 3, compare B with munoblotting (Fig. 1, A and B). Therefore, we investigated the D; endogenous Htt and EGFP-Rab5: 43 ± 6% overlap, n = 10; function of HAP40 and Htt with respect to Rab5. HAP40 and EGFP-Rab5: 31 ± 7% overlap, n = 10; Fig. 3, We fi rst tested whether HAP40, Htt, or both interact compare C with D). We experimentally verifi ed that the HAP40 directly and specifi cally with Rab5. To this end, full-length fl uorescence signals (Fig. 3 D) were not caused by bleed- HAP40 was cloned from a rat brain cDNA library and in vitro through of the Htt signals (because of extended AlexaFluor568 translated. Because of its large molecular mass (348 kD; Fig. emission in the Cy5 channel) and that swapping these fl uores- 2 A), fragments of wild-type Htt were translated to facilitate cent dyes on the secondary antibodies resulted in similar distri- the analysis. In vitro translation yielded major products of pre- bution patterns of Htt and HAP40 (see Cell culture procedures). dicted size as well as lower molecular mass bands presumably The endosomal colocalization of endogenous HAP40 and as a result of internal initiation (Fig. 2 B). Immobilized Rab5- Htt was even more striking upon expression of the activated GST fusion protein preloaded with either GDP or GTPγS were EGFP-Rab5Q79L mutant (Htt: 52 ± 7% overlap, n = 8), incubated with the translation products and washed, and bound which caused the characteristic swelling of early endosomes proteins were eluted with glutathione analyzed by SDS-PAGE (Fig. 4 A; Stenmark et al., 1994). and autoradiography (Christoforidis et al., 1999). Similar to Given the dif culties detecting endogenous HAP40 and fi early endosome antigen 1 (EEA1), which served as a positive Htt on early endosomes in untreated cells (Fig. 3, B and C) we control, HAP40 displayed specifi c binding to GTP γS- versus sought to verify the changes in the localization of both proteins GDP-bound Rab5 (Fig. 2 C, compare lanes 1 and 11 with lanes upon the overexpression of HAP40 biochemically. To this end, 2 and 12). In contrast, none of the Htt fragments exhibited sig- we prepared early endosomes from HeLa cells for Western blot nifi cant binding to Rab5 (Fig. 2 C, lanes 3–10). Because Htt analysis. Indeed, we found that the levels of HAP40 and Htt in- was purifi ed on the GST-Rab5 af nity column, we ne fi xt tested creased on early endosomes from the HAP40 overexpressor whether binding of Htt to Rab5 occurs indirectly and requires compared with untreated cells (Fig. 3 E). Early endosome HAP40 as a bridge. HAP40 and Htt fragments were cotranslated (EEA1 and transferrin receptor [Tfr]) as well as lysosomal in vitro (Fig. 2 B, lanes 6–9) and applied on the Rab5 columns. (lysosome-associated membrane protein (LAMP); Eskelinen Indeed, the COOH-terminal part of Htt (Fig. 2 A, Htt4) was et al., 2003) and Golgi (GM130; Nakamura et al., 1995) markers 606 JCB • VOLUME 172 • NUMBER 4 • 2006 remained unchanged, confi rming equal loading and the speci- ficity of changes through elevated HAP40 on early endosomes. To confi rm the requirement of HAP40 for the recruitment of Htt onto early endosomes, we transfected HeLa cells with short interfering RNA (siRNA) duplexes against HAP40 and un- related siRNA (against GFP) as control. The HAP40 siRNA spe- cifi cally and ef ciently reduced the protein le fi vels by 90% (Fig. 4 C), whereas the level of EEA1 remained unchanged. When cells were cotransfected with the expression vector for EGFP- Rab5Q79L and HAP40 siRNA, Htt was no longer detectable on the enlarged endosomes (8 ± 6% overlap, n = 9; Fig. 4 B), con- fi rming HAP40 as a prerequisite to bridge Htt to active Rab5. Collectively, these data suggest that active Rab5 and HAP40 are rate limiting for the recruitment of Htt onto early endosomes. The Htt–HAP40 complex inhibits the binding and motility of early endosomes on microtubules Htt has previously been shown to bind microtubules and reg- ulate microtubule–motor interactions (Block-Galarza et al., 1997; Engelender et al., 1997; Hoffner et al., 2002; Gauthier et al., 2004). Because Rab5 regulates endosome motility along microtubules (Nielsen et al., 1999; Hoepfner et al., 2005), we explored the role of the Htt–HAP40 complex in this process. First, by using a cell- and cytosol-free assay that recapitulates the Rab5-dependent movement of early endosomes along mi- crotubules (Hoepfner et al., 2005), we found that Htt–HAP40 inhibited microtubule-dependent early endosome motility. Addition of the GTPγS- (containing Htt and HAP40; Fig. 1 B) but not the GDP-loaded Rab5 column eluate reduced the motility compared with control conditions (Fig. 5 A). This inhibition was Figure 2. Htt–HAP40 complex is a novel Rab5 effector. (A) Schematic 23 glutamine residues (23Q), the adjacent proline-rich sequence (P), the caspase-3 cleavage site, and the four recombinant fragments (Htt1–4) cloned in pcDNA3.1 using the indicated restriction sites. (B) Autoradio- graph of in vitro–translated Htt fragments (Htt1–4) and HAP40. Full-length HAP40 cDNA and Htt fragments were in vitro translated in the presence of [ S]methionine, separated by SDS-PAGE, and autoradiographed. Given their large size, the bands of strongest intensity (arrows) and correspond- ing to the predicted masses for HAP40 (40 kD, lane 1), Htt1 (65 kD, lane 2), Htt2 (97 kD, lane 3), Htt3 (84 kD, lane 4), and Htt4 (102 kD, lane 5) were accompanied by multiple products because of either initiation of translation at internal sites or premature termination. Lanes 6–9: each Htt fragment was cotranslated with HAP40. (C) Autoradiograph of the in vitro–translated proteins in B eluted from immobilized GST-Rab5 preloaded with GTPγS (T) or GDP (D). (D) Autoradiograph of Htt fragments cotrans- lated in vitro with HAP40 and eluted from various immobilized Rab pro- teins as indicated. The experiment was performed as described in C, but Htt fragments and HAP40 were cotranslated (see B, lanes 6–9) and ap- plied onto immobilized Rab proteins. Besides HAP40, the COOH-terminal Htt fragment (Htt4) was eluted from GST-Rab5 beads (compare lanes 7 with 8), whereas Htt1 (lanes 1 and 2), Htt2 (lanes 3 and 4), and Htt3 (lanes 5 and 6) did not show signifi cant specifi c association with Rab5. Moreover, none of the Htt fragments or HAP40 displayed specifi c interac- tions with GST-Rab4, 6, 7, or 11. Positive controls (POS, lanes 9 and 10) for each Rab protein were EEA1 (170 kD) for Rab5, Rabenosyn-5 (89 kD) for Rab4 (de Renzis et al., 2002), VPS39 (100 kD) for Rab7 (Rink et al., 2005), and GapCenA (115 kD) for Rab6 and 11 (Cuif et al., 1999 and unpublished data). (E) Htt and HAP40 elute in equimolar amounts from the Rab5 column. A mixture of Htt4 and HAP40 was applied onto Rab5 columns and eluted as in D but with a 10-fold excess of Htt4 to prevent HAP40 from binding freely to Rab5. (C–E) Arrows point to correct transla- tion products as in B. THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 607 Figure 3. Overexpressed HAP40 recruits Htt onto early endosomes. Immunofl uorescence microscopy analysis of HeLa cells expressing EGFP-Rab5 and/or HAP40 and immuno- stained for HAP40 and Htt as indicated. (A) The anti-HAP40 antibody specifi cally recognizes its antigen (green). Cells were stained with the antibody alone (anti-HAP40) or pre- mixed with HAP40-GST fusion (anti-HAP40 & GST-HAP40) or GST protein (anti-HAP40 & GST). The image after specifi c de- pletion of the antibody (anti-HAP40 & GST-HAP40) was ob- tained at sixfold prolonged exposure time compared with the others. (B) Endogenous Htt (red) and endogenous HAP40 (green); <1% overlapping (n = 10). (C) Endogenous Htt (red) and EGFP-Rab5 (green); 7% overlapping (SD ± 5%, n = 10). (D) Overexpressed HAP40 (red) recruits endogenous Htt (red) onto endosomes labeled with EGFP-Rab5WT (green); 43% overlapping of endogenous Htt with EGFP-Rab5 (merge Rab5 & Htt; SD ± 6%, n = 10) and 31% overlapping of overex- pressed HAP40 with EGFP-Rab5 (merge Rab5 & HAP40; SD ± 7%, n = 10). Cells were cotransfected with HAP40 and GFP-Rab5WT expression constructs. Arrowheads in insets (magnifi ed images of boxed areas) highlight colocalization. Bar, 10 μm. (E) Western blot analysis of early endosomes prepared from untreated (WT) or HeLa cells overexpressing HAP40 (HAP40). Blots were probed for HAP40, Htt, EEA1, transferrin receptor (Tfr), LAMP1, and GM130 as indicated. specifi cally rescued with anti-Htt but not unrelated antibodies. assay as described previously (Nielsen et al., 1999) with The addition of 1 μM GST-HAP40 fusion protein completely some modifi cations to improve the quantitative assessment. blocked the in vitro motility (Fig. 5, A and B). Second, we An early endosome–enriched fraction was prepared from performed a biochemical early endosome–microtubule- binding HeLa cells pulsed with rhodamine–transferrin, incubated with 608 JCB • VOLUME 172 • NUMBER 4 • 2006 Figure 4. Htt is recruited onto early endo- somes in a Rab5- and HAP40-dependent fashion. Immunofl uorescence microscopy anal- ysis of HeLa cells transfected with expression vectors for EGFP-Rab5Q79L alone or cotrans- fected with siRNA duplexes against HAP40 as indicated. (A) EGFP-Rab5Q79L (green) recruits endogenous Htt (red) onto endosomes; 52% overlapping (SD ± 7%, n = 8). Cells were transfected with EGFP-Rab5Q79L expression construct alone. (B) RNAi against HAP40 (siHAP40) leads to the loss of endogenous Htt (red) from endosomes; 8% overlapping (SD ± 6%, n = 9). Cells were cotransfected with HAP40 siRNA and EGFP-Rab5Q79L (green) expression constructs. Bar, 10 μm. (C) Knockdown of HAP40 protein by RNAi as shown by Western blot analysis. EEA1 and HAP40 expression levels in untransfected cells (control) and cells transfected with unrelated (siGFP) or siRNA against HAP40 (siHAP40). taxol- stabilized microtubules, ATP, and factors to be tested, compare lane 5 with lanes 4 and 2). To rule out the idea that the and centrifuged through a sucrose cushion. The resulting pellet observed differences result from the bundling of microtubules of microtubule-associated material was analyzed by immuno- causing unspecifi c cosedimentation of any membranous struc- blotting (Fig. 6 A) and fl uorimetrically for the rhodamine– ture, we probed the pellets for nonendosomal contaminants in transferrin content (Fig. 6 D). In a dilution series for calibration, the fraction. Both the lysosomal (LAMP1) and Golgi marker we verifi ed that the amount of endosomes and fl uorescence in- (GM130) pelleted with similar effi ciency in all samples. Collec- tensity correlated linearly (see Microtubule and actin spin-down tively, these data indicate that Htt–HAP40 specifi cally lowers assays). Western blotting revealed that the β-tubulin content was the binding of early endosomes to microtubules. similar between samples, ruling out secondary effects on micro- Having validated the assay, we next quantifi ed the amount tubule stability (Fig. 6 A, compare lanes 2–5 with lane 1). The of endosomes bound to microtubules fl uorimetrically. Omission addition of 1 μM GST-HAP40 protein decreased the amount of either microtubules or early endosomes reduced the fl uores- of early endosomes in the pellet as revealed by EEA1 and Tfr cence signal to background levels (Fig. 6 D). As reported previ- (Fig. 6 A, compare lane 3 with lane 2). The GST-Rab5–GTPγS ously (Nielsen et al., 1999), the association of early endosomes column eluate adding 0.3 μM HAP40 (Fig. 6 C) caused a with microtubules was energy dependent and required active similar inhibition (Fig. 6A, compare lane 4 with lane 2) that was Rab5. Omitting ATP or substituting it with the nonhydrolyzable rescued through the addition of antiserum against Htt (Fig. 6 A, adenylyl-imidodiphosphate analogue resulted in an 50% THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 609 reduction in microtubule binding. The requirement for ATP can be explained by the role of PI3-K in the assembly and mainte- nance of a functional Rab5 domain on endosomes (Zerial and McBride, 2001) and in recruitment of the endosomal kinesin KIF16B (Hoepfner et al., 2005). Extraction of Rab proteins from membranes by the addition of 1 μM of recombinant Rab–GDP dissociation inhibitor (GDI; Ullrich et al., 1994) or treatment with RN-tre, a GTPase-activating protein for Rab5 (Lanzetti et al., 2000), caused an 40% reduction of endosomes in the pellet (Fig. 6 D). As for endosome motility (Fig. 5 A), Htt and HAP40 inhibited endosome–microtubule binding. The GST-Rab5–GTPγS column eluate led to an 25% reduc- tion in binding (Fig. 6 D), but supplementing the reaction with antibodies against Htt restored binding to control levels. The addition of 1 μM HAP40-GST fusion protein decreased the binding by 60%, whereas GST alone did not have any effect. Collectively, these data underpin the ability of HAP40 and Htt to destabilize endosome–microtubule association. We next performed time-lapse video microscopy studies to correlate these biochemical fi ndings in vitro with the regula- tion of early endosome dynamics in vivo. We detected a dras- tic reduction in motility of EGFP-Rab5–positive endosomes in HeLa cells overexpressing HAP40 (Fig. 7 A) compared with cells transfected with EGFP-Rab5 alone (Fig. 7 A and Vid- eos 1 and 2, available at http://www.jcb.org/cgi/content/full/ jcb.200509091/DC1). Whereas some residual motility activ- ity was observed in the cell periphery, early endosomes in the perinuclear region appeared static, with frequent short-range movements almost completely impaired in long-range motility (Nielsen et al., 1999, Rink et al. 2005). Collectively, these data suggest that the Rab5-dependent recruitment of Htt onto endo- somes by HAP40 disrupts early endosome–microtubule inter- actions, thus leading to a reduction in organelle motility. HD cells display increased levels of HAP40 and are impaired in early endosome motility We next asked whether alterations of endosome motility could occur in cells bearing the HD mutation. Primary fi broblasts from fi ve healthy individuals and fi ve unrelated HD patients were transfected with EGFP-Rab5 to compare the motility of early endosomes. The identity of the HD cell lines was con- fi rmed by Western blotting to detect the polyQ-expanded Htt. Because the cells were derived from patients heterozygous for Figure 5. Htt and HAP40 decrease in vitro reconstituted motility of early the HD gene, they express both normal and mutant Htt. endosomes along microtubules. (A) Purifi ed early endosomes labeled by internalization of rhodamine–transferrin were mixed with buffer (control) The latter is known to display a lower mobility in SDS-PAGE alone or with various eluates of bovine brain cytosol proteins that were af- (Trottier et al., 1995), thus causing a doublet on the blot (Fig. 7 D). fi nity purifi ed on GST-Rab5 columns. Eluate obtained from columns loaded Strikingly, we observed a severe reduction in early endosome with GDP was added directly to the sample (+GDP-eluate). Eluate ob- tained from columns loaded with GTPγS was added directly to the sample motility in all HD cell lines compared with fi broblasts from (+GTP-eluate) or after preincubation with antiserum against the cytoplas- healthy individuals (Fig. 7 B and Videos 3 and 4, available mic domain of Tfr (+eluate + anti-Tfr), antiserum against Htt (+eluate at http://www.jcb.org/cgi/content/full/jcb.200509091/DC1). + anti-Htt), or without eluate but with 1 μM of recombinant GST-HAP40 fu- sion protein (+HAP40). In vitro motility of early endosomes along micro- The similarity between this phenotype and the alterations in- tubules recorded using time-lapse fl uorescence video microscopy (see duced upon HAP40 overexpression in HeLa cells (Fig. 7 A and Materials and methods) was quantifi ed by counting motility events per Videos 1 and 2) hinted to a common molecular basis. Interest- video. Error bars show the SD of 10 videos. (B) Videos recorded under control conditions (control) or with 1 μM GST-HAP40 fusion protein ingly, we discovered an 10-fold up-regulation of endogenous (HAP40) used for the analysis in A are displayed as merged stacks of over- HAP40 levels by Western blotting in all HD cell lines com- laid images collected at 2-s intervals over 2 min. When represented in this pared with normal fi broblasts (Fig. 7 D). As a control, the lev- manner, a moving object will generate a trajectory consisting of a linear series of overlapping spots. Bar, 10 μm. els of EEA1 remained unchanged. Moreover, we found that 610 JCB • VOLUME 172 • NUMBER 4 • 2006 HAP40 protein levels were signifi cantly elevated in striatal tissue (caudate, putamen, accumbens, and globus pallidus) from hu- man postmortem brains affected by HD (adult onset grade) compared with control brains (Fig. 8 B). Our data suggest that the motility block in HD cells may be caused by elevated HAP40 levels, as mimicked by overexpression of HAP40 in HeLa cells. Consistently, endogenous Htt localized to EGFP- Rab5–labeled endosomes in fi broblasts from human HD pa- tients as well as striatal STHdhQ cells from a HD mouse model (Trettel et al., 2000) but not in cells lacking the mutant Htt (Fig. 9). This phenotype is also caused by overexpressed HAP40 in HeLa cells (Fig. 3 D). Our data do not exclude the possibility that the observed inhibition of early endosome motility may have other under- lying causes. As a test to our hypothesis, we attempted to rescue the inhibition of early endosome motility by specifi cally ab- lating HAP40 from the HD fi broblasts by RNA interference (RNAi). Transfection of HAP40 siRNA effi ciently reduced the HAP40 protein levels in these cells (Fig. 7 E) as in HeLa cells (Fig. 4 C). Indeed, endosome motility was restored by RNAi against HAP40 (Fig. 7 C and Video 5, available at http://www. jcb.org/cgi/content/full/jcb.200509091/DC1) but not against GFP, suggesting that the up-regulation of HAP40 is indeed the underlying mechanism of the motility defect in HD fi broblasts. Given the ability of HAP40 to reduce endosome– microtubule binding in vitro (Fig. 6, A and D), we investigated whether the observed motility block in HD cell lines was caused by a release of endosomes from microtubules in vivo. Immunofl uorescence analysis on cells transfected with EGFP-Rab5 showed a consid- erable alignment of early endosomes to microtubules in healthy fi broblasts (82 ± 9% overlap, n = 10) but to a much lesser extent in HD cell lines (15 ± 6% overlap, n = 9; Fig. 7, compare F with G). In contrast, early endosomes were strikingly aligned with fi lamentous actin (F-actin) in all fi ve HD cell lines (44 ± 8% overlap, n = 10) but not in healthy fi broblasts (2 ± 1% overlap, n = 10; Fig.7, compare G with F). A similar in samples prepared for the early endosome– microtubule/actin-binding assays. Samples were prepared as described for A and B either with GST- HAP40 fusion (lane 1) or GST-Rab5–GTPγS column eluate (lane 2). 10-μl aliquots of each sample were separated by SDS-PAGE and immunoblotted for HAP40. Bands corresponded to the correct masses of GST-HAP40 or HAP40 as indicated on the right. Nonrelevant lanes on the same blot were sliced out in Adobe Photoshop to juxtapose the lanes shown. (D) Quantifi - cations of early endosomes bound to microtubules. Binding was performed as described for A, but the resulting pellet of microtubule- associated mate- rial was lysed to release the rhodamine–transferrin label of early endo- some. Fluorescence (arbitrary units) served as a direct measure for the Figure 6. Modulation of binding of early endosomes to microtubules and amount of early endosomes bound to microtubules. Error bars represent F-actin by Htt and HAP40. (A) Reduction of binding of early endosomes the SD of samples in triplicate. Binding was performed in the presence (EE) to microtubules in vitro caused by Htt and HAP40. A spin-down assay of 15 μg of early endosomes and 16 μg of microtubules (control), in was performed (see Materials and methods), and the resulting pellet of mi- the absence of microtubules (−MT), in the absence of early endosomes crotubule-associated material was analyzed by immunoblotting with anti- (−EE), in the absence of ATP (−ATP), with all three components omitted bodies against proteins indicated on the right. (B) Stimulation of binding of (buffer), with 2 mM adenylyl-imidodiphosphate (+AMP-PNP), with 1 μM early endosomes to F-actin in vitro caused by Htt and HAP40. A spin-down of recombinant RN-tre (+RN-tre), with 1 μM Rab-GDI (+GDI), with 1 μM assay was performed as described for A but with 10 μg of freshly polymer- HAP40-GST fusion protein (+HAP40), with 1 μM GST (+GST), with 10 μg ized F-actin replacing the microtubule. (A and B) Microtubules (A) or F-actin GST-Rab5–GTPγS column eluate (+eluate), or with eluate preincubated (B) spun alone (−EE, lane 1), with early endosomes (control; +EE, lane 2), with anti-Htt antiserum (+eluate+anti-Htt). (E) Quantifi cations of early en- with early endosomes and 1 μM GST-HAP40 fusion protein (+EE+HAP40, dosomes bound to F-actin. Binding was performed as described for B, and lane 3), with early endosomes and 10 μg GST-Rab5–GTPγS column elu- quantifi cations were made as in D. Binding was performed in the presence ate (+EE+eluate, lane 4), or eluate preincubated with anti-Htt antiserum of 15 μg of early endosomes, 10 μg of F-actin (control), and with the (+EE+eluate+anti-Htt, lane 5). (C) Comparison of HAP40 protein levels addition of various components as in D. THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 611 Figure 7. Elevated protein levels of HAP40 shift early endosomes from microtubules to actin fi laments, causing a severe decrease of motility in vivo. HeLa cells and human primary fi broblasts expressing EGFP-Rab5 were imaged using time-lapse video microscopy. (A–C) Images generated by merging a stack of overlaid images collected at 300-ms intervals over 2 min, as for Fig. 5 B. Videos corresponding to A–C are available as online supplemental materials (Videos 1–5, available at http://www.jcb.org/cgi/content/full/jcb.200509091/DC1). (A) In HeLa cells, coexpression of EGFP-Rab5 and HAP40 (right) led to a drastic reduction in endosome motility compared with EGFP-Rab5 alone (WT, left). (B) In fi broblasts from HD patients (HD), such reduction was also evident from the comparison with fi broblasts from healthy individuals (WT). (C) RNAi against HAP40 in these HD fi broblasts (HAP40 RNAi) restores endo- some motility. HD fi broblast cell lines were cotransfected with EGFP-Rab5 expression vector and siRNA against HAP40. (D) Similar to HeLa cells overex- pressing HAP40 (compare lane 1 with lane 2), endogenous protein levels of HAP40 were elevated in fi ve HD fi broblast cell lines (lanes 8–12) compared with fi broblasts from fi ve healthy individuals (lanes 3–7). Blots were also probed with anti-EEA1 and anti-Htt antiserum to confi rm equal loading and the identity of the HD cell lines (see Results). (E) Knockdown of HAP40 protein levels in fi broblasts from all fi ve HD cell lines by RNAi. Western blot analysis of EEA1 and HAP40 in untransfected cells (lanes 1, 4, 7, 10, and 13), cells transfected with unrelated siRNA against GFP (lanes 2, 5, 8, 11, and 14), or HAP40 siRNA (lanes 3, 6, 9, 12, and 15). (F) EGFP-Rab5–labeled endosomes align primarily with microtubules in healthy fi broblasts. Primary human fi broblasts from healthy individuals were transfected with EGFP-Rab5 (green) and fi xed and immunostained for microtubules and F-actin. The same cell is shown with its β-tubulin staining (red) on the left and for F-actin (red) on the right, as indicated. Arrowheads in the insets (magnifi cations of the boxed areas) point to endosomes aligned with microtubules. Overlap of EGFP-Rab5 with tubulin signals was 82% (SD ± 9%, n = 10) for healthy fi broblasts and 15% (SD ± 6%, n = 9) for HD cell lines (see G). (G) The cell processed as in F shows that EGFP-Rab5–labeled endosomes align strikingly with F-actin in fi bro- blasts from HD patients. Arrowheads in the insets point to endosomes (green) aligned with F-actin (red). Overlap of EGFP-Rab5 with actin was 2% (SD ± 1%, n = 10) for healthy fi broblasts (see F) and 44% (SD ± 8%, n = 10) for HD cell lines. Bar, 10 μm. 612 JCB • VOLUME 172 • NUMBER 4 • 2006 phenotype was obtained by the overexpression of HAP40 in apparently occur in peripheral tissues such as fi broblasts as well HeLa cells (not depicted). as in neuronal systems and, therefore, are of potential relevance for HD. The Htt–HAP40 complex enhances the binding of early endosomes to actin Increased HAP40 levels cause alterations The alignment of early endosomes on F-actin in HD cell lines in endocytic traffi cking could be a secondary effect from the inhibition of endosome– To gain some insights into possible alterations of endocytic microtubule interactions. To directly test the role of HAP40 in transport caused by increased levels of HAP40, we tested the the association between early endosomes and actin fi laments uptake of transferrin in HeLa cells overexpressing HAP40 and (Fig. 6, B and E), we modifi ed the biochemical sedimentation in fi broblasts from healthy and HD patients. Fig. 10 A shows assay used to study endosome–microtubule interactions (Fig. 6, that the uptake of transferrin was reduced by 30% in HeLa cells A and D) by replacing taxol-stabilized microtubules with freshly overexpressing HAP40 compared with mock-transfected or in vitro–polymerized F-actin. Unlike for endosome–microtubule control cells. Consistent with this result, fi broblasts from HD binding, depletion of active Rab5 from endosomal membranes patients displaying higher levels of endogenous HAP40 (Fig. 7 D) by treatment with either Rab-GDI or RN-tre did not decrease displayed a similarly reduced uptake of transferrin compared endosome–actin interactions (Fig. 6 E). Thus, a basal level of with normal fi broblasts (Fig. 10 B). As for the block of endo- endosome–F-actin binding activity was independent of Rab5. some motility, the inhibitory effect on transferrin uptake was However, the addition of HAP40-GST fusion protein stimulated rescued by the depletion of HAP40 by RNAi. These data sug- binding (260%) over control levels, whereas GST alone was gest that the inhibitory effects on endosome motility caused by ineffective. Evidently, endosome binding to microtubules and up-regulation of HAP40 also result in defects in cargo transport F-actin is reciprocally regulated through HAP40 because con- through the endocytic pathway. centrations (1 μM) inhibiting binding to microtubules (Fig. 6 D) clearly stimulated binding to F-actin (Fig. 6 E). This effect Discussion was Rab5 dependent because when Rab-GDI or RN-tre were added together with HAP40, the stimulation was nearly abol- The key fi nding of this study is that the complex between ished (Fig. 6 E). Again, immunoblotting confi rmed that the HAP40 and Htt is a direct downstream effector of Rab5 that amount of actin in the pellets was unaffected by any added pro- regulates the dynamics of early endosomes through a switch tein and that all changes in pelletable fl uorescence corresponded from microtubules to F-actin. These fi ndings provide important consistently to altered band intensities of early endosomal new insights into how the motility of early endosomes is regu- markers (EEA1 and TFr) but not to others (LAMP1 and GM130), lated under physiological and pathological conditions. Htt has indicating specifi c effects of Htt–HAP40 on endosome–actin been implicated in clathrin-mediated endocytosis, regulation of binding (Fig. 6 B). the actin cytoskeleton, and microtubule-dependent transport along the endocytic pathway via interactions with its numerous Alterations in early endosome motility binding partners (Harjes and Wanker, 2003). Such multiplicity in a HD mouse model of striatal cells of roles implies that the activities of Htt in endocytic membrane Next, we investigated the role of HAP40 in Rab5 dynamics in an traffi cking need to be spatially and temporally coordinated. experimental system that is more relevant for HD using immor- In showing for the fi rst time that a complex between Htt and one talized STHdhQ striatal cells (Trettel et al., 2000). These cells of its binding partners, HAP40, can be recruited onto endo- were established from embryonic normal or HD knock-in mice somes by interacting directly with Rab5, our data provide novel 7/7 and either express normal (STHdhQ ) or mutant Htt as a result insights into the mechanisms governing the targeting of Htt to of a CAG expansion inserted into the endogenous Htt gene (het- early endosomes and its regulatory activity on cytoskeleton- 7/111 111/111 erozygous STHdhQ and homozygous STHdhQ ). Thus, dependent dynamics. they refl ect the closest situation to HD patients as normal, and Under normal physiological conditions, early endosomes mutant Htt are expressed at endogenous levels. Remarkably, undergo frequent short-range movements on actin but also we again found endogenous HAP40 protein levels elevated in long-range bidirectional movements along microtubules 7/111 111/111 7/7 STHdhQ and STHdhQ compared with STHdhQ cells (Nielsen et al., 1999; Gasman et al., 2003; Rink et al. 2005). (Fig. 8 B), which is consistent with the data on HD fi broblasts Plus end movement of early endosomes along microtubules is (Fig. 7 D) and brain tissue from HD patients (Fig. 8 B). Live cell propelled by KIF16B (Hoepfner et al. 2005). Overexpression imaging revealed EGFP-Rab5–positive organelles moving bidi- of HAP40, which is rate limiting for the recruitment of Htt rectionally in neuronlike outgrowths as well as in the cell body on the membrane, caused the detachment of early endosomes 7/7 of normal STHdhQ cells (Fig. 8 A and Video 6, available at from microtubules and their preferential association with actin http://www.jcb.org/cgi/content/full/jcb.200509091/DC1). Con- fi laments, thus limiting both their velocity and range of 111/111 7/111 versely, STHdhQ and STHdhQ cells clearly displayed movements. Therefore, the Htt–HAP40–Rab5 complex is a key a drastic reduction of endosome dynamics (Fig. 8 A and Videos regulator of the switch from one type of fi laments to another. 7 and 8), which is consistent with the observations on HD Our data are consistent with previous studies documenting fi broblasts (Fig. 7 B and Videos 3 and 4). Collectively, defects alterations in microtubule-dependent motility in HD model of Rab5 dynamics caused by pathogenic excess of HAP40 systems (Block-Galarza et al., 1997; Engelender et al., 1997; THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 613 Figure 8. Elevated endogenous HAP40 pro- tein levels impair Rab5 dynamics in vivo in 111/111 7/111 STHdhQ and STHdhQ striatal cells from a HD mouse model. (A) Cells expressing 7/7 111/111 normal (STHdhQ ) or mutant (STHdhQ 7/111 and STHdhQ ) Htt were transfected with EGFP-Rab5 plasmid, differentiated, and im- aged using time-lapse video microscopy. Over- laid images were generated as described for Fig. 5 B. Videos corresponding to A are avail- able as online supplemental material (Videos 6–8, available at http://www.jcb.org/cgi/ content/full/jcb.200509091/DC1). Motility of Rab5 compartments was drastically reduced in 111/111 7/111 STHdhQ and STHdhQ compared 7/7 with STHdhQ cells. Insets show magnifi ca- tions of boxed areas. Bar, 10 μm. (B) Endog- enous protein levels of HAP40 are elevated in 111/111 7/111 STHdhQ (lane 1) and STHdhQ (lane 2) 7/7 compared with STHdhQ cells (lane 3) as well as in striatal tissue from fi ve human postmortem brains affected by HD (lanes 4–8) compared with healthy control brains (lanes 9–13). Blots were also probed with anti-EEA1 and anti-Htt antiserum to confi rm equal load- ing and the expression of wild-type and mutant Htt as described for Fig. 7 D. Gauthier et al., 2004). However, these studies have exclusively nucleus (Landles and Bates, 2004). The majority of HAP40 is implicated alterations between mutant Htt and HIP/HAP also nuclear under normal conditions (Fig. 3; Peters and Ross, effectors. For example, wild-type Htt has been shown to interact 2001), although the functional signifi cance of this localization Glued with the dynactin subunit p150 via HAP1 and mutant Htt to is unclear at present. Our data indicate that HAP40 fulfi lls a disrupt the dynein motor complex in axonal transport (Gauthier function in the cytoplasm. It is interesting in this respect to note et al., 2004). In this study, we have uncovered a different mech- that HAP40 adds to the increasing list of proteins implicated anism based on the up-regulation of an Htt adaptor, HAP40. in a dual role in endocytic traffi cking and nuclear signaling Compelling evidence in support of this mechanism was pro- (Miaczynska et al., 2004). The molecular mechanism underly- vided by the rescue of the motility block upon depletion of ing the up-regulation of HAP40 unexpectedly observed in cells HAP40 by RNAi both in HD fi broblasts and in striatal cells. and brain tissue from HD patients remains to be determined. Because the COOH-terminal part of Htt is responsible for the The most likely explanation is that it arises as a consequence of underlying interactions with HAP40 and Rab5, this endosomal alterations in gene expression caused by mutated Htt in the recruitment affects normal as well as mutant Htt. Consistently, nucleus. Messenger RNA microarray studies have revealed wild-type (overexpression of HAP40 in HeLa cells), heterozy- many transcriptional abnormalities in HD (Chan et al., 2002; 7/111 111/111 gous (STHdhQ ), and homozygous (STHdhQ ) HD cells Sipione et al., 2002; Sugars and Rubinsztein, 2003), although display very similar phenotypes. In this way, functional compe- no changes for HAP40 or any other Htt-interacting partners tition with the Rab5-dependent endosomal kinesin KIF16B and have been reported so far. Up-regulation of HAP40 at the pro- disruption of the dynein–dynactin complex (Gauthier et al., 2004) tein level might thus serve as a new diagnostic indicator for the could account for the compromised bidirectional microtubule- disease. However, it is premature to judge to what extent this based motility. Therefore, our data suggest that multiple defects abnormality and the consequent alterations in membrane dy- may contribute to the block of vesicular transport in HD cells, namics contribute to the pathogenetic mechanism of HD as which implicates various HIP/HAPs interacting with different compared with nuclear activities of Htt and HAP40. Although regions of Htt. endosome motility is not perturbed in HD cells to the extent of Previous fi ndings have shown that Htt is also linked to compromising cell viability, we nevertheless uncovered defects Rab8 through FIP-2 (Hattula and Peranen, 2000) and that this in endocytic transport, specifi cally a decrease in transferrin interaction regulates cell polarization and morphogenesis. uptake. These observations may be of particular functional rel- Whereas the mechanism underlying the latter process is unclear, evance in neurons given the importance of long-range transport in light of our observation, we propose that Htt is a multifunc- (Block-Galarza et al., 1997; Engelender et al., 1997; Gauthier tional protein that may be recruited by different Rab GTPases et al., 2004). Therefore, it is possible that alterations in endo- through different adaptors at different intracellular locations to some motility and transport may further compromise the patho- regulate organelle dynamics. logical state induced by mutated Htt on the survival and function The primary pathological cause of HD is attributed to the of neurons or some selected populations of neurons where en- abnormal activity of Htt and its interacting partners in the dosomal transport is particularly rate limiting in vivo. Addressing 614 JCB • VOLUME 172 • NUMBER 4 • 2006 7/111 Figure 9. Htt and HAP40 are recruited onto Rab5 vesicles in primary fi broblasts from human HD patients and STHdhQ striatal cells from a HD mouse model. (A) Image gallery of the same fi broblast cell from a healthy individual transfected with EGFP-Rab5 (green) and fi xed and immunostained for endog- enous HAP40 (red) and Htt (red). Boxed areas are magnifi ed in the top panels. (B) The same analysis of a fi broblast from a HD patient. Yellow (arrowheads) indicates colocalization of EGFP-Rab5 and Htt (merge Rab5 and Htt,) or of EGFP-Rab5 and HAP40 (merge Rab5 and HAP40). Overlapping of EGFP-Rab5 with Htt was 6% (SD ± 3%, n = 10) for fi broblasts from healthy individuals (A) and 46% (SD ± 8%, n = 10) from HD patients (B). Overlapping of EGFP- Rab5 with HAP40 was 4% (SD ± 3%, n = 10) for fi broblasts from healthy individuals (A) and 51% (SD ± 9%, n = 10) from HD patients (B). (C) Image gal- 7/7 lery of the same differentiated STHdhQ cell (homozygous for normal Htt) transfected with EGFP-Rab5 (green) and immunostained for endogenous HAP40 7/111 (red) and Htt (red). (D) The same analysis of a differentiated STHdhQ cell (heterozygous for mutant Htt). Overlapping of EGFP-Rab5 with Htt was 5% 7/7 7/111 (SD ± 4%, n = 10) for STHdhQ cells (C) and 83% (SD ± 12%, n = 10) for STHdhQ cells (D). Overlapping of EGFP-Rab5 with HAP40 was 4% 7/7 7/111 (SD ± 3%, n = 10) for STHdhQ cells (C) and 86% (SD ± 15%, n = 10) for STHdhQ cells (D). The analysis was restricted to cellular outgrowths. 111/111 Essentially the same results were obtained for STHdhQ cells (homozygous for mutant Htt; not depicted). Bar, 10 μm. this hypothesis requires a more thorough investigation of Materials and methods HAP40 and Htt function in the transport of different types of Rab5 affi nity chromatography and HAP40 and Htt cloning cargo along the endocytic/recycling as well as degradative GST-Rab5 affi nity chromatography with bovine brain cytosol or in vitro– pathway in neuronal cells rather than fi broblasts. A deeper anal- translated proteins was performed as described previously (Christoforidis ysis of the role of HAP40 on the molecular level will hopefully et al., 1999). HAP40 was cloned from rat brain cDNA by PCR, Htt frag- ments were cloned by PCR on full-length Htt cDNA in pBlueScript (a gift improve our understanding of both HD pathogenesis and the from M. Sherman, Boston University School of Medicine, Boston, MA), mechanisms coordinating organelle–cytoskeleton interactions and all PCR products were cloned into pcDNA3.1 (Invitrogen) and se- during membrane traffi cking. quenced to confi rm their identity. To estimate the stoichiometry of the THE HTT–HAP40 COMPLEX IS A NOVEL RAB5 EFFECTOR • PAL ET AL. 615 Cell culture procedures HeLa and human primary fi broblast cells were grown according to stan- dard procedures. Immortalized STHdhQ striatal cell lines from control and HD knock-in mice were cultured and differentiated as described previously (Trettel et al., 2000). For transient expression studies, cells were transfected using LipofectAMINE 2000 (Invitrogen) and used 12 h after transfection for immunoblot analysis or intracellular localization studies according to standard protocols. Fixed cells were analyzed using a 100×/NA 1.40 plan-Apochromat oil immersion lens (Carl Zeiss MicroImaging, Inc.) on a microscope (Axiovert S100TV; Carl Zeiss MicroImaging, Inc.). Illumination was performed with a 100-W mercury lamp with fi lter sets for GFP, Alexa- Fluor568, DAPI, and Cy5 fl uorescence. Images were acquired at 1,300 × 1,000 pixels with a digital camera (SP 1.4.4; Diagnostic Instruments) con- trolled by the MetaVue 6.1 software package (Universal Imaging Corp.). Raw images from various color channels were assembled and colorized in Adobe Photoshop 7.0. Brightness and contrast were adjusted for visual clarity. To validate immunofl uorescence microscopy studies using Alexa- Fluor568- and Cy5-conjugated secondary antibodies, HAP40 was overex- pressed in HeLa cells and cells stained with a mixture of anti-HAP40 and -Htt antisera. Single addition of secondary antibody against mouse IgG (for Htt) conjugated with AlexaFluor568 resulted in no detectable bleed- through into the Cy5 channel at the exposure time and fi lter settings used for HAP40 detection with Cy5. Moreover, signals for HAP40 that were ob- tained with Cy5 looked very similar in the presence or absence of Alexa- Fluor568. Finally, when these dyes were swapped on the secondary antibodies, very similar distribution patterns for HAP40 and Htt were obtained. Collectively, the colocalization between HAP40 and Htt revealed in Figs. 3 and 9 is not caused by bleed-through. The percentage of colocal- ization between immunofl uorescence signals was determined as follows: raw signals from discrete membrane structures were manually counted and inspected for overlapping with signals in other channels to calculate the percentage of colocalization. Only signals above value 70 on the eight-bit tonal scale were considered specifi c and used for the analysis. Typically, Figure 10. Elevated levels of HAP40 reduces transferrin uptake. (A) HeLa 10 cells (n = 10) were counted to calculate the mean colocalization cells transfected with HAP40 expression (HAP40), empty plasmid (mock), and SD. For overlapping studies of EGFP-labeled early endosomes with cy- or untreated (WT) were serum starved, allowed to internalize biotinylated toskeletal fi laments, these organelles were considered colocalized to either transferrin (Tf) for the indicated times, washed, and lysed, and internalized microtubules or F-actin if at least three early endosomes in a row were transferrin was quantifi ed. The mean values of triplicate samples from one clearly aligned to a fi lament. In areas where microtubules and F-actin con- representative experiment out of three with SD (error bars, often omitted by verged (e.g., close to the cell edge), early endosomes could often not be plot symbols) are shown. (B) The same uptake for primary human fi bro- assigned to either type of fi lament. The sum of early endosomes aligned to blasts untreated or transfected with siRNA duplexes against HAP40 microtubules, F-actin, and unassignable early endosomes (100%) was (siHAP40) or unrelated GFP (siGFP). The mean values of different cell lines used to calculate percentages of colocalization. Internalized biotinylated from fi ve healthy persons (5× WT), fi ve HD patients (5× HD), the same but transferrin was detected with ruthenium-labeled antitransferrin antibodies treated with siHAP40 (5× HD siHAP40), or the mean values of triplicate and subsequently quantifi ed by ECL analysis as described previously sample obtained from one HD line treated with unrelated siGFP (1× HD (Hoepfner et al. 2005). siGFP) are shown. Error bars (mostly omitted by plot symbols) represent the SD between cell lines (5× WT, 5× HD, and 5× HD siHAP40) or of tripli- cate samples (1× HD siGFP). Cell lines and tissue samples The primary human fi broblast cell lines GM00023, GM00024, GM00037, GM00038, and GM00041 (Apparently Healthy cell collection) and GM04281, GM04723, GM03621, GM04869, and GM04847 (Hun- bound complex, GST-Rab5 was incubated in the presence of a 10-fold ex- tington Disease cell collection) were obtained from Coriell Cell Repositories. cess of the COOH-terminal fragment of Htt over HAP40. Band intensities Cell lines were established from both genders from healthy, unrelated obtained under these conditions were quantifi ed in ImageJ software individuals by the age of 3–31 yr or from clinically affected onset HD (National Institutes of Health; Htt4/HAP40 = 6:1) and corrected for the patients by the age of 19–32 yr. Immortalized striatal cell lines established number of [ S]methionine residues incorporated (Htt4/HAP40 = 22:4). from mouse embryos (STHdhQ) were gifts from M. MacDonald (Richard B. The resulting ratio was 1:1. Assuming a 1:1 ratio of Rab protein to Simches Research Center, Boston, MA; Trettel et al., 2000). Human effector, the stoichiometry of the Rab5/HAP40/Htt interaction was esti- postmortem brain samples (CAP; Globus Pallidus) from healthy and HD mated at 1:1:1. grades 3 and 4 donors were provided by the Harvard Brain Tissue Resource Center. Antibodies and recombinant proteins Monoclonal mouse antibodies against Htt were obtained from Chemicon Protein identifi cation by mass spectrometry and Y. Trottier (Institut de Génétique et de Biologie Moléculaire et Cellu- Gel-separated Coomassie-stained proteins were excised from the gel slab laire, Université Louis Pasteur, Illkirch, France), and polyclonal rabbit anti- and in-gel digested with trypsin as described previously (Shevchenko et al., body against HAP40 was obtained from Chemicon. Microtubules were 1996). Tryptic peptides were sequenced by nanoelectrospray tandem revealed with mouse monoclonal anti–β-tubulin antibody (BD Biosciences), mass spectrometry on hybrid quadrupole time-of-fl ight mass spectrometers and F-actin was revealed with AlexaFluor568-conjugated phalloidin (Invit- (Q-TOF I; Micromass, Ltd. and QSTAR Pulsar I; MDS Sciex) as described rogen). Secondary anti–mouse and rabbit IgG for immunofl uorescence mi- previously (Shevchenko et al., 1997). Database searching was performed croscopy were conjugated to AlexaFluor568 and Cy5 (Invitrogen). Rabbit by Mascot software (Matrix Science, Ltd.). polyclonal anti-EEA1 antibody was described previously (Christoforidis et al. 1999), mouse monoclonal antibody against the cytoplasmic tail of Tfr was purchased from Invitrogen, mouse monoclonal anti-LAMP1 antibody In vitro motility of early endosomes was obtained from Becton Dickinson, and mouse monoclonal anti-GM130 The in vitro motility assay was essentially performed as described previ- antibody was obtained from Abcam. Rab-GDI and RN-tre were prepared ously (Nielsen et al., 1999) with the following modifi cations (Hoepfner as described previously (Ullrich et al., 1994; Lanzetti et al., 2000). GST et al., 2005). KHMG (110 mM KCl, 50 mM Hepes-KOH, pH 7.4, 2 mM and HAP40-GST fusion proteins were affi nity purifi ed from Escherichia coli MgCl2, and 10% glycerol) was used as an assay buffer. For preparation lysates according to standard procedures. of the antifade solution, BRB80 buffer was substituted with KHMG, and the 616 JCB • VOLUME 172 • NUMBER 4 • 2006 solution was further supplemented with 10% serum. The energy mix con- Center is acknowledged for providing human postmortem brain samples from sisted of 75 mM creatine phosphate, 10 mM ATP, 10 mM GTP, and 20 mM healthy and HD grades 3 and 4 donors. MgCl2 in BRB80. For the assay, taxol-stabilized microtubules were per- This work was supported by the Max Planck Society and grants from fused in a microscopy chamber and allowed to bind to the coverslip. Next, the Human Frontier Science Program (RG-0260/1999-M) and the European 10 μl of 10% nonspecifi c rabbit serum in antifade solution was perfused in Union (HPRN-CT-2000-00081). A. Pal, F. Severin, and B. Lommer are long- the chamber followed by 10 μl of the assay mixture (2 μl of 5 mg/ml fl uo- term Max Planck fellows. rescently labeled early endosome, 1 μl of energy mix, 6 μl of the antifade solution, and 1 μl of saturated hemoglobin solution in KHMG) and was in- Submitted: 15 September 2005 cubated for 5 min at RT. At least three videos per sample were recorded Accepted: 10 January 2006 (60 frames with 2-s intervals) and analyzed as described previously (Nielsen et al., 1999). References In vivo motility of early endosomes Cells were grown on glass coverslips, transfected with EGFP-Rab5 alone Block-Galarza, J., K.O. Chase, E. Sapp, K.T. Vaughn, R.B. Vallee, M. DiFiglia, or together with siRNA against HAP40, and transferred to custom-built and N. Aronin. 1997. Fast transport and retrograde movement of huntingtin aluminum microscope slide chambers (Nielsen et al., 1999) just before and HAP 1 in axons. Neuroreport. 8:2247–2251. observation. 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Cell. 121:437–450. as described for microtubules. Hoffner, G., P. Kahlem, and P.J. Djian. 2002. Perinuclear localization of hun- tingtin as a consequence of its binding to microtubules through an inter- Online supplemental material action with beta-tubulin: relevance to Huntington’s disease. J. Cell Sci. 115:941–948. All videos show EGFP-Rab5 dynamics, and images were acquired as described in In vivo motility of early endosomes. Video 1 shows a wild- Landles, C., and G.P. Bates. 2004. Huntingtin and the molecular pathogenesis type HeLa cell transfected with plasmid for EGFP-Rab5 expression only. of Huntington’s disease. EMBO Rep. 5:958–963. Video 2 shows a HeLa cell cotransfected with plasmids for EGFP-Rab5 and Lanzetti, L., V. Rybin, M.G. Malabarba, S. Christoforidis, G. Scita, M. Zerial, HAP40. Video 3 shows a primary human fi broblast from a healthy person and P.P. Di Fiore. 2000. The Eps8 protein coordinates EGF receptor sig- transfected with plasmid for EFGP-Rab5 expression only, whereas Video 4 nalling through Rac and traffi cking through Rab5. Nature. 408:374–377. shows the fi broblast from a HD patient. Video 5 shows a primary human Miaczynska, M., L. Pelkmans, and M. Zerial. 2004. Not just a sink: endosomes fi broblast from a HD patient cotransfected with plasmid for EGFP-Rab5 in control of signal transduction. Curr. Opin. Cell Biol. 16:400–406. transfection and siRNA duplexes against HAP40. Video 6 shows a striatal Nakamura, N., C. Rabouille, R. Watson, T. Nilsson, N. Hui, P. Slusarewicz, 7/7 STHdhQ cell transfected with plasmid for EGFP-Rab5 expression only, T.E. Kreis, and G. Warren. 1995. Characterization of a cis-Golgi matrix 7/111 Video 7 shows the same for an STHdhQ cell, and Video 8 shows this protein, GM130. J. Cell Biol. 131:1715–1726. 111/111 for an STHdhQ cell. Online supplemental material is available at Nielsen, E., F. Severin, J.M. 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Journal

The Journal of Cell BiologyPubmed Central

Published: Feb 13, 2006

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