Abstract

Article10 July 2008free access Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons Emilie Colin Emilie Colin Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Diana Zala Diana Zala Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Géraldine Liot Géraldine Liot Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Hélène Rangone Hélène Rangone Institut Curie, Orsay, France CNRS UMR146, Orsay, FrancePresent address: Department of Genetics, University of Cambridge, Cambridge CB23EH, UK Search for more papers by this author Maria Borrell-Pagès Maria Borrell-Pagès Institut Curie, Orsay, France CNRS UMR146, Orsay, FrancePresent address: Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain Search for more papers by this author Xiao-Jiang Li Xiao-Jiang Li Department of Human Genetics, Emory University, Atlanta, GA, USA Search for more papers by this author Frédéric Saudou Corresponding Author Frédéric Saudou Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Sandrine Humbert Corresponding Author Sandrine Humbert Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Emilie Colin Emilie Colin Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Diana Zala Diana Zala Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Géraldine Liot Géraldine Liot Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Hélène Rangone Hélène Rangone Institut Curie, Orsay, France CNRS UMR146, Orsay, FrancePresent address: Department of Genetics, University of Cambridge, Cambridge CB23EH, UK Search for more papers by this author Maria Borrell-Pagès Maria Borrell-Pagès Institut Curie, Orsay, France CNRS UMR146, Orsay, FrancePresent address: Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu I Sant Pau, Barcelona, Spain Search for more papers by this author Xiao-Jiang Li Xiao-Jiang Li Department of Human Genetics, Emory University, Atlanta, GA, USA Search for more papers by this author Frédéric Saudou Corresponding Author Frédéric Saudou Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Sandrine Humbert Corresponding Author Sandrine Humbert Institut Curie, Orsay, France CNRS UMR146, Orsay, France Search for more papers by this author Author Information Emilie Colin1,2,‡, Diana Zala1,2,‡, Géraldine Liot1,2, Hélène Rangone1,2, Maria Borrell-Pagès1,2, Xiao-Jiang Li3, Frédéric Saudou 1,2 and Sandrine Humbert 1,2 1Institut Curie, Orsay, France 2CNRS UMR146, Orsay, France 3Department of Human Genetics, Emory University, Atlanta, GA, USA ‡These authors contributed equally to this work *Corresponding authors: CNRS UMR146, Institut Curie, bat.110, centre universitaire, Orsay F-91400, France. Tel.: +33 169 863 024; Fax: +33 169 863 051; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2008)27:2124-2134https://doi.org/10.1038/emboj.2008.133 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The transport of vesicles in neurons is a highly regulated process, with vesicles moving either anterogradely or retrogradely depending on the nature of the molecular motors, kinesins and dynein, respectively, which propel vesicles along microtubules (MTs). However, the mechanisms that determine the directionality of transport remain unclear. Huntingtin, the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport. Huntingtin is phosphorylated at serine 421 by the kinase Akt but the role of this modification is unknown. Here, we demonstrate that phosphorylation of wild-type huntingtin at S421 is crucial to control the direction of vesicles in neurons. When phosphorylated, huntingtin recruits kinesin-1 to the dynactin complex on vesicles and MTs. Using brain-derived neurotrophic factor as a marker of vesicular transport, we demonstrate that huntingtin phosphorylation promotes anterograde transport. Conversely, when huntingtin is not phosphorylated, kinesin-1 detaches and vesicles are more likely to undergo retrograde transport. This also applies to other vesicles suggesting an essential role for huntingtin in the control of vesicular directionality in neurons. Introduction The proper intracellular transport of membranous organelles and of other cargoes is determined by the nature of the molecular motors that propel vesicles along microtubules (MTs). Kinesins generally move vesicles anterogradely from the cell body to the tip of the neurites, whereas cytoplasmic dynein leads to the retrograde transport of vesicles back towards the cell body. In vitro studies have demonstrated that dynein and kinesins can function independently and that the direction of transport of a given cargo along MTs depends on the nature of the molecular motor that is present (Vale, 2003). However, the situation is more complex in vivo. A high level of coordination is necessary to ensure the proper transport of a given cargo to the right place. One key multiprotein complex that could have an important function in the coordination of bidirectional transport of vesicles is dynactin, which is required for attaching motors to cargoes (Holleran et al, 1998; Martin et al, 1999; Deacon et al, 2003; Schroer, 2004; Welte, 2004). In particular, the p150Glued subunit of dynactin physically interacts with MTs, the intermediate chains of dynein (DIC) and kinesin. However, the mechanisms by which the net directionality of organelle movement is achieved remain unclear and the proteins or factors that control directly or indirectly directionality remain to be identified. The huntingtin (htt) protein is a positive regulatory factor for vesicular transport. htt associates with vesicles and MTs (Borrell-Pages et al, 2006) and facilitates vesicular transport in both anterograde and retrograde directions in vitro, in mammalian neuronal cells and in Drosophila (Gunawardena et al, 2003; Gauthier et al, 2004; Caviston et al, 2007). This mechanism involves the direct interaction of htt with dynein intermediate chain (Caviston et al, 2007) and with huntingtin-associated protein-1 (HAP1), which in turn interacts with the p150Glued subunit of dynactin and kinesin (Engelender et al, 1997; Li et al, 1998; Gauthier et al, 2004; McGuire et al, 2006). We have previously described an assay to study intracellular transport by analysing the dynamics of brain-derived neurotrophic factor (BDNF)–eGFP-containing vesicles using fast 3D videomicroscopy followed by deconvolution (Gauthier et al, 2004; Dompierre et al, 2007). BDNF is synthesized from a large precursor protein, pre-pro-BDNF, that is proteolytically processed and trafficked through the Golgi apparatus to the trans-Golgi network where it is packaged into secretory vesicles (Thomas and Davies, 2005). BDNF is important to study htt's function, and its dysfunction in disease as the striatal source of BDNF that is crucial for the survival of striatal neurons is dependent on the proper synthesis and transport of BDNF from the cortico-striatal-projecting neurons (Altar et al, 1997; Saudou et al, 1998; Zuccato et al, 2001; Baquet et al, 2004; Gauthier et al, 2004). Most of the aforementioned studies have been conducted in cells rather than in primary cultures of neurons and the role of htt in the control of anterograde and retrograde transport has not been investigated. Therefore, to further characterize the function of htt in axonal transport, we studied the regulatory role of htt in transport in primary cultures of cortical neurons, the neurons that transport BDNF in vivo (Altar et al, 1997). We show here that htt is a facilitator of anterograde and retrograde transport in neurons and that this function is regulated by the phosphorylation of htt at serine 421. When htt is phosphorylated, kinesin-1 is recruited to vesicles and MTs, facilitating anterograde transport. By contrast, retrograde transport is favoured in the absence of htt phosphorylation at S421. This study reveals that vesicular directionality can be regulated by phosphorylation of htt and identifies htt as the first regulator of the direction of transport in neurons. Results Huntingtin regulates anterograde and retrograde transport in neurons To analyse transport in neurons and avoid overexpression artefacts or toxicity due to the long-term expression or downregulation of the genes studied, experimental conditions require the electroporation technique and the analysis of the neurons about 3 days after electroporation and plating. However, at this stage, neurons are not fully mature and a cautious interpretation of the transport system needs to be done. Previous studies established that the non-uniform organization of MTs in dendrites is progressively acquired during maturation and is achieved when the neurons are fully polarized (Baas et al, 1989). In contrast, in 3 days in vitro (DIV) neurons, most if not all distinct neurites show an MT organization with the plus ends distal to the cell body. We analysed in our cultured cortical neurons the directionality of MT growth in the neurites using end-binding protein 3 (EB3) coupled to GFP. EB3 is a neuron-specific plus end-tracking protein whose dynamics reflect the 'plus' end MT polymerization state (Stepanova et al, 2003). We found that most of the movements of the EB3–GFP comets (90–95%) were directed distal to the cell body, indicating that outward movements correspond to 'plus' end-directed anterograde movements in three DIV cortical neurons (data not shown) (Stepanova et al, 2003; Gauthier et al, 2004). Having determined the MT orientation in our experimental system, we specifically tracked the BDNF vesicles in the neurites of three DIV cortical neurons and classified the intracellular movements as anterograde and retrograde transport according to the direction moved by the vesicles relative to the cell body (see Materials and methods in Supplementary data). We used a 480 amino-acid N-terminal fragment of wild-type htt that has the same subcellular distribution as the full-length htt in neurons (Humbert et al, 2002) and increases the mean velocity of BDNF vesicles as efficiently as the full-length protein in neuronal cells (Supplementary Figure S1). htt increased velocity in both directions using the 480 amino-acid N-terminal fragment of wild-type htt (Figure 1A; Supplementary Movie 1). Conversely, the reduction of htt protein levels by RNA interference (RNAi) decreased both anterograde and retrograde velocity of moving vesicles. A scramble RNA in the same conditions had no effect on BDNF anterograde and retrograde velocities (data not shown). We further validated the specificity of this assay by analysing the consequences of decreasing the levels of certain components of the molecular motors, such as p150Glued, KHC (kinesin-1 heavy chain) and DHC (dynein heavy chain) by RNAi (Figure 1A). As we were unable to detect DHC using anti-DHC antibodies, we analysed the effect of DHC targeting on the protein levels of DIC. As previously shown, the targeting of DHC through RNAi induces the specific downregulation of DIC (Caviston et al, 2007). Similar to the results obtained by antibody-blocking strategies (Waterman-Storer et al, 1997), reducing p150Glued and dynein levels each induced a considerable decrease in the movement of vesicles in both directions. Also, decreasing the levels of KIF5B and KIF5C, the predominant isoforms of kinesin-1 in cortical neurons (Kanai et al, 2000), reduced anterograde and retrograde transport. These results are in agreement with a previous study showing that silencing kinesin-1 but not kinesin-2 affects the transport of BDNF-containing vesicles (Dompierre et al, 2007). Figure 1.Phosphorylation of htt regulates BDNF transport directionality. (A) Anterograde and retrograde BDNF vesicular velocities are stimulated by htt in cortical neurites and are inhibited by siRNA targeting htt, p150Glued, KHC or DHC. The reduction of protein levels by siRNA is shown by immunoblotting. (B) Expression of htt-S421A stimulates retrograde transport, whereas htt-S421D stimulates anterograde transport as shown by kymographs. (C) Mean velocity, (D) scatter plots of translocation velocities as a function of the distance travelled between two pauses are shown. (E) IGF-1 promotes anterograde transport of BDNF vesicles in the scramble RNA condition. This effect is lost in the absence of htt (siRNA-htt). Control: corresponding empty vector (*P<0.05, **P<0.01, ***P<0.001; see Supplementary data for detailed statistical analyses and number of measures). Download figure Download PowerPoint htt is subjected to post-translational modifications. This is the case of htt phosphorylation by the serine/threonine kinase Akt at the S421. Although this phosphorylation is crucial to regulate the toxicity of the mutant pathogenic htt that contains the abnormal polyglutamine expansion, it was found that wild-type htt, the non-toxic form of htt, is also a physiological substrate of Akt in primary cultures of neurons and in rodent brains (Humbert et al, 2002; Pardo et al, 2006). However, the consequences of wild-type htt phosphorylation remain to be established, as phosphorylation of wild-type htt had no obvious effect on protein stability, degradation, solubility or toxicity in cells (Humbert et al, 2002). As wild-type htt controls both anterograde and retrograde transport, we asked whether phosphorylation at S421 could regulate htt's function in axonal transport. We analysed the consequences of htt phosphorylation on BDNF transport in neurites by expressing a form of htt that cannot be phosphorylated, htt-S421A, or a form of htt that mimicks constitutive phosphorylation, htt-S421D, in cortical neurons. Strikingly, when htt contained an unphosphorylatable S421, the vesicles moved faster in the retrograde direction than in the anterograde direction (Figure 1B and C; Supplementary Movie 2). By contrast, when the neurons were transfected with a constitutively S421-phosphorylated htt, the vesicles moved significantly faster in the anterograde direction than in the retrograde direction. These differences in velocities were not due to differences in expression levels (data not shown). We also calculated the effect of htt phosphorylation on the distance travelled (run lengths) by vesicles between two pauses and found an increase in the travelled distances by retrograde-moving vesicles when htt was not phosphorylated (Figure 1D). Conversely, htt phosphorylation at S421 increased the run lengths of anterograde but not retrograde-moving vesicles. These results show that the phosphorylation state of S421 regulates the efficiency of transport in one direction over the other. To establish that the observed effect is physiological and specifically due to htt, we studied the dynamics of BDNF-containing vesicles in the neurites of neurons in the presence of IGF-1. This treatment leads to phosphorylation of htt at S421 (Humbert et al, 2002; Pardo et al, 2006). In the presence of IGF-1, anterograde transport was favoured (Figure 1E). We next decreased htt levels by siRNA and observed that in this condition, IGF-1 was not able to promote its effect on anterograde transport. This shows unequivocally that IGF-1 specifically increases BDNF anterograde transport along MTs through htt. S421 stimulation of transport is an MT-based mechanism but does not affect the MT binding of huntingtin and BDNF or MT dynamics BDNF vesicular transport is inhibited by the treatment of cells with nocodazole, an MT-depolymerizing drug, indicating that this transport requires an intact MT network (Gauthier et al, 2004). However, a report revealed that htt regulates the motility of endosomes by redistributing them from an MT localization to a preferential association with actin filaments (Pal et al, 2006). To test whether the modification of BDNF transport directionality observed in our assay could result from a switch to actin filaments, we treated cells with latrunculin B, a known actin-depolymerizing agent, and analysed BDNF transport (Figure 2A). Whereas nocodazole disrupted BDNF movement in neurons, latrunculin B treatment had no obvious effect on htt localization with respect to actin filaments and the movement of BDNF vesicles in neurites could still be detected (Figure 2A; Supplementary Figure S2A; Supplementary Movies 3 and 4). We also investigated whether the change in BDNF vesicular dynamics upon phosphorylation might be due to a change in the distribution of BDNF with markers of the endocytic pathway such as LAMP1, which labels late endosomes as well as lysosomes, and EEA1, which is specific to the early endosomal compartment. We observed a very low colocalization of BDNF-containing vesicles with such markers, and the relative distribution of BDNF vesicles to these markers was not affected by the phosphorylation state of htt at S421 (Supplementary Figure S2B). We next investigated whether htt phosphorylation regulates the association of htt to MTs or modifies the attachment of BDNF-containing vesicles to MTs and found no significant effect of htt phosphorylation on the association of htt and BDNF to MTs (Supplementary Figure S3A). Figure 2.Phosphorylation of htt increases localization of kinesin-1 to microtubules. (A) Huntingtin-mediated control of BDNF trafficking is MT dependent. Time projection and velocity show that nocodazole treatment of cortical neurons significantly reduces motility of BDNF vesicles, whereas latrunculin B has no marked effect. (B) S421-phosphorylated htt is present on MTs in COS7 cells. Cells are immunostained using anti-phospho-htt-S421–714 and anti-α-tubulin antibodies. (C) Compared with unphosphorylatable htt (htt-S421A), expression of S421 constitutively phosphorylated htt (htt-S421D) in neuronal cells significantly increases the percentage of KHC-positive dots decorating microtubules. The localization of DIC is unchanged in both conditions (**P<0.01; see Supplementary data for detailed statistical analyses and number of measures). Download figure Download PowerPoint To test the possibility that htt phosphorylation alters MT dynamics, we analysed the effect of htt phosphorylation on MT growth in mouse neuronal cells using EB3–eGFP. Transfecting cells with htt, htt-S421A and htt-S421D constructs had no significant effect on the patterning of MT growth in cells (Supplementary Figure S3B). We next measured the MT polymerization rate and found that htt phosphorylation state had no detectable effect on MT growth and on the nucleation of MTs (growth of new MTs) in cells (Supplementary Figure S3C and D). Taken together, these results indicate that, though the change in BDNF dynamics upon htt phosphorylation depends on MTs, the phosphorylation status of htt has no effect on the binding of htt and BDNF to MTs or on MT dynamics. Kinesin-1 is recruited on MTs upon S421 huntingtin phosphorylation Previous studies have reported a localization of htt on MTs (Gutekunst et al, 1995). However, these studies used antibodies that recognized the protein whatever its phosphorylation status. To make sure that htt phosphorylation at S421 is relevant to the mechanism studied, we examined by immunofluorescence the localization of phosphorylated htt with respect to the MT cytoskeleton using a previously described anti-phospho-htt-S421–714 antibody raised against the phospho-S421 epitope (Pardo et al, 2006). We observed phospho-specific immunostaining along MTs, as shown by the partial colocalization with α-tubulin (Figure 2B). We next analysed the localization of DIC and kinesin-1 to MTs in htt-S421A- or htt-S421D-transfected cells. Whereas htt phosphorylation had no profound effect on the colocalization of DIC with α-tubulin, we found a marked increase in the localization of kinesin-1 to MTs in cells expressing phosphorylated htt (Figure 2C). We quantified in a random and blinded manner the recruitment of the motor proteins to MTs and found a statistically significant increase in the association of KHC but not DIC to the MTs (Figure 2C). In agreement, we also observed a preferential colocalization of endogenous KHC with S421-phosphorylated htt on structures that could correspond to MT stretches (data not shown) using conditions in which S421 of htt is phosphorylated (Humbert et al, 2002). By contrast, in serum-starved cells treated with wortmannin, a condition that leads to loss of phosphorylation at S421, we did not observe such colocalization (data not shown). Huntingtin phosphorylation at S421 leads to kinesin-1 recruitment to vesicles Having shown that htt phosphorylation leads to an increased recruitment of KHC to MTs, we analysed the association of molecular motors with BDNF-containing vesicles. We transfected mouse neuronal cells with htt-S421A or htt-S421D constructs and performed subcellular fractionation obtaining a cytosolic (S3) and a pellet (P3) fraction that is enriched in small vesicles and vesicle-associated proteins. This fraction contains htt and BDNF-containing vesicles (Figure 3A) (Gauthier et al, 2004). The P3 fraction was also enriched in kinesin-1 and in the dynein–dynactin complex. Interestingly, compared with cells transfected with the unphosphorylatable form of htt, cells transfected with htt-S421D showed a statistically significant higher level of kinesin-1 in the P3 fraction compared with the S3 fraction (P3/S3 ratio) (Figure 3A). In contrast, htt phosphorylation status had no significant effect on the P3/S3 ratio of DIC (data not shown). To confirm these findings, we performed subcellular fractionation in mouse neuronal cells that were cultured in conditions in which the PI3K/Akt pathway was either inhibited (1% serum+100 nM wortmannin) or activated (20% serum+50 ng/ml IGF-1). These conditions lead to the phosphorylation of endogenous htt at S421 as shown by immunoblotting experiments with the anti-phospho-htt-S421–714 antibody (Figure 3B). After IGF-1 treatment, kinesin-1 was enriched in the P3 fraction, whereas after serum starvation and wortmannin treatment, most of the kinesin-1 was found in the S3 fraction corresponding to the cytosol (Figure 3C). Thus, when htt is phosphorylated at S421, kinesin-1 is enriched in the fraction that contains synaptic vesicles. To make sure that the observed changes are due to htt, we analysed the consequences of decreasing the levels of htt by RNAi on the distribution of kinesin-1 after S3/P3 subcellular fractionation (Figure 3D). In cells with low htt levels, the kinesin-1 levels in the P3 fraction were significantly reduced compared with the scramble situation. Taken together, these results demonstrate that htt phosphorylation at S421 leads to the recruitment of kinesin-1 to vesicles. Figure 3.Phosphorylation of htt leads to a recruitment of kinesin-1 to vesicles. (A) Subcellular fractionation of mouse neuronal cell extracts by successive centrifugation steps is analysed by immunoblotting for the presence of kinesin-1 (KHC), dynein (DIC), htt, p150Glued, GM130 (Golgi marker) and synaptophysin (small vesicles marker). Quantitative assessment of the optical density of KHC is expressed as P3/S3 ratio and shows a recruitment of KHC from cytosol to vesicles in cells expressing htt-S421D. (B) Phosphorylation of endogenous htt at S421 is increased by IGF-1 treatment. Total extracts from mouse neuronal cells treated with wortmannin (wort)/1% serum or IGF-1/20% serum are immunoprobed with the anti-phospho-htt-S421–714 and anti-htt antibodies. Quantification of phosphorylated htt (P-htt) is expressed as a ratio of P-htt/total htt optical densities. (C) Phosphorylation of endogenous htt leads to kinesin-1 recruitment to vesicles. Extracts treated as in (B) are subjected to subcellular fractionation, analysed and quantified as in (A). (D) Loss of htt reduces kinesin-1 recruitment to vesicles. Mouse neuronal cells treated with siRNA targeting htt or control scramble RNA (scRNA) were subjected to subcellular fractionation, analysed and quantified as in (A). (***P<0.001; see Supplementary data for detailed statistical analyses and number of measures). Download figure Download PowerPoint S421-phosphorylated huntingtin co-sediments with kinesin-1 We studied the nature of the motor complexes upon htt phosphorylation through sucrose gradient fractionation. We and others have previously used this approach to show that a fraction of soluble wild-type htt co-sediments with the p150Glued–dynein complex (Li et al, 1998; Gauthier et al, 2004). We examined whether an increased phosphorylation of htt modifies the sedimentation pattern of htt, p150Glued, dynein or kinesin-1 in linear sucrose gradients. Mouse neuronal cells were cultured as in Figure 3B and C. Cell extracts were fractionated on a linear sucrose gradient (7.5–25%) revealing that the htt and kinesin-1 sedimentation pattern was different between low and high htt phosphorylation conditions (Figure 4). S421-phosphorylated htt co-migrated with kinesin-1 in IGF-1/high serum condition, whereas htt and kinesin-1 were not present in the same fractions in wortmannin/low serum condition. We obtained similar results with the human neuroblastoma SH-SY5Y cell line (data not shown). This indicates that the nature of the cytoplasmic complex of htt and molecular motors is modified upon serum treatment. The preferential co-sedimentation of S421-phosphorylated htt with kinesin-1 suggests that htt and kinesin-1 may be present in the same native cytoplasmic complex upon htt phosphorylation. Taken together, the results of our biochemical and immunofluorescence experiments demonstrate that when htt is phosphorylated at S421, kinesin-1 colocalizes with htt and MTs and is associated with BDNF-containing vesicles at MTs. Conversely, in conditions that decrease the fraction of phosphorylated htt, KHC is weakly associated with htt, MTs and vesicles. Figure 4.Phosphorylation of htt modifies soluble molecular motor complexes. The co-sedimentation of htt with kinesin in sucrose gradients is modified by phosphorylation at S421. Total extracts from mouse neuronal cells treated with wortmannin/1% serum or IGF-1/20% serum (as in Figure 3B) are fractionated by sucrose gradient and analysed by immunoblotting. The distribution of the optical density of endogenous KHC and htt in the different fractions is shown in the graphs. Download figure Download PowerPoint Phosphorylation of huntingtin increases the level of kinesin-1 in motor complex How does htt phosphorylation lead to the recruitment of kinesin-1 to MTs and vesicles? One mechanism could involve the association of htt with HAP1. Indeed, htt associates with HAP1, and HAP1 associates with the p150Glued subunit of dynactin and the light chain of kinesin-1 (Engelender et al, 1997; Li et al, 1998; Gauthier et al, 2004; McGuire et al, 2006). We have shown that this complex is essential for stimulating MT-based transport and requires the HAP1 protein. In agreement, the first exon of htt, which does not contain the HAP1-binding domain cannot stimulate transport, whereas a longer fragment does (Figure 1A) (Gauthier et al, 2004). Furthermore, HAP1-deficient cells have an impaired capacity to transport BDNF- and amyloid precursor protein (APP)-containing vesicles efficiently (Gauthier et al, 2004; McGuire et al, 2006). We first investigated whether the phosphorylation of htt at S421 regulates the interaction between HAP1 and htt. GST–HAP1 pull-down assays revealed no differences in the association of HAP1 to htt using extracts from cells transfected with either the htt-S421A or the htt-S421D construct (Figure 5A). As previously reported, in these conditions, polyQ-expanded htt has a higher affinity to HAP1 (Figure 5A) (Li et al, 1995). We next determined the association between HAP1 and p150Glued or kinesin-1. We used a full-length HAP1A construct as the HAP1A isoform interacts with the kinesin light chain-2 (KLC2) C-terminal domain (McGuire et al, 2006). However, htt phosphorylation at S421 had no effect on the association of p150Glued or kinesin-1 to HAP1A (Figure 5B). Immunoprecipitation experiments using anti-HAP1A antibodies further confirmed that htt phosphorylation does not modify the ability of HAP1 to bind to kinesin-1 (Figure 5C). We next tested whether htt phosphorylation could affect the association of kinesin-1 to the other components of the motor complex (Ligon et al, 2004). In cells expressing htt-S421D, the ability of p150Glued subunit of dynactin to co-immunoprecipitate KHC was significantly greater than in cells expressing htt-S421A (Figure 5D and E). This difference was not due to differences in the expression levels of htt as these were similar (data not shown). We confirmed this increased co-immunoprecipitation by the complementary experiment. When htt contained the S421D mutation, KHC antibodies co-immunoprecipitated p150Glued more efficiently (Figure 5F and G). Although the exact nature of the interaction between kinesin-1 and p150Glued that is modified upon htt phosphorylation remains to be determined (direct versus indirect), our results suggest a mechanism by which kinesin-1 is recruited to the BDNF-containing vesicles on MTs. Figure 5.Phosphorylation of huntingtin at S421 modifies the nature of the molecular motor complexes. (A) GST–HAP1 pull-down experiments of protein extracts from H