The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport
2007; Springer Nature; Volume: 26; Issue: 6 Linguagem: Inglês
10.1038/sj.emboj.7601609
ISSN1460-2075
AutoresYoichi Araki, Takanori Kawano, Hidenori Taru, Yuhki Saito, Sachiyo Wada, Kanako Miyamoto, Hisako Kobayashi, Hiroyuki Ishikawa, Yu Ohsugi, Tohru Yamamoto, Kenji Matsuno, Masataka Kinjo, Toshiharu Suzuki,
Tópico(s)Plant Molecular Biology Research
ResumoArticle1 March 2007free access The novel cargo Alcadein induces vesicle association of kinesin-1 motor components and activates axonal transport Yoichi Araki Yoichi Araki Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Takanori Kawano Takanori Kawano Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Hidenori Taru Hidenori Taru Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Yuhki Saito Yuhki Saito Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Sachiyo Wada Sachiyo Wada Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Kanako Miyamoto Kanako Miyamoto Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan Search for more papers by this author Hisako Kobayashi Hisako Kobayashi Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan Search for more papers by this author Hiroyuki O Ishikawa Hiroyuki O Ishikawa Genome and Drug Research Center, Tokyo University of Science, Noda, Japan Search for more papers by this author Yu Ohsugi Yu Ohsugi Laboratory of Supramolecular Biophysics, Research Institute for Electric Science, Hokkaido University, Sapporo, Japan Search for more papers by this author Tohru Yamamoto Tohru Yamamoto Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Kenji Matsuno Kenji Matsuno Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan Search for more papers by this author Masataka Kinjo Masataka Kinjo Laboratory of Supramolecular Biophysics, Research Institute for Electric Science, Hokkaido University, Sapporo, Japan Search for more papers by this author Toshiharu Suzuki Corresponding Author Toshiharu Suzuki Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Yoichi Araki Yoichi Araki Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Takanori Kawano Takanori Kawano Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Hidenori Taru Hidenori Taru Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Yuhki Saito Yuhki Saito Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Sachiyo Wada Sachiyo Wada Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Kanako Miyamoto Kanako Miyamoto Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan Search for more papers by this author Hisako Kobayashi Hisako Kobayashi Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan Search for more papers by this author Hiroyuki O Ishikawa Hiroyuki O Ishikawa Genome and Drug Research Center, Tokyo University of Science, Noda, Japan Search for more papers by this author Yu Ohsugi Yu Ohsugi Laboratory of Supramolecular Biophysics, Research Institute for Electric Science, Hokkaido University, Sapporo, Japan Search for more papers by this author Tohru Yamamoto Tohru Yamamoto Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Kenji Matsuno Kenji Matsuno Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan Search for more papers by this author Masataka Kinjo Masataka Kinjo Laboratory of Supramolecular Biophysics, Research Institute for Electric Science, Hokkaido University, Sapporo, Japan Search for more papers by this author Toshiharu Suzuki Corresponding Author Toshiharu Suzuki Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Search for more papers by this author Author Information Yoichi Araki1, Takanori Kawano1, Hidenori Taru1, Yuhki Saito1, Sachiyo Wada1, Kanako Miyamoto2, Hisako Kobayashi2, Hiroyuki O Ishikawa3, Yu Ohsugi4, Tohru Yamamoto1, Kenji Matsuno2, Masataka Kinjo4 and Toshiharu Suzuki 1 1Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan 2Department of Biological Science and Technology, Tokyo University of Science, Noda, Japan 3Genome and Drug Research Center, Tokyo University of Science, Noda, Japan 4Laboratory of Supramolecular Biophysics, Research Institute for Electric Science, Hokkaido University, Sapporo, Japan *Corresponding author. Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita12-Nishi6, Kita-ku, Sapporo 060-0812, Japan. Tel.: +81 11 706 3250; Fax: +81 11 706 4991; E-mail: [email protected] The EMBO Journal (2007)26:1475-1486https://doi.org/10.1038/sj.emboj.7601609 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Alcadeinα (Alcα) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alcα strongly associates with kinesin light chain (KD≈4–8 × 10−9 M) through a novel tryptophan- and aspartic acid-containing sequence. Alcα can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alcα, and the kinesin-1 motor complex dissociates from Alcα-containing vesicles in a JIP1 concentration-dependent manner. Alcα-containing vesicles were transported with a velocity different from that of amyloid β-protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alcα- and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alcα with kinesin-1 blocked transport of APP-containing vesicles and increased β-amyloid generation. Inappropriate interactions of Alc- and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease. Introduction Conventional kinesin (kinesin-1) is composed of two heavy (KHC) and two light (KLC) chains (Hirokawa, 1998). The N-terminal half of KLC interacts with KHC, and the C-terminus of KLC links to membrane cargo either directly or via cytosolic adaptor proteins such as the JNK-interacting protein 1 (JIP1) (Gauger and Goldstein, 1993; Gindhart and Goldstein, 1996; Verhey et al, 2001). The combinations of cargoes, adaptor proteins, and motor molecules contribute to the diversity of vesicular transport systems (Hirokawa, 1998), the dysfunction of which may underlie some neurodegenerative disorders (Guzik and Goldstein, 2004) such as Alzheimer's disease (AD) (Stokin et al, 2005). Amyloid β-protein precursor (APP) has been implicated in the development and progression of AD (Gandy, 2005) and is proposed to be a kinesin-1 cargo. APP can bind to kinesin-1 directly (Kamal et al, 2000; Gunawardena and Goldstein, 2001) or through JIP1 (Inomata et al, 2003; Matsuda et al, 2003). Although the cargo hypothesis has been challenged (Lazarov et al, 2005) and it is unclear how APP functions mechanistically in vesicle transport, disruption of APP-containing vesicle transport may alter APP metabolism, thereby increasing levels of neurotoxic β-amyloid (Aβ) (Gunawardena and Goldstein, 2001; Stokin et al, 2005). Alcadein (Alc) is an evolutionarily conserved type I membrane protein, which has four isoforms, Alcα1, Alcα2, Alcβ, and Alcγ, in mammals (Araki et al, 2003). These proteins were isolated independently as postsynaptic Ca2+-binding proteins called the calsyntenins (Vogt et al, 2001). Alc associates with APP in neurons through cytoplasmic interactions of both proteins with X11-like (X11L) (Araki et al, 2003), a neuron-specific adaptor protein (Tomita et al, 1999). Formation of a tripartite APP/X11L/Alc complex stabilizes both APP and Alc proteins metabolically. Dissociation of X11L from the complex induces coordinated cleavages of APP and Alc, generating the Aβ fragment from APP and the β-Alc peptide from Alc, in addition to the release of their respective cytoplasmic-domain fragments, AICD and AlcICD (Araki et al, 2004). Thus, Alc may be similar to APP in function as well as metabolism. Here, we present evidence that Alc is a kinesin-1 cargo and exhibits novel functions. The transport of Alc-containing vesicles competes with that of APP-containing vesicles for kinesin-1, and disruption of transport of APP-containing vesicles increases Aβ generation. This analysis contributes to our understanding of both the pathogenesis of AD and the physiological function of Alc. Results Direct interaction of Alc with KLC A yeast two-hybrid screen using the cytoplasmic domain of Alcα (amino acids 872–969 of mouse Alcα1) as bait isolated 11 full-length and partial cDNA clones encoding KLC1 (see Figure 1A and Supplementary Figure S1 for the schematic structures of KLC1 encoded by the isolated cDNA clones; Supplementary data is shown in Sup_1.pdf.) and one partial cDNA clone encoding KLC2. The interaction between Alcα1 and KLC1 was confirmed by co-immunoprecipitation in HEK293 cells overexpressing Alcα1 and KLC1 (Figure 1A and B, right). Immunoprecipitates obtained with an anti-Alcα antibody contained KLC1, indicating that Alcα interacts with KLC1. To identify the Alcα-binding domain, FLAG-tagged amino- (KLC1-N or N) and carboxy- (KLC1-C or C) terminal halves of KLC1 (Figure 1A) were overexpressed. An anti-FLAG antibody co-immunoprecipitated Alcα1 with KLC1 and with KLC1-C, but not with KLC1-N (Figure 1B, left). These data indicate that Alcα interacts with the KLC1 carboxy-terminus, which includes the tetratrico-peptide repeat (TPR; see Figure 1A), with which other KLC-binding proteins interact (Gindhart and Goldstein, 1996). Alcα1 was also co-immunoprecipitated with FLAG-KLC2 (data not shown). Figure 1.Interaction of Alcα with KLC and determination of the KLC-binding site on Alcα. (A) Structure of the cytoplasmic domains of Alcα, KLC1, KLC1-N (N), KLC1-C (C), and KLC2. Numbers represent amino-acid sequences. TM, transmembrane domain; WD, tryptophan- and aspartic acid-containing sequence (see 'G'); NP, Asn-Pro motif; AR, acidic region; CC, coiled-coil domain; TPR, tetratrico-peptide repeat. Epitopes for the UT83, UT109, and UT110 antibodies are indicated with bars. (B) (Left panel) Co-immunoprecipitation of Alcα1 with amino-terminal FLAG-tagged KLC1 and deletion constructs. HEK293 cells were transiently cotransfected with pcDNA3-hAlcα1 and pcDNA3.1-FLAG-mKLC1 (KLC1), pcDNA3.1-FLAG-mKLC1-N (N), pcDNA3.1-FLAG-mKLC1-C (C), or pcDNA3.1-FLAG (−). Cell lysates were immunoprecipitated with an anti-FLAG (M2) antibody. The immunoprecipitates (IP) and lysate were analyzed by Western blotting with M2 and UT83. (Right panel) Co-immunoprecipitation of KLC1 with Alcα1. HEK293 cells were transiently transfected with pcDNA3-hAlcα1 and pcDNA3.1-mKLC1. Transfection with plasmid (+) or vector alone (−) is indicated. Cell lysates were immunoprecipitated with an anti-Alcα (UT83) antibody. The immunoprecipitates (IP) and lysate were analyzed by Western blotting with UT83 and UT109. (C) Localization of Alcα1 and KLC1. CAD cells were transiently cotransfected with the indicated combinations of pcDNA3-hAlcα1, pcDNA3.1-FLAG-mKLC1, and pcDNA3.1-FLAG-mKLC1-C for 48 h and differentiated for 24 h by depleting serum. Alcα1 and FLAG-tagged KLC1 were immunostained with UT83 (green) and M2 (red) antibodies, respectively. Nuclei were stained using 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI, blue). Merged signals are shown at the left. Scale bar, 5 μm. (D) Membrane fractionation of brain tissues. The post-nuclear supernatant of adult mouse brains was fractionated by 0–28% iodixanol density gradient centrifugation (Araki et al, 2003). (Upper panel) The density (blue circles) and protein concentration (pink squares) are indicated. Fraction numbers are indicated along the abscissa. (Lower panel) The fractions were analyzed by Western blotting with antibodies to Alcα (UT83), KHC (H2), KLC1 (UT109), KLC2 (UT110), and the Golgi-resident GM130 (clone no. 35) and the ER-resident PDI (1D3). The dotted square indicates the fractions containing the highest levels of Alcα, KHC, KLC1, and KLC2. (E) Co-immunoprecipitation of Alcα with KLC. The mouse brain membrane fraction (500 μg protein) was solubilized, and Alcα1, KLC1, and KLC2 were immunoprecipitated with the UT83, UT109, and UT110 antibodies, respectively, or a control non-immune antibody. The membrane fraction (Membrane, 10 μg protein) and immunoprecipitates (IP) were analyzed by Western blotting with antibodies specific for Alcα (UT83), KLC1 (UT109), KLC2 (UT110), KHC (H2), and SYT (clone no. 41). IgG(H) indicates the IgG heavy chain (rabbit). (F) Determination of the KLC-binding site on Alcα. (Left) Various GST-fusion proteins of the Alcα cytoplasmic domain (shown schematically, numbers are amino-acid residues) were incubated with HEK293 cell lysates expressing FLAG-KLC1. FLAG-KLC1 bound to GST-Alcαcyt was recovered using glutathione beads. (Right) The bound and unbound FLAG-KLC1 were analyzed by Western blotting with the M2 antibody. GST-Alcα1 protein constructs were detected by Western blotting with an anti-GST antibody. In the left panel, '+' indicates positive binding and '−' indicates negative binding. (G) Amino-acid sequences of WD1 and WD2 of Alcα1. Numbers indicate amino-acid residues. Identical (*) and similar (:) amino-acid residues are indicated. Download figure Download PowerPoint Alcα1 appears as two bands on SDS–PAGE gels. The more slowly migrating band increased in intensity in the presence of KLC1 and had a mobility similar to that of brain Alcα (Figure 1B and Supplementary Figure S2). HEK293 cells express substantial amounts of endogenous KHC and KLC2 but lower levels of endogenous KLC1 (Supplementary Figure S2A), as do undifferentiated CAD cells (data not shown), suggesting that the expression of exogenous KLC1 may increase the formation of functional kinesin-1 in both cell types because increased number of transporting Alcα vesicles were observed (data not shown for HEK293 cells and Figure 1C for CAD cells). We determined that Alcα1 was post-translationally modified with both high-mannose (arrow 2) and complex (arrow 1) N-glycans (Supplementary Figure S2B). The predominance of the slow-migrating Alcα1 increased in the presence of KLC1 but not KLC1-C (Figure 1B, left), which does not bind to KHC (Gauger and Goldstein, 1993), suggesting that trafficking of Alcα-containing vesicles by kinesin-1 facilitates complex N-glycosylation. Alcα colocalized with KLC1 in CAD cells (Figure 1C). CAD cells overexpressing Alcα1 in the presence (Figure 1C, third rows) or absence (first rows) of KLC overexpression began to extend processes that were not observed in cells not expressing Alcα1 (second rows) after serum depletion. Alcα1 protein localized to neurite tips, as well as the ER and Golgi (third rows), whereas FLAG-KLC1 was located in the cytoplasm (second rows). A few small vesicles were observed in cells expressing Alcα1 alone (first rows), but a large number of small vesicles appeared when Alcα1 was coexpressed with FLAG-KLC1, and the proteins were colocalized (third rows), suggesting that Alcα binds the active kinesin-1 motor complex and uses it to transport vesicles to neurite tips. Such vesicles were not observed in cells coexpressing KLC1-C with Alcα1 (fourth rows), as KLC1-C is not able to bind KHC. Alcα was included in a protein complex containing KLC and KHC in vivo. Mouse brain membranes were fractionated by iodixanol density gradient centrifugation, and Alcα, KHC, KLC1, KLC2, the Golgi-resident protein GM130, and the ER-resident protein disulfide isomerase (PDI) were detected in these membranes (Figure 1D). The complex N-glycosylated Alcα was recovered with kinesin-1 subunits from vesicle fractions that were less dense than the Golgi membrane fractions. The faster-migrating high-mannose N-glycosylated Alcα was largely recovered in the ER. The membranes resulted by 100 000 g centrifugation were solubilized, immunoprecipitated with anti-Alcα, anti-KLC1, anti-KLC2, or nonimmune control antibodies, and subjected to Western blot analysis (Figure 1E). Alcα, KLC1, KLC2, and KHC were immunoprecipitated by anti-Alcα, anti-KLC1, as well as the anti-KLC2 antibody. As a control, synaptotagmin (SYT) was not co-immunoprecipitated by any of the antibodies. These results suggest that Alcα associates with kinesin-1 in vivo. A pull-down assay with recombinant KLC1 and a GST-Alcα cytoplasmic domain fusion protein (GST-Alcαcyt) demonstrated a direct interaction between KLC1 and Alcα (Supplementary Figure S3A). KLC1 bound to GST-Alcαcyt with a dissociation constant (KD) of ≈7.9 × 10−9 M. Surface plasmon resonance (SPR) analysis also demonstrated a strong affinity (KD≈4.3 × 10−9 M) between GST-Alcαcyt and KLC1: the interaction had an association rate (Kass) of approximately 1.3 × 103 M/s and a dissociation rate (Kdiss) of approximately 5.5 × 10−6 M/s (Supplementary Figure S3B). This low dissociation rate indicates a very strong binding between Alcα and KLC1. The region of Alcα that binds KLC1 was determined by a pull-down assay with glutathione beads bearing GST fusion proteins (Figure 1F). KLC bound to the Alcα fusion protein containing amino acids 873–971, but did not bind to fusion proteins lacking both WD motifs, WD1=amino acids 891–900 and WD2=amino acids 962–971. Each WD motif contains the consensus, D/E-W-D-D-S-A/T-L-T/S (Figure 1G). Deletion of either WD1 or WD2 preserved KLC1 binding, indicating that one WD motif is sufficient for KLC binding. KLC binding to the WD motifs was completely abolished when Ala was substituted for the conserved amino-acid residues in the WD motifs (unpublished observations and Figure 6A), demonstrating a highly specific interaction between Alcα and KLC. These findings in vitro strongly suggest that Alcα1 can function as a kinesin-1 cargo. Figure 2.Kinesin-1-dependent anterograde transport of Alcα cargo in living neuronal cells. (A, B) Anterograde movement of Alcα1 cargo vesicles in an axon. (A) Differentiating CAD cells expressing Alcα1-GFP were observed using TIRF microscopy (panel 1). (B) Kinesin-1-dependent transport of Alcα1 cargo in neuronal cells. Differentiating CAD cells expressing Alcα1-GFP in which KLC1 and KLC2 expression has been knocked down using siRNA were observed using TIRF microscopy (panel 1). Vesicle movements in the dotted square were tracked with time-lapse imaging and are indicated with colored lines and numbers (see Supplementary Movie 1, parts 1 and 2 in Sup_2.mov). Scale bar, 5 μm. (C, D) Inhibition of anterograde transport of Alcα1 cargo by expression of JIP1b. Differentiating CAD cells expressing Alcα1-GFP in the presence of JIP1b (C) and JIP1bΔC11 (D) were observed using TIRF microscopy (panel 1). Vesicle movements in the dotted square were tracked with time-lapse imaging and are indicated with colored lines and numbers (see Supplementary Movie 1, parts 3 and 4 in Sup_2.mov). Scale bar, 5 μm. (A–D) Red lines indicate tracks of anterograde transport, blue lines indicate tracks of retrograde transport, and green spots indicate stationary vesicles moving at less than 0.4 μm/s (panel 1). Alcα1 and APP cargo vesicles transported anterogradely ('A') and retrogradely ('R'), and stationary vesicles ('S') in 25 cells were counted with Metamorph software and the fraction of the total number of vesicles (%) is indicated (panel 2). Distribution (%) of anterograde (red) and retrograde (blue) transport velocity of Alcα1 cargo and of stationary vesicles (green) is shown (panel 3). Download figure Download PowerPoint Alc is a novel cargo for kinesin-1 and can mediate the vesicle association of kinesin-1 motor components To investigate the characteristics of Alcα1 as a cargo, an Alcα1–GFP fusion protein was expressed in differentiating CAD cells and assayed for vesicular transport. Fast anterograde transport of Alcα1 cargo was observed, together with a minor amount of retrograde movement (Figure 2A, panel 1, and Supplementary Movie 1, part 1 in Sup_2.mov). Approximately 80% of the total Alcα1 cargo vesicles (375 vesicles in 25 cells) showed anterograde transport and 10–15% showed retrograde movement (Figure 2A, panel 2). The anterograde vesicles were transported at a velocity of 1.48±0.45 μm/s (mean±s.d.), which was calculated from the movement of 4125 vesicles (Figure 2A, panel 3) and was consistent with the velocity of 1.32±0.58 μm/s observed in primary cultured mouse cortical neurons expressing Alcα1-Venus (Supplementary Figure S4A and Movie 2, part 1 in Sup_3.mov) and with the velocity of 1.47±0.76 μm/s observed in non-neuronal HEK293 cells expressing Alcα1-Venus and KLC1 (Supplementary Movie 3, part 1 in Sup_4.mov). Alcα1-Venus transport was suppressed by treatment with nocodazole and Alcα1-mRFP-containing vesicles moved on microtubule tracks composed of YFP-tubulin in HEK293 cells (Supplementary Movie 3, parts 2 and 3 in Sup_4.mov). These observations indicate that Alcα is transported on microtubule tracks. Figure 3.Vesicle association of kinesin-1 components mediated by Alcα1 cargo in axonal transport. (A, B) Vesicule association of KLC1 induced by Alcα1. Differentiating CAD cells expressing GFP-KLC1 with (B) or without (A) Alcα1 were observed with TIRF microscopy. (C, D) Inhibition of vesicular association of KHC by KLC. Differentiating CAD cells expressing GFP-KHC with (D) or without (C) KLC1 were observed with TIRF microscopy. (E, F) Vesicular association of KHC mediated by Alcα1 (E) but not by JIP1b and APP (F) in the presence of KLC1. (G) KLC is not vesicle associated in the presence of JIP1b and APP. (A–G) Vesicle movements were tracked with time-lapse imaging and are indicated with colored lines and numbers (panel 1; see Supplementary Movie 4, in Sup_5.mov). Red lines indicate tracks of anterograde vesicle transport, blue lines indicate tracks of retrograde vesicle transport, and green spots indicate stationary vesicles moving at less than 0.4 μm/s. Scale bar, 5 μm. Vesicles containing KLC or KHC transported anterogradely ('A') and retrogradely ('R'), and stationary vesicles ('S') in 25 cells were counted with Metamorph software and the fraction of the total number of vesicles (%) is indicated (panel 2 of B, C, E). Distribution (%) of anterograde (red) and retrograde (blue) transport velocity of Alcα1 cargo, as well as stationary vesicles (green), is indicated (panel 3 of B, C, E). Download figure Download PowerPoint To determine whether transport required kinesin-1, endogenous KLC1 and KLC2 expression was knocked down. Differentiating CAD cells expressing siRNA directed against KLC1 or KLC2 showed almost no expression of the respective KLC (data not shown). When siRNA treatment against both KLC1 and KLC2 was performed in differentiating CAD cells, fast anterograde transport of vesicles containing Alcα1-GFP was dramatically inhibited, whereas retrograde transport was largely unaffected (Figure 2B, panel 1, and Supplementary Movie 1, part 2 in Sup_2.mov). Approximately 60% of vesicles (375 vesicles in 25 cells) were stationary (Figure 2B, panels 2 and 3), which is likely to result from interruption of anterograde transport owing to lack of KLC. Identical results were obtained in primary cultured mouse cortical neurons (Supplementary Figure S4B and Movie 2, part 2 in Sup _3.mov). Interestingly, Alcα showed a striking effect on the vesicle association of kinesin-1 motor components. GFP-KLC1 was expressed in differentiating CAD cells in the presence or absence of Alcα1 expression. GFP-KLC alone showed a diffuse distribution, as described previously (Verhey et al, 1998), which also agreed with KLC1 immunostaining (Figure 1C). In contrast, in the presence of Alcα1-, KLC1-containing vesicles were visible and underwent anterograde transport (Figure 3A and B, panels 1 and 2, and Supplementary Movie 4, parts 1 and 2 in Sup _5.mov). The velocity of GFP-KLC1 in anterograde transport was distributed among two populations of vesicles moving at 1.2–1.4 μm/s or ∼3.0 μm/s (Figure 3B, panel 3). These values are consistent with the velocities of Alcα1- and APP cargo-containing vesicles, respectively. Faster moving vesicles at ∼3.0 μm/s may be transported with APP/JIP1b or another cargo following the vesicle association of kinesin-1 component by Alcα1. When GFP-KHC was expressed, vesicles containing GFP-KHC were detected and underwent anterograde transport in the absence of exogenously expressed Alcα1 (Figure 3C and Supplementary Movie 4, part 3 in Sup_5.mov). The velocity of GFP-KHC agreed with that of Alcα1 (∼1.5 μm/s) and APP (∼3 μm/s), indicating that kinesin-1 composed of GFP-KHC and endogenous KLC was associated to membrane via endogenous APP/JIP1b and Alcα1(Figure 3C, panel 3). The vesicle association of GFP-KHC was suppressed by coexpression of unlabeled KLC (Figure 3D and Supplementary Movie 4, part 4 in Sup_5.mov), consistent with the previous observation that overexpression of KLC inhibits binding of KHC to microtubules (Verhey et al, 1998). However, GFP-KHC-containing vesicles were again visible and underwent anterograde transport when Alcα1 was coexpressed in the KLC-overexpressing cells (Figure 3E and Supplementary Movie 4, part 5 in Sup_5. mov). This result cannot be simply due to the quenching and/or masking of overexpressed KLC1 by Alcα1, as overexpression of APP or JIP1b does not cause GFP-KHC to reassociate with vesicles under the same conditions (Figure 3F and Supplementary Movie 4, part 6 in Sup_5.mov). JIP1b and APP did not show any ability to induce vesicle association of GFP-KLC (Figure 3G and Supplementary Movie 4, part 7 in Sup_5.mov). These findings indicate that Alcα modulates the vesicle association of kinesin-1 motor components in axonal cargo transport. Figure 4.Transport of APP-containing vesicles and suppression of transport by expression of Alcα1 and AlcαICD in living neuronal cells. (A) Anterograde transport of APP-containing vesicles in an axon. Differentiating CAD cells expressing APP-GFP were observed using TIRF microscopy (panel 1). (B) Kinesin-1-dependent transport of APP-containing vesicles in neuronal cells. Differentiating CAD cells expressing APP-GFP and in which KLC1 and KLC2 expression has been knocked down using siRNA were observed using TIRF microscopy (panel 1). (C–E) Anterograde transport of APP-containing vesicles in differentiating CAD cells expressing APP-GFP in the presence or absence of Alcα1 or AlcαICD. Axonal transport of APP-GFP in the presence of Alcα1 (C), AlcαICD (D), or AlcαICD(AWAA) (E) was observed with TIRF microscopy. Vesicle movements in the dotted square were tracked with time-lapse imaging and are indicated with colored lines and numbers (see Supplementary Movie 5, in Sup_6.mov). Red lines indicate vesicles transported anterogradely ('A'), blue lines indicate vesicles transported retrogradely ('R'), and green spots indicate stationary vesicles moving at less than 0.5 μm/s ('S'). Scale bar, 5 μm. The vesicles in 25 cells were counted with Metamorph software and the fraction of the total number of vesicles (%) is indicated (panel 2). Distribution (%) of anterograde (red) and retrograde (blue) transport velocity of APP-containing vesicles and of stationary vesicles (green) is indicated (panel 3). Download figure Download PowerPoint Alc blocks transport of APP-containing vesicles and JIP1b blocks transport of Alc cargo vesicles APP is transported by the kinesin-1 motor system (Kaether et al, 2000; Kamal et al, 2000; Gunawardena and Goldstein, 2001). Transport of APP-containing vesicles by kinesin-1 was observed by movement of APP-GFP in differentiating CAD cells (Figure 4A, panel 1, and Supplementary Movie 5, part 1 in Sup_6.mov). The anterograde transport velocity of APP-containing vesicles was extremely fast; most vesicles moved at a speed of 3.0–3.6 μm/s with an average of 3.03±1.07 μm/s (Figure 4A, panel 3). Transport of APP-containing vesicles was suppressed when endogenous KLC1 and KLC2 expression was knocked down (Figure 4B and Supplementary Movie 5, part 2 in Sup_6.mov), suggesting that kinesin-1 transports APP cargoes at a velocity distinct from that of Alcα1 cargoes. Figure 5.Separate transport of APP- and Alcα-containing vesicles by kinesin-1. (A) Interaction of Alcα1 with adaptor proteins. HEK293 cells were transiently transfected with pcDNA3-hAlcα1 (+) in the presence of pcDNA3-FLAG-hX11L, pcDNA3-N-FLAG-FE65, pcDNA3-FLAG-JIP1b, pcDNA3.1-FLAG-mKLC1, or vector alone (−). Cells were lysed and immunoprecipitated with the anti-FLAG M2 antibody. Immunoprecipitates (IP) and cell lysates (Lysate) were analyzed by Western blotting with the M2 and anti-Alcα UT83 antibodies. (B) Effect of wild-type and mutant JIP1b on the Alcα1-KLC1 interaction. (Left) HEK293 cells were transiently cotransfected
Referência(s)