Ndel1 Controls the Dynein-mediated Transport of Vimentin during Neurite Outgrowth
2008; Elsevier BV; Volume: 283; Issue: 18 Linguagem: Inglês
10.1074/jbc.m710200200
ISSN1083-351X
AutoresSu Yeon Shim, Benjamin A. Samuels, Jian Wang, Gernot Neumayer, Camille Belzil, Ramsés Ayala, Yang Shi, Yujiang Geno Shi, Li‐Huei Tsai, Minh Dang Nguyen,
Tópico(s)Cellular transport and secretion
ResumoNdel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1Kamiya A. Kubo K. Tomoda T. Takaki M. Youn R. Ozeki Y. Sawamura N. Park U. Kudo C. Okawa M. Ross C.A. Hatten M.E. Nakajima K. Sawa A. Nat. Cell Biol. 2005; 7: 1167-1178Crossref PubMed Scopus (445) Google Scholar, 2Kamiya A. Tomoda T. Chang J. Takaki M. Zhan C. Morita M. Cascio M.B. Elashvili S. Koizumi H. Takanezawa Y. Dickerson F. Yolken R. Arai H. Sawa A. Hum. Mol. Genet. 2006; 15: 3313-3323Crossref PubMed Scopus (142) Google Scholar, 3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 4Ozeki Y. Tomoda T. Kleiderlein J. Kamiya A. Bord L. Fujii K. Okawa M. Yamada N. Hatten M.E. Snyder S.H. Ross C.A. Sawa A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 289-294Crossref PubMed Scopus (342) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar, 6Liang Y. Yu W. Li Y. Yang Z. Yan X. Huang Q. Zhu X. J. Cell Biol. 2004; 164: 557-566Crossref PubMed Scopus (104) Google Scholar). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein αCOP. Ndel1 promotes the interaction between Lis1, αCOP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/αCOP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration. Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, is emergently viewed as an integrator of the cytoskeleton. By regulating the dynamics of microtubules and assembly of neuronal intermediate filaments (IFs), Ndel1 promotes neurite outgrowth, neuronal migration, and cell integrity (1Kamiya A. Kubo K. Tomoda T. Takaki M. Youn R. Ozeki Y. Sawamura N. Park U. Kudo C. Okawa M. Ross C.A. Hatten M.E. Nakajima K. Sawa A. Nat. Cell Biol. 2005; 7: 1167-1178Crossref PubMed Scopus (445) Google Scholar, 2Kamiya A. Tomoda T. Chang J. Takaki M. Zhan C. Morita M. Cascio M.B. Elashvili S. Koizumi H. Takanezawa Y. Dickerson F. Yolken R. Arai H. Sawa A. Hum. Mol. Genet. 2006; 15: 3313-3323Crossref PubMed Scopus (142) Google Scholar, 3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 4Ozeki Y. Tomoda T. Kleiderlein J. Kamiya A. Bord L. Fujii K. Okawa M. Yamada N. Hatten M.E. Snyder S.H. Ross C.A. Sawa A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 289-294Crossref PubMed Scopus (342) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar, 6Liang Y. Yu W. Li Y. Yang Z. Yan X. Huang Q. Zhu X. J. Cell Biol. 2004; 164: 557-566Crossref PubMed Scopus (104) Google Scholar). To further understand the roles of Ndel1 in cytoskeletal dynamics, we performed a tandem affinity purification of Ndel1-interacting proteins. We isolated a novel Ndel1 molecular complex composed of the IF vimentin, the molecular motor dynein, the lissencephaly protein Lis1, and the cis-Golgi-associated protein αCOP. Ndel1 promotes the interaction between Lis1, αCOP, and the vimentin-dynein complex. The functional result of this complex is activation of dynein-mediated transport of vimentin. A loss of Ndel1 functions by RNA interference fails to incorporate Lis1/αCOP in the complex, reduces the transport of vimentin, and culminates in IF accumulations and altered neuritogenesis. Our findings reveal a novel regulatory mechanism of vimentin transport during neurite extension that may have implications in diseases featuring transport/trafficking defects and impaired regeneration. The cytoskeleton constitutes a highly organized structure formed by the interconnection of three filamentous networks (microtubules (MTs), 3The abbreviations used are: MT, microtubule; IF, intermediate filament; NF, neuronal IF; DISC1, Disrupted-in-Schizophrenia protein 1; GFP, green fluorescent protein; PBS, phosphate-buffered saline; RNAi, RNA interference; DIC, dynein intermediate; DHC, dynein heavy chain; COP, coat protein; RFP, red fluorescent protein. 3The abbreviations used are: MT, microtubule; IF, intermediate filament; NF, neuronal IF; DISC1, Disrupted-in-Schizophrenia protein 1; GFP, green fluorescent protein; PBS, phosphate-buffered saline; RNAi, RNA interference; DIC, dynein intermediate; DHC, dynein heavy chain; COP, coat protein; RFP, red fluorescent protein. intermediate filaments (IFs), and microfilament) and their associated proteins (7Chang L. Goldman R.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 601-613Crossref PubMed Scopus (301) Google Scholar, 8Dent E.W. Gertler F.B. Neuron. 2003; 40: 209-227Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar, 9Etienne-Manneville S. Traffic. 2004; 5: 470-477Crossref PubMed Scopus (273) Google Scholar, 10Guzik B.W. Goldstein L.S. Curr. Opin. Cell Biol. 2004; 16: 443-450Crossref PubMed Scopus (105) Google Scholar). The dynamics of these networks regulate essential intracellular functions such as transport and trafficking and, therefore, impact on cell division, morphology, and integrity (7Chang L. Goldman R.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 601-613Crossref PubMed Scopus (301) Google Scholar, 8Dent E.W. Gertler F.B. Neuron. 2003; 40: 209-227Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar, 9Etienne-Manneville S. Traffic. 2004; 5: 470-477Crossref PubMed Scopus (273) Google Scholar, 10Guzik B.W. Goldstein L.S. Curr. Opin. Cell Biol. 2004; 16: 443-450Crossref PubMed Scopus (105) Google Scholar). Named according to their intermediate diameter (10 nm), IFs constitute the largest family of cytoskeletal proteins. Up to 60 genes encoding for IFs are expressed in a tissue-specific and spatio-temporal manner in mammals (7Chang L. Goldman R.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 601-613Crossref PubMed Scopus (301) Google Scholar, 11Fuchs E. Cleveland D.W. Science. 1998; 279: 514-519Crossref PubMed Scopus (832) Google Scholar, 12Lariviere R.C. Julien J.P. J. Neurobiol. 2004; 58: 131-148Crossref PubMed Scopus (276) Google Scholar). After synthesis in the cytoplasm, a fraction of soluble IF proteins is rapidly incorporated into the polymeric filamentous network, referred to as the "insoluble" fraction (7Chang L. Goldman R.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 601-613Crossref PubMed Scopus (301) Google Scholar, 12Lariviere R.C. Julien J.P. J. Neurobiol. 2004; 58: 131-148Crossref PubMed Scopus (276) Google Scholar). In parallel, individual IF subunits, dimers, or small oligomers that constitute the "soluble" fraction translocate rapidly along MTs via molecular motors in structures termed particles (13Helfand B.T. Chang L. Goldman R.D. Annu. Rev. Cell Dev. Biol. 2003; 19: 445-467Crossref PubMed Scopus (85) Google Scholar, 14Helfand B.T. Chang L. Goldman R.D. J. Cell Sci. 2004; 117: 133-141Crossref PubMed Scopus (213) Google Scholar). This model is particularly relevant for the assembly of the IF vimentin (15Prahlad V. Yoon M. Moir R.D. Vale R.D. Goldman R.D. J. Cell Biol. 1998; 143: 159-170Crossref PubMed Scopus (271) Google Scholar, 16Helfand B.T. Mikami A. Vallee R.B. Goldman R.D. J. Cell Biol. 2002; 157: 795-806Crossref PubMed Scopus (142) Google Scholar) and more controversial in the case of neuronal IFs (NFs) (17Yan Y. Brown A. J. Neurosci. 2005; 25: 7014-7021Crossref PubMed Scopus (40) Google Scholar, 18Wang L. Ho C.L. Sun D. Liem R.K. Brown A. Nat. Cell Biol. 2000; 2: 137-141Crossref PubMed Scopus (268) Google Scholar, 19Shah J.V. Flanagan L.A. Janmey P.A. Leterrier J.F. Mol. Biol. Cell. 2000; 11: 3495-3508Crossref PubMed Scopus (147) Google Scholar). The neurofilament proteins (NF-L, NF-M, and NF-H) are the most abundant IFs in mature central nervous system neurons (12Lariviere R.C. Julien J.P. J. Neurobiol. 2004; 58: 131-148Crossref PubMed Scopus (276) Google Scholar). We recently demonstrated that Ndel1, the mammalian homologue of the Aspergillus nidulans NudE, a protein implicated in nuclear distribution during hyphal growth, binds directly to the key subunit NF-L, thereby regulating NF assembly (5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar). Ndel1 also interacts with the Disrupted-in-Schizophrenia protein 1 (DISC1) to regulate MT dynamics during neurite outgrowth in neuroblastoma PC-12 cells and during neuronal migration in the developing cortex. Interestingly, Ndel1 associates with the molecular motors dynein/dynactin (20Sasaki S. Shionoya A. Ishida M. Gambello M.J. Yingling J. Wynshaw-Boris A. Hirotsune S. Neuron. 2000; 28: 681-696Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 21Niethammer M. Smith D.S. Ayala R. Peng J. Ko J. Lee M.S. Morabito M. Tsai L.H. Neuron. 2000; 28: 697-711Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 22Wynshaw-Boris A. Gambello M.J. Genes Dev. 2001; 15: 639-651Crossref PubMed Scopus (143) Google Scholar), and numerous independent reports documented the dynein transport of the vimentin, an IF important for glial activation and neurite formation and exhibiting pro- and anti-regenerative properties following axonal injury (13Helfand B.T. Chang L. Goldman R.D. Annu. Rev. Cell Dev. Biol. 2003; 19: 445-467Crossref PubMed Scopus (85) Google Scholar, 14Helfand B.T. Chang L. Goldman R.D. J. Cell Sci. 2004; 117: 133-141Crossref PubMed Scopus (213) Google Scholar, 15Prahlad V. Yoon M. Moir R.D. Vale R.D. Goldman R.D. J. Cell Biol. 1998; 143: 159-170Crossref PubMed Scopus (271) Google Scholar, 16Helfand B.T. Mikami A. Vallee R.B. Goldman R.D. J. Cell Biol. 2002; 157: 795-806Crossref PubMed Scopus (142) Google Scholar, 23Boyne L.J. Fischer I. Shea T.B. Int. J. Dev. Neurosci. 1996; 14: 739-748Crossref PubMed Scopus (49) Google Scholar, 24Dubey M. Hoda S. Chan W.K. Pimenta A. Ortiz D.D. Shea T.B. J. Neurosci. Res. 2004; 78: 245-249Crossref PubMed Scopus (34) Google Scholar, 25Yoon M. Moir R.D. Prahlad V. Goldman R.D. J. Cell Biol. 1998; 143: 147-157Crossref PubMed Scopus (210) Google Scholar, 26Perlson E. Hanz S. Ben-Yaakov K. Segal-Ruder Y. Seger R. Fainzilber M. Neuron. 2005; 45: 715-726Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 27Shea T.B. Beermann M.L. Fischer I. J. Neurosci. Res. 1993; 36: 66-76Crossref PubMed Scopus (55) Google Scholar, 28Pekny M. Nilsson M. Glia. 2005; 50: 427-434Crossref PubMed Scopus (1246) Google Scholar, 29Pekny M. Prog. Brain Res. 2001; 132: 23-30Crossref PubMed Scopus (74) Google Scholar). Whether Ndel1 associates with the dynein-vimentin complex during neurite outgrowth and regulates vimentin dynamics remains elusive. Using a proteomic approach of tandem affinity purification, we now identify Ndel1 as a key component in the vimentindynein complex in cells and discover that this complex is formed during neurite extension. Nde11 participates in the formation of a quint-partite complex by promoting interaction between the lissencephaly protein Lis1, the vesicular protein αCOP, and the vimentin-dynein complex. The results of this complex are activation of dynein-mediated transport of vimentin and functional consequences on neurite outgrowth. Ndel1 Complex Purification—The purification procedure was performed as previously described (30Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (639) Google Scholar). Briefly, recombinant retroviruses expressing a bicistronic messenger RNA containing open reading frames of FLAG-HA-tagged human Ndel1 and interleukin-2 receptor-α were constructed and transduced into suspension HeLa cells. The infected HeLa cells were selected twice using anti-interleukin-2 receptor monoclonal antibody-conjugated magnetic beads, and the resulting FLAG-HA-Ndel1-stable cell lines were grown in suspension. Soluble extract was made from 40 liters of cells. The Ndel1 complex was tandem-purified in detergent-free buffers by using anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Sigma) and then anti-HA 12CA5 monoclonal antibody-conjugated beads. FLAG-HA double purified material was separated by 4–20% gradient SDS-PAGE and stained with Coomassie Blue. The various protein bands were excised and analyzed by mass spectrometry at the Harvard Medical School Taplin Biological Mass Spectrometry Facility. Western Blots and Immunoprecipitations—Total protein extracts of CAD cells were obtained by homogenization in SDS-urea β-mercaptoethanol (0.5% SDS, 8 m urea in 7.4 phosphate buffer). Soluble fractions were prepared in Triton X-100 (10 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA (pH 8.0), and 1% Triton), HEPES (50 mm HEPES, pH 7.0, 150 mm NaCl, 1 mm EDTA, 0.1% Nonidet P-40, 25 mm NaF, 10 mm Na3VO4 and 1 mm dithiothreitol) or E1A lysis buffer (50 mm Tris-HCl (pH 7.5), 250 mm NaCl, 5 mm EDTA (pH 8.0), and 0.1% Nonidet P-40) with a mixture of protease inhibitors (leupeptin, pepstatin, and aprotinin). The protein concentration was estimated by the Bradford procedure (Bio-Rad). Proteins (20–50 μg) were fractionated on 7.5% SDS-PAGE and blotted onto a nitrocellulose or polyvinylidene difluoride membrane for Western blot analysis. Membranes were incubated with antibodies against Ndel1 (210 and 211) (3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar), Lis1 (486) (21Niethammer M. Smith D.S. Ayala R. Peng J. Ko J. Lee M.S. Morabito M. Tsai L.H. Neuron. 2000; 28: 697-711Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar) dynein intermediate chain (MAB 1618, Chemicon), actin (C4, MAB1501, Chemicon), vimentin (Abcam, Chemicon), αCOP I (Abcam), p58 (Sigma-Aldrich), FAK (N-20, Santa Cruz Biotechnology), GFP (Molecular Probes), acetylated tubulin (Abcam), tyrosinated tubulin (Abcam), GAP-43 (Abcam), and DISC1 (a kind gift from Dr. Brandon, Wyeth). The Western blots were revealed by chemiluminescence (Renaissance, Western blot kit, PerkinElmer Life Sciences). Quantifications were corrected with levels of actin or α-tubulin and performed with the Labscan program (Image Master, two-dimensional software, v. 3.10, Amersham Biosciences). Yeast Two-hybrid Interactions—Cloning of human cDNA for full-length (amino acid residues 1–345) of Ndel1 in pPC97 vector (pDBLeu, Invitrogen) for expression as a GAL4 DNA binding domain fusion protein was described previously (5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar, 21Niethammer M. Smith D.S. Ayala R. Peng J. Ko J. Lee M.S. Morabito M. Tsai L.H. Neuron. 2000; 28: 697-711Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). pPC86-NFL rod construct for expression of NFL rod domain (77–364) as a GAL4 activation domain was a kind gift from Drs. Michael Ehlers (Duke University) and Richard Huganir (Johns Hopkins University). pPC86-vimentin, pPCα-COP, and pPC86-DISC1 fragment constructs for expression as a GAL4 activation domain was a kind gift from Dr. Wallace Ip (University of Cincinnati), Blanche Schwappach (University of Heidelberg), and Dr. Akira Sawa (Johns Hopkins University), respectively. Yeast two-hybrid assays were performed as described previously (5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar). Cell Culture, Immunofluorescence, and Live Imaging—Cultures of CAD cells were performed as described previously (3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar). Following transfection with Lipofectamine 2000 (Invitrogen) in serum-free medium, CAD cells stop proliferating and exhibit morphological characteristics and molecular signatures of primary neurons such as expression of class III β-tubulin and enzymatically active tyrosine hydroxylase (31Qi Y. Wang J.K. McMillian M. Chikaraishi D.M. J. Neurosci. 1997; 17: 1217-1225Crossref PubMed Google Scholar). After transfection, CAD cells were washed once in 37°c PBS and then fixed with 4% paraformaldehyde/PBS for 10 min at room temperature. After fixation, the cells were washed twice with PBS and then blocked with PBS, 5% skimmed milk (or 3% bovine serum albumin), 0.1% Triton X-100 for 1 h. After fixation, cells were incubated with primary antibody (vimentin, Ndel1, Tubulin) diluted in the blocking buffer for 1 h at 37°c or overnight in a cold room. Following three washes with PBS, secondary antibodies (Alexa 488, 594, Cy2, Cy3) diluted in blocking buffer were added to the cells for 1 h at 37 °C or 2 h at 25 °C. The cells were finally washed three times with PBS followed by a wash with water before mounting with Immuno-Mount. Pictures were taken with a C-1 eclipse Nikon confocal microscope. For live imaging, 24 h after plating, CAD cells were first transfected with a construct encoding a random sequence without homology to any known mRNA (control) or Ndel1 RNAi, and an RFP-centrin construct to mark the cells that have integrated the control or Ndel1 RNAi (see supplemental Fig. S2). After 24 h, when Ndel1 RNAi efficiently knocks down the levels of Ndel1 protein (3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar), the same cells were transfected a second time with a GFP-vimentin construct. 6–8 h after the second transfection, live imaging of GFP-vimentin dots (a pattern distinct of the soluble pool of IF subunits) was performed. A picture was taken every 30 s for a total of 10 min per cell; up to 56 cells were recorded during four independent experiments for a total of 190 vimentin dots. Note that beyond 10 h of post-transfection, the GFP-vimentin subunits were readily found in filamentous form (supplemental Fig. S2). To quantify the movements of GFP-vimentin, only uni-nuclear cells that expressed one RFP-centrin dot (one centrosome), had Ndel1 depletion, and expressed mobile GFP-vimentin dots (non filamentous) were taken. Clear and distinct moving vimentin dots were picked randomly and far apart from any GFP-vimentin aggregates to avoid eventual blockage of transport. Dots with no lateral, ascendant, and descendant movements were considered immobile. Generation, Characterization of siRNA Sequences, and RNAi Vectors—RNAi sequences were selected based on the criteria proposed previously (32Sui G. Soohoo C. Affar el B. Gay F. Shi Y. Forrester W.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar). Complementary hairpin sequences or oligonucleotides were commercially synthesized and cloned into pSilencer 2.0 under promoter U6 (Ambion). The sequence for Ndel1 comprises bp 276–294 (GCAGGTCTCAGTGTTAGAA) (3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar). A random sequence without homology to any known mRNA was used for control RNAi. All RNAi constructs were tested in 3T3, COS-7, and CAD cells by both Western blot and immunofluorescent staining. Sucrose and Glycerol Gradients—High speed supernatants were made from spinal cord and nerve homogenates of 4-month-old wild-type littermates or cell lysates in buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA) or a modified version of radioimmune precipitation assay buffer without detergents plus protease inhibitors and fractionated on sucrose gradients. 200 μl of homogenates was overlaid onto a 12-ml, 5–20% sucrose gradient and centrifuged at 32,000 × g for 18 h at 4 °C in TiSW41 swing buckets in a Beckman L8–70M Ultra-centrifuge. Sucrose gradients were previously made in Beckman Ultra-Clear tubes using a gradient maker Hoeffer 15 as described by the manufacturer's protocol (Amersham Biosciences). During all centrifugations, gradients loaded with standards (1 mg) such as bovine serum albumin, catalase, or thyroglobulin were processed simultaneously. Following centrifugation, 12 fractions of 1 ml were collected for each sample. Glycerol gradients were performed according to a previous study (3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Novel Ndel1 Interacting Partners in Cells—A proteomicbased tandem affinity purification for Ndel1 was employed to determine fine molecular complexes involved in cytoskeletal dynamics and rearrangement. Briefly, Ndel1 was tagged at the N terminus with two affinity tags (FLAG and HA epitopes) and stably expressed in HeLa cells in suspension. Exogenous Ndel1 was expressed at a physiological level, i.e. 1.2-fold the level of endogenous Ndel1 (data not shown). Following several detergent-free fractionations and co-immunoprecipitations (see "Experimental Procedures" and Ref. 30Shi Y. Sawada J. Sui G. Affar el B. Whetstine J.R. Lan F. Ogawa H. Luke M.P. Nakatani Y. Nature. 2003; 422: 735-738Crossref PubMed Scopus (639) Google Scholar), the soluble cytoplasmic fraction was isolated and Ndel1 interacting proteins were identified by mass spectrometry. From the screen, some of the proteins recovered were: endogenous Ndel1, dynein intermediate (DIC), and heavy chains (DHC), the lissencephaly gene product, Lis1, the IF vimentin, and the trafficking-associated protein αCOP (Fig. 1A). In agreement with previous studies (3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 21Niethammer M. Smith D.S. Ayala R. Peng J. Ko J. Lee M.S. Morabito M. Tsai L.H. Neuron. 2000; 28: 697-711Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 22Wynshaw-Boris A. Gambello M.J. Genes Dev. 2001; 15: 639-651Crossref PubMed Scopus (143) Google Scholar, 33Sasaki S. Mori D. Toyo-oka K. Chen A. Garrett-Beal L. Muramatsu M. Miyagawa S. Hiraiwa N. Yoshiki A. Wynshaw-Boris A. Hirotsune S. Mol. Cell. Biol. 2005; 25: 7812-7827Crossref PubMed Scopus (142) Google Scholar, 34Tarricone C. Perrina F. Monzani S. Massimiliano L. Kim M.H. Derewenda Z.S. Knapp S. Tsai L.H. Musacchio A. Neuron. 2004; 44: 809-821Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), our results indicated that Ndel1 can form a complex with itself, DIC, DHC, and Lis1. Remarkably, we also uncovered that Ndel1 is present in complexes with vimentin, an IF important for neurite extension with pro- and anti-regeneration properties (7Chang L. Goldman R.D. Nat. Rev. Mol. Cell Biol. 2004; 5: 601-613Crossref PubMed Scopus (301) Google Scholar, 23Boyne L.J. Fischer I. Shea T.B. Int. J. Dev. Neurosci. 1996; 14: 739-748Crossref PubMed Scopus (49) Google Scholar, 24Dubey M. Hoda S. Chan W.K. Pimenta A. Ortiz D.D. Shea T.B. J. Neurosci. Res. 2004; 78: 245-249Crossref PubMed Scopus (34) Google Scholar, 26Perlson E. Hanz S. Ben-Yaakov K. Segal-Ruder Y. Seger R. Fainzilber M. Neuron. 2005; 45: 715-726Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 28Pekny M. Nilsson M. Glia. 2005; 50: 427-434Crossref PubMed Scopus (1246) Google Scholar, 29Pekny M. Prog. Brain Res. 2001; 132: 23-30Crossref PubMed Scopus (74) Google Scholar, 35Wilhelmsson U. Li L. Pekna M. Berthold C.H. Blom S. Eliasson C. Renner O. Bushong E. Ellisman M. Morgan T.E. Pekny M. J. Neurosci. 2004; 24: 5016-5021Crossref PubMed Scopus (347) Google Scholar, 36DePianto D. Coulombe P.A. Exp. Cell Res. 2004; 301: 68-76Crossref PubMed Scopus (75) Google Scholar), as well as with αCOP, a subunit of the non-clathrin-coated vesicular coat proteins (COPs) coatomer COP I, which is essential for the retrieval and transport of vesicles from the cis-Golgi back to the endoplasmic reticulum (37Lee M.C. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (704) Google Scholar, 38McMahon H.T. Mills I.G. Curr. Opin. Cell Biol. 2004; 16: 379-391Crossref PubMed Scopus (234) Google Scholar). Because the soluble fraction was isolated, the IFs obtained were likely in a non-filamentous form, and αCOP was not membrane-associated. It is noteworthy that, under our experimental conditions, Erk and importins, which have been shown to associate with dynein and vimentin (26Perlson E. Hanz S. Ben-Yaakov K. Segal-Ruder Y. Seger R. Fainzilber M. Neuron. 2005; 45: 715-726Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), were not found in the tandem affinity purification. Neither were other Ndel1-interacting partners such as Fez-1 and DISC1 (1Kamiya A. Kubo K. Tomoda T. Takaki M. Youn R. Ozeki Y. Sawamura N. Park U. Kudo C. Okawa M. Ross C.A. Hatten M.E. Nakajima K. Sawa A. Nat. Cell Biol. 2005; 7: 1167-1178Crossref PubMed Scopus (445) Google Scholar, 2Kamiya A. Tomoda T. Chang J. Takaki M. Zhan C. Morita M. Cascio M.B. Elashvili S. Koizumi H. Takanezawa Y. Dickerson F. Yolken R. Arai H. Sawa A. Hum. Mol. Genet. 2006; 15: 3313-3323Crossref PubMed Scopus (142) Google Scholar, 39Mackie S. Millar J.K. Porteous D.J. Curr. Opin. Neurobiol. 2007; 17: 95-102Crossref PubMed Scopus (86) Google Scholar, 40Morris J.A. Kandpal G. Ma L. Austin C.P. Hum. Mol. Genet. 2003; 12: 1591-1608Crossref PubMed Scopus (331) Google Scholar). Ndel1 Interacts Directly with Lis1 but Not with vimentin and αCOP in the Yeast Two-hybrid System—To further understand the interactions between members of the complex, we used the yeast two-hybrid system. In the assay, the association between Ndel1 and Lis1 is direct while the interactions between Ndel1 and vimentin, Ndel1 and αCOP, and αCOP and vimentin were not direct (Fig. 1B). Neurofilament light chain (NF-L) and a Ndel1-interacting fragment of DISC1 (c1), which have been reported to interact directly with Ndel1 in the same system (4Ozeki Y. Tomoda T. Kleiderlein J. Kamiya A. Bord L. Fujii K. Okawa M. Yamada N. Hatten M.E. Snyder S.H. Ross C.A. Sawa A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 289-294Crossref PubMed Scopus (342) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar) (Fig. 1B), were used as positive controls. Because Lis1 is known to interact directly with dynein (3,20–22,41), which transports vimentin cargo (16Helfand B.T. Mikami A. Vallee R.B. Goldman R.D. J. Cell Biol. 2002; 157: 795-806Crossref PubMed Scopus (142) Google Scholar, 26Perlson E. Hanz S. Ben-Yaakov K. Segal-Ruder Y. Seger R. Fainzilber M. Neuron. 2005; 45: 715-726Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), Ndel1 may associate with vimentin via Lis1/dynein. It is noteworthy that we cannot rule out the possibility that Ndel1 and vimentin, Ndel1 and αCOP, and αCOP and vimentin may interact directly in other systems. Formation of the Novel Ndel1 Complex in Neuronal Cells Extending Neurites—To validate the existence of the quint-partite complex Ndel1-vimentin-dynein-Lis1-αCOP in cells, we performed additional co-immunoprecipitations on soluble lysates of differentiating neuroblastoma CAD cells (see "Experimental Procedures") transfected with a random RNAi sequence (used as a control) and undergoing neurite outgrowth. Lis1 and αCOP co-immunoprecipitated with Ndel1 in differentiating CAD cells (Fig. 2A). Furthermore, vimentin, Lis1, Ndel1, and αCOP also co-immunoprecipitated with DIC (Fig. 2B). Finally, Ndel1, Lis1, and vimentin co-immunoprecipitated with αCOP (Fig. 2C). These results confirmed the existence of the quint-partite complex in neuronal cells undergoing neurite outgrowth. To define the role of Ndel1 in the formation of the complex during neurite outgrowth, we knocked down the levels of Ndel1 protein in CAD cells extending neurite using a specific RNAi sequence (3Shu T. Ayala R. Nguyen M.D. Xie Z. Gleeson J.G. Tsai L.H. Neuron. 2004; 44: 263-277Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 5Nguyen M.D. Shu T. Sanada K. Lariviere R.C. Tseng H.C. Park S.K. Julien J.P. Tsai L.H. Nat. Cell Biol. 2004; 6: 595-608Crossref PubMed Scopus (82) Google Scholar) and assessed the interactions between vim
Referência(s)