Cytoplasmic dynein has been implicated in numerous aspects of intracellular movement. We recently found dynein inhibitors to interfere with the reorientation of the microtubule cytoskeleton during healing of wounded NIH3T3 cell monolayers. We now find that dynein and its regulators dynactin and LIS1 localize to the leading cell cortex during this process. In the presence of serum, bright diffuse staining was observed in regions of active ruffling. This pattern was abolished by cytochalasin D, and was not observed in cells treated with lysophosphatidic acid, conditions which allow microtubule reorientation but not forward cell movement. Under the same conditions, using total internal reflection fluorescence microscopy, clear punctate dynein/dynactin containing structures were observed along the sides and at the tips of microtubules at the leading edge. Overexpression of dominant negative dynactin and LIS1 cDNAs or injection of antidynein antibody interfered with the rate of cell migration. Together, these results implicate a leading edge cortical pool of dynein in both early and persistent steps in directed cell movement.
A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.
Heat shock transcription factors (HSFs) maintain protein homeostasis through regulating expression of heat shock proteins, especially in stressed conditions. In addition, HSFs are involved in cellular differentiation and development by regulating development-related genes, as well as heat shock genes. Here, we showed chronic sinusitis and mild hydrocephalus in postnatal HSF1-null mice, which are associated with impaired mucociliary clearance and cerebrospinal flow, respectively. Analysis of ciliary beating revealed that the amplitude of the beating was significantly reduced, and ciliary beat frequencies were lower in the respiratory epithelium, ependymal cells, oviduct, and trachea of HSF1-null mice than those of wild-type mice. Cilia possess a common axonema structure composed of microtubules of α- and β-tubulin. We found a marked reduction in α- and ciliary βiv-tubulin in the HSF1-null cilia, which is developmentally associated with reduced Hsp90 expression in HSF1-null mice. Treatment of the respiratory epithelium with geldanamycin resulted in rapid reduction of ciliary beating in a dose-dependent manner. Furthermore, Hsp90 was physically associated with ciliary βiv-tubulin, and Hsp90 stabilizes tubulin polymerization in vitro. These results indicate that HSF1 is required to maintain ciliary beating in postnatal mice, probably by regulating constitutive expression of Hsp90 that is important for tubulin polymerization. Heat shock transcription factors (HSFs) maintain protein homeostasis through regulating expression of heat shock proteins, especially in stressed conditions. In addition, HSFs are involved in cellular differentiation and development by regulating development-related genes, as well as heat shock genes. Here, we showed chronic sinusitis and mild hydrocephalus in postnatal HSF1-null mice, which are associated with impaired mucociliary clearance and cerebrospinal flow, respectively. Analysis of ciliary beating revealed that the amplitude of the beating was significantly reduced, and ciliary beat frequencies were lower in the respiratory epithelium, ependymal cells, oviduct, and trachea of HSF1-null mice than those of wild-type mice. Cilia possess a common axonema structure composed of microtubules of α- and β-tubulin. We found a marked reduction in α- and ciliary βiv-tubulin in the HSF1-null cilia, which is developmentally associated with reduced Hsp90 expression in HSF1-null mice. Treatment of the respiratory epithelium with geldanamycin resulted in rapid reduction of ciliary beating in a dose-dependent manner. Furthermore, Hsp90 was physically associated with ciliary βiv-tubulin, and Hsp90 stabilizes tubulin polymerization in vitro. These results indicate that HSF1 is required to maintain ciliary beating in postnatal mice, probably by regulating constitutive expression of Hsp90 that is important for tubulin polymerization. Heat shock response is characterized by induction of a set of heat shock proteins (Hsps) 2The abbreviations used are:Hspheat shock proteinCBFciliary beat frequencyHSFheat shock transcription factorPBSphosphate-buffered salineGFPgreen fluorescent proteinBSAbovine serum albuminDH2dynein heavy chain 2LIC3dynein light intermediate chain 3. and is a fundamental adoptive response in all organisms from bacteria to humans. This response is regulated mostly at the level of transcription by heat shock transcription factors that bind to the heat shock element in eukaryotes (1Wu C. Annu. Rev. Cell Biol. 1995; 11: 441-469Crossref Scopus (991) Google Scholar, 2Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1556) Google Scholar). Among four HSF family members (HSF1-4), HSF1 plays a key role in heat shock response in mammals, whereas HSF3 does so in avians (3Nakai A. Cell Stress Chaperones. 1999; 4: 86-93Crossref PubMed Scopus (82) Google Scholar, 4Pirkkala L. Nykanen N. Sistonen L. FASEB J. 2001; 15: 1118-1131Crossref PubMed Scopus (842) Google Scholar). This HSF-mediated induction of Hsps is required for acquisition of thermotolerance (5Tanabe M. Kawazoe Y. Takeda S. Morimoto R.I. Nagata K. Nakai A. EMBO J. 1998; 17: 1750-1758Crossref PubMed Scopus (87) Google Scholar, 6McMillan D.R. Xiao X. Shao L. Graves K. Benjamin I.J. J. Biol. Chem. 1998; 273: 7523-7528Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 7Inouye S. Katsuki K. Izu H. Fujimoto M. Sugahara K. Yamada S. Shinkai Y. Oka Y. Katoh Y. Nakai A. Mol. Cell. Biol. 2003; 23: 5882-5895Crossref PubMed Scopus (69) Google Scholar) and protection of cells from various pathophysiological conditions (8Zou Y. Zhu W. Sakamoto M. Qin Y. Akazawa H. Toko H. Mizukami M. Takeda N. Minamino T. Takano H. Nagai T. Nakai A. Komuro I. Circulation. 2003; 108: 3024-3030Crossref PubMed Scopus (71) Google Scholar, 9Wirth D. Christians E. Li X. Benjamin I.J. Gustin P. Toxicol. Appl. 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In addition to the role in heat shock response, HSFs play critical functions in developmental processes such as gamatogenesis and neurogenesis (15Jedlicka P. Mortin M.A. Wu C. EMBO J. 1997; 16: 2452-2462Crossref PubMed Scopus (240) Google Scholar, 16Christians E. Davis A.A. Thomas S.D. Benjamin I.J. Nature. 2000; 407: 693-694Crossref PubMed Scopus (235) Google Scholar, 17Kallio M. Chang Y. Manuel M. Alastalo T.P. Rallu M. Gitton Y. Pirkkala L. Loones M.T. Paslaru L. Larney S. Hiard S. Morange M. Sistonen L. Mezger V. EMBO J. 2002; 21: 2591-2601Crossref PubMed Scopus (141) Google Scholar, 18Wang G. Ying Z. Jin X. Tu N. Zhang Y. Phillips M. Moskophidis D. Mivechi N.F. Genesis. 2004; 38: 66-80Crossref PubMed Scopus (94) Google Scholar, 19Chang Y. Ostling P. Akerfelt M. Trouillet D. Rallu M. Gitton Y. El Fatimy R. Fardeau V. Crom Le S. Morange M. Sistonen L. Mezger V. Genes Dev. 2006; 20: 836-847Crossref PubMed Scopus (72) Google Scholar, 20Santos S.D. Saraiva M.J. Neuroscience. 2004; 126: 657-663Crossref PubMed Scopus (55) Google Scholar), in maintenance of the sensory organs (21Bu L. Jin Y. Shi Y. Chu R. Ban A. Eiberg H. Andres L. Jiang H. Zheng G. Qian M. Cui B. Xia Y. Liu J. Hu L. Zhao G. Hayden M.R. Kong X. Nat. Genet. 2002; 31: 276-278Crossref PubMed Scopus (245) Google Scholar, 22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (198) Google Scholar, 23Min J.N. Zhang Y. Moskophidis D. Mivechi N.F. Genesis. 2004; 40: 205-217Crossref PubMed Scopus (102) Google Scholar, 24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and in immune response (25Inouye S. Izu H. Takaki E. Suzuki H. Shirai M. Yokota Y. Ichikawa H. Fujimoto M. Nakai A. J. Biol. Chem. 2004; 279: 38701-38709Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 26Zheng H. Li Z. J. Immunol. 2004; 173: 5929-5933Crossref PubMed Scopus (45) Google Scholar). Although the precise mechanisms of how HSFs act in these physiological processes are still unclear, genetic evidence shows that HSFs regulate constitutive gene expression in unstressed cells and tissues (27Nakai A. Ishikawa T. EMBO J. 2001; 20: 2885-2895Crossref PubMed Scopus (72) Google Scholar, 28Yan L.J. Christians E.S. Liu L. Xiao X. Sohal R.S. Benjamin I.J. EMBO J. 2002; 21: 5164-5172Crossref PubMed Scopus (197) Google Scholar). Furthermore, it was revealed in sensory and immune cells that HSFs not only maintain protein homeostasis by regulating constitutive expression of Hsps, but are also involved in cell growth and differentiation by regulating expression of cytokines such as interleukin-6, fibroblast growth factors, and LIF (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (198) Google Scholar, 24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 25Inouye S. Izu H. Takaki E. Suzuki H. Shirai M. Yokota Y. Ichikawa H. Fujimoto M. Nakai A. J. Biol. Chem. 2004; 279: 38701-38709Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). However, we do not know any client protein stabilized by Hsps that is under the control of HSFs in unstressed conditions. heat shock protein ciliary beat frequency heat shock transcription factor phosphate-buffered saline green fluorescent protein bovine serum albumin dynein heavy chain 2 dynein light intermediate chain 3. We previously showed abnormal nasal cavities in HSF1-null adult mice, which is associated with atrophy of the olfactory epithelium and sinusitis characterized by accumulation of mucus (24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and demonstrated that HSF1 is required to maintain olfactory neurogenesis. However, the mucus accumulation cannot be explained by this function of HSF1. Here, we examined the molecular mechanisms underlining mucus accumulation in the HSF1-null nasal cavity, and demonstrated that HSF1 is required to maintain ciliary beating in many organs, probably by regulating constitutive expression of Hsp90 that facilitates tubulin polymerization. This is the first demonstration that HSF1 plays a role in maintaining dynamic movement of a microstructure in cells. Histopathology and Immunohistochemistry—HSF1-null (7Inouye S. Katsuki K. Izu H. Fujimoto M. Sugahara K. Yamada S. Shinkai Y. Oka Y. Katoh Y. Nakai A. Mol. Cell. Biol. 2003; 23: 5882-5895Crossref PubMed Scopus (69) Google Scholar) mice were maintained by crossing with ICR mice. Mice were systemically anesthetized with ketamine (16 mg/kg, intraperitoneal) and xylazine (16 mg/kg, intraperitoneal), and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS). Tissues were removed and soaked in 4% paraformaldehyde for 12 h at 4 °C. After washing with PBS, the blocks were dehydrated, embedded in paraffin, and cut into sections 5-μm thick. To prepare nasal sections, the fixed tissues were incubated in K-CX decalcification solution (Fujisawa, Osaka, Japan) for 24 h, and then neutralized in 5% Na2SO4 for 24 h. The sections were stained with hematoxylin and eosin. To identify mucus, sections were stained with periodic acid-Schiff and counterstained with hematoxylin. Immunostaining of the paraffin sections was performed as described previously (12Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar). Antibodies used were antisera for mouse Hsp110 (αHsp110a), human Hsp90 (αHsp90c), human Hsp70 (αHsp70-1), human Hsp40 (αHsp40-1) (24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 29Fujimoto M. Takaki E. Hayashi T. Kitaura Y. Tanaka Y. Inouye S. Nakai A. J. Biol. Chem. 2005; 280: 34908-34916Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), mouse Hsp27 (αmHsp27c), mouse TCP-1α (αmTCP-1α) (both were generated by immunizing rabbits with recombinant mouse Hsp27 or TCP-1α), DH2 and LIC3 (30Mikami A. Tynan S.H. Hama T. Luby-Phelps K. Saito T. Crandall J.E. Besharse J.C. Vallee R.B. J. Cell Sci. 2002; 115: 4801-4808Crossref PubMed Scopus (88) Google Scholar), or monoclonal antibodies for α-tubulin (fluorescein isothiocyanate-conjugated mouse IgG, F2168, Sigma) and βiv-tubulin (T7941, Sigma). Peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG was used as a second antibody. Signals were detected using a DAB substrate kit (Vector Laboratories, Inc.). Sections were counterstained with methyl green. To perform double staining, fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Cappel) and Alexa Fluor 568-conjugated anti-mouse IgG (Invitrogen) were used as second antibodies, and the sections were mounted in a Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories), and visualized using fluorescence microscopy (Axioplan 2, Zeiss). Estimation of Mucociliary Clearance—Analysis of the nasal mucociliary clearance was performed as described previously with some modifications (31Foster W.M. Walters D.M. Longphre M. Macri K. Miller L.M. J. Appl. Physiol. 2001; 90: 1111-1117Crossref PubMed Scopus (128) Google Scholar). Pelican ink (5 μl) was injected into the nasal vestibules (a region 5 mm from the naris) using a micropipette with a fine catheter. The nose was divided into two through nasal septum, and the clearance of ink was examined. Assay of the Cerebrospinal Fluid Flow—The flow of cerebrospinal fluid was examined as described previously with modifications (32Sawamoto K. Wichterle H. Gonzalez-Perez O. Cholfin J.A. Yamada M. Spassky N. Murcia N.S. Garcia-Verdugo J.M. Marin O. Rubenstein J.L. Tessier-Lavigne M. Okano H. Alvarez-Buylla A. Science. 2006; 311: 629-632Crossref PubMed Scopus (642) Google Scholar). The lateral wall of the lateral ventricle was dissected using a fine scalpel and forceps, and immediately soaked in a culture dish containing PBS at room temperature. For visualization of the flow, a small amount of pelican ink was placed on the surface of the lateral wall of the dissected ventricle. Movement of pelican ink was observed with a stereomicroscope (MZ6, Leica), and recorded with a digital camera and software (PowerShot S50, Canon). Analysis of Ciliary Movement—The nasal mucosa, trachea, oviducts, and inner surface of the lateral ventricle were removed from anesthetized mice, cut into small pieces (∼5mm square blocks), and suspended in control solution (121 mm NaCl, 4.5 mm KCl, 1 mm CaCl2, 1.5 mm NaHCO3, 1.5 mm NaHepes, 5 mm HHepes, and 5 mm glucose) at 4 °C. Each tissue block was cut into thin pieces by two adherent razor blades, and was placed on a coverslip precoated with Cell-Tak (Becton Dickinson Labware, Bedford, MA) to adhere slices firmly on the coverslip. The coverslip with slices was set in the perfusion chamber, the volume of which was ∼20 μl, and the rate of perfusion 200 μl/min (33Kawakami M. Nagira T. Hayashi T. Shimamoto C. Kubota T. Mori H. Yoshida H. Nakahari T. Exp. Physiol. 2004; 89: 739-751Crossref PubMed Scopus (19) Google Scholar, 34Hayashi T. Kawakami M. Sasaki S. Katsumata T. Mori H. Yoshida H. Nakahari T. Exp. Physiol. 2005; 90: 535-544Crossref PubMed Scopus (61) Google Scholar). The chamber was mounted on an differential interference contrast microscope (E600-FN, Nikon, Tokyo, Japan), which was connected to a high-speed digital video camera (FASTCAM 512PCI, Photoron, Tokyo). The sampling rate of the high-speed camera was 500 Hz. The ciliary beat frequency (CBF) of each tissue in a slice preparation was calculated from the time for 10 beating cycles (33Kawakami M. Nagira T. Hayashi T. Shimamoto C. Kubota T. Mori H. Yoshida H. Nakahari T. Exp. Physiol. 2004; 89: 739-751Crossref PubMed Scopus (19) Google Scholar, 34Hayashi T. Kawakami M. Sasaki S. Katsumata T. Mori H. Yoshida H. Nakahari T. Exp. Physiol. 2005; 90: 535-544Crossref PubMed Scopus (61) Google Scholar). Transmission Electron Microscopy—Mice were systemically anesthetized and transcardially perfused with a fixative (2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 m cacodylate buffer, pH 7.4). Blocks of bone containing paranasal sinuses and trachea were removed and soaked in the fixative for 16 h. Samples were then incubated for 10 days in a decalcification solution by changing the solution every day. Transmission electron microscopy was performed as described previously (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (198) Google Scholar). Reverse Transcriptase-PCR Analysis—Total RNA was isolated from dissected tissues by using TRIzol reagent (Invitrogen). cDNAs were synthesized as described previously (24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Twenty-five to 35 cycles of PCR were performed using 1 μl from each reverse transcription product. Primers used for amplifying tubulin genes were: tuba-F, 5′-agaattccagaccaacctgg-3′; tuba-R, 5′-gtgttgctcagcatgcacac-3′; tubb3-F, 5′-attggcaacagcacg-3′; tubb3-R, 5′-tcacttgggcccctg-3′; tubb4-F; 5′-tcggagcagttcacc-3′; and tubb4-R, 5′-ttaagccacctcctct-3′. The primers tuba-F and tuba-R can amplify all α-tubulin genes (α1 to α8) (Mouse Genome Informatics). The S16 ribosomal protein gene was amplified as a control (35Tanabe M. Sasai N. Nagata K. Liu X.D. Liu P.C. Thiele D.J. Nakai A. J. Biol. Chem. 1999; 274: 27845-27856Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The amplified DNA was stained with ethidium bromide and photographed using an Epi-Light UV FA1100 (Aisin Cosmos R&D Co., Japan). Western Blot Analysis—Whole cell extracts from tissues were prepared and Western blot analysis was performed as described previously (24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Chromatin Immunoprecipitation—Mice were systemically anesthetized as described above. Tissues were dissected and immediately soaked in 1% formaldehyde in PBS at 37 °C for 10 min. Chromatin immunoprecipitation was performed using a chromatin immunoprecipitation assay kit (Upstate, New York) essentially according to the manufacturer's instructions (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (198) Google Scholar). Thirty-three cycles of PCR were performed to amplify a DNA fragment of the mouse Hsp90α (Hsp86) gene (-209 to +56 from a transcription start site) containing a canonical heat shock element (36Dale E.C. Yang X. Moore S.K. Shyamala G. Cell Stress Chaperones. 1997; 2: 87-93Crossref PubMed Scopus (16) Google Scholar). Primers used to amplify chromatin immunoprecipitation-enriched DNA were: Hsp90-CHIP-5, 5′-GCTGTGGAGGAGGGGCTTGCGTTCGTT-3′, and Hsp90-CHIP-3, 5′-GTGGCTGAATGAACACGCACGAGACGTGA-3′. Immunoprecipitation—HEK293 cells were plated in 100-mm dishes for 16 h, then transfected with an expression vector pEGFP2, pGFP2-βiv-tubulin, or pGFP2-βiii-tubulin (20 μg) by the calcium phosphate method as described previously (14Hayashida N. Inouye S. Fujimoto M. Tanaka Y. Izu H. Takaki E. Ichikawa H. Rho J. Nakai A. EMBO J. 2006; 25: 4773-4783Crossref PubMed Scopus (89) Google Scholar). At 4 h after the transfection, cells were washed with PBS and incubated further for 44 h in normal medium. Cells were then washed with PBS, harvested, suspended in five packed cell pellet volumes of RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 50 mm Tris (pH 7.5), 0.5 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin) for 10 min on ice. The supernatants were collected after centrifugation at 15,000 × g for 10 min, the 1 μl of preimmune serum or antiserum for Hsp90 (αhHsp90c) was added at 4 °C overnight, and mixed with 40 μl of protein A-Sepharose beads (1:1 suspension in PBS) (Amersham Biosciences) by rotating at 4 °C for 1 h in the presence or absence of 1 μm geldanamycin. The complexes were washed five times with RIPA buffer, suspended in SDS sample buffer, and boiled for 3 min. The samples were loaded on SDS-PAGE, and transferred onto nitrocellulose membranes. The membranes were immunoblotted using a mouse monoclonal antibody for GFP (GF200, Nacalai Tesque, Kyoto, Japan). Alternatively, immunoprecipitation was performed using antibody for GFP, and immunoblot analysis was performed using antiserum for Hsp90. In Vitro Tubulin Polymerization Assay—Hsp90 was purified from mouse L5178Y cells as described previously (37Miyata Y. Yahara I. Biochemistry. 1995; 34: 8123-8129Crossref PubMed Scopus (104) Google Scholar), and stored in a buffer containing 50 mm Tris-HCl, 100 mm NaCl, 2 mm EDTA, 1 mm dithiothreitol, 10% glycerol at -80 °C until use. A tubulin polymerization assay was performed according to the manufacturer's instructions for a fluorescence-based tubulin polymerization assay kit (BK011, Cytoskeleton, Inc.). Briefly, a tubulin reaction mixture (50 μl) containing tubulin and a fluorescent reporter was mixed with bovine serum albumin (BSA, Fraction V, Nacalai Tesque), Hsp90 (5 μl), or paclitaxel that promotes microtubule polymerization and stability (38Schiff P.B. Fant J. Horwitz S.B. Nature. 1979; 277: 665-667Crossref PubMed Scopus (3198) Google Scholar) and incubated at 37 °C until 40 min. Fluorescence emission at 460 nm (excitation wavelength is 360 nm) was measured by using a CytoFluor II Fluorescence Multi-well Plate Reader (Per-Septive Biosystems, Inc.). Statistical Analysis—Significant values were determined by analyzing data with the Mann-Whitney's U test using StatView version 4.5J for Macintosh (Abacus Concepts, Berkley, CA). A level of p < 0.05 was considered significant. Mucus Accumulation in the Nasal Cavity and Hydrocephalus in HSF1-null Mice—Previously, we showed abnormal nasal cavity in HSF1-null adult mice, which is associated with atrophy of the olfactory epithelium and the accumulation of mucus (Fig. 1A, a and d) (24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). This revealed that HSF1 is required for maintenance of olfactory neurogenesis. However, the mucus accumulation cannot be explained by this function of HSF1. Furthermore, the accumulation of mucus was not ameliorated in mice deficient for both HSF1 and HSF4, whereas the atrophied olfactory epithelium was partially restored in the same mice (24Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). We examined the mucociliary clearance in the nasal cavity by monitoring movement of Pelican ink (31Foster W.M. Walters D.M. Longphre M. Macri K. Miller L.M. J. Appl. Physiol. 2001; 90: 1111-1117Crossref PubMed Scopus (128) Google Scholar). The ink injected into the nasal vestibules was cleared after 1 h, and moved to the posterior turbinates (Fig. 1A, b and c). In contrast, the ink was hardly removed from the vestibules of HSF1-null mice even after 3 h (Fig. 1A, e and f), indicating that the mucociliary clearance was severely impaired in HSF1-null mice. Therefore, HSF1 may have a direct role in mucociliary clearance in the nasal cavity. The whipping movement of the cilia generates moving force of the mucus in the respiratory epithelium. As cilia are present in many organs in the body, any dysfunction caused by genetic mutations of their components cause many phenotypes known as immotile ciliary syndrome or primary ciliary dyskinesia (39Ibanez-Tallon I. Heintz N. Omran H. Hum. Mol. Genet. 2003; 12: R27-R35Crossref PubMed Google Scholar, 40Afzelius B.A. Mossberg B. Scriver C.R. Braudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1994: 3943-3954Google Scholar). Therefore, we examined the morphology of other organs such as the tracheae, oviducts, and ventricles. Histological examination showed grossly normal morphology in the tracheae and oviducts in 6-week-old HSF1-null mice. However, we noticed hydrocephalus in HSF1-null 6-week-old mice (Fig. 1B). All of the ventricles, including the aqueduct and fourth ventricle, were enlarged, indicating communicating hydrocephalus. Hydrocephalus was observed even in 3-week-old HSF1-null mice, but not in 2-week-old mice (data not shown). Degeneration of neural cells was not found in the brains of 6-week-old HSF1-null mice (data not shown) (20Santos S.D. Saraiva M.J. Neuroscience. 2004; 126: 657-663Crossref PubMed Scopus (55) Google Scholar). One possible reason for this phenotype may be impaired flow of cerebrospinal fluid (41Ibanez-Tallon I. Pagenstecher A. Fliegauf M. Olbrich H. Kispert A. Ketelsen U.P. North A. Heintz N. Omran H. Hum. Mol. Genet. 2004; 13: 2133-2141Crossref PubMed Scopus (284) Google Scholar). Therefore, we examined the ependymal flow in isolated ventricles by dropping a small amount of Pelican ink onto the exposed surfaces of dissected walls of the lateral ventricles (Fig. 1C). The ink moved along the expected cerebrospinal fluid flow on the wall in wild-type mice, but stayed in the same place and hardly moved in HSF1-null mice. These results indicate that the ependymal flow is impaired in HSF1-null mice, and this is consistent with the notion of ciliary dysfunction in both the respiratory epithelium and ependymal cells. Ciliary Beating Is Severely Impaired in HSF1-null Mice—To examine whether or not ciliary movement is impaired in HSF1-null mice, we monitored ciliary beating in the freshly isolated respiratory epithelium of the nasal cavity. The differential interference contrast image showed beating cilia located on the surface of respiratory cells, and each cilium movement was detected by video (supplementary Movies 1 and 2) (33Kawakami M. Nagira T. Hayashi T. Shimamoto C. Kubota T. Mori H. Yoshida H. Nakahari T. Exp. Physiol. 2004; 89: 739-751Crossref PubMed Scopus (19) Google Scholar, 34Hayashi T. Kawakami M. Sasaki S. Katsumata T. Mori H. Yoshida H. Nakahari T. Exp. Physiol. 2005; 90: 535-544Crossref PubMed Scopus (61) Google Scholar). In wild-type mice, the whipping movement of the cilia was observed and CBF was 13.27 ± 1.53 Hz (Fig. 2A). In marked contrast, the beating amplitude was significantly low, and CBF reduced to 8.70 ± 1.72 Hz in HSF1-null mice. Similarly, CBF in the ventricles of wild-type mice was 18.65 ± 2.54 Hz, whereas that of HSF1-null mice was 8.60 ± 1.82 Hz. Interestingly, the whipping movement of the cilia was markedly impaired even in the tracheae and oviducts (data not shown), and CBF was decreased from 14.38 ± 0.90 and 10.90 ± 0.96 Hz to 11.50 ± 1.82 and 7.52 ± 0.32 Hz in the HSF1-null tracheae and oviducts, respectively (Fig. 2A). These results indicate that HSF1 deficiency causes severe impairment of ciliary beating in many organs. As the cilium is composed of an axoneme structure that normally consists of nine peripheral microtubule doublets arranged around two central microtubules (39Ibanez-Tallon I. Heintz N. Omran H. Hum. Mol. Genet. 2003; 12: R27-R35Crossref PubMed Google Scholar), we next examined the ultrastructure of the cilia by electron microscopy. In the HSF1-null respiratory epithelium, dynein arms and radial spokes were observed, but almost 10% of the cilia possessed abnormal central microtubules and microtubule doublets such as deletion or transposition (Fig. 2B). These abnormalities were characteristic of some types of ciliary dyskinesia (42Sturgess J.M. Chao J. Turner J.A. N. Engl. J. Med. 1980; 303: 318-322Crossref PubMed Scopus (152) Google Scholar). Although it is unclear whether the abnormal structures in HSF1-null mice may be responsible for ciliary dyskinesia, these results suggest that assembly or organization of microtubules might be impaired. Tubulin Expression Decreases in the Motile Cilia of HSF1-null Mice—As HSF1-null mice have abnormal microtubule ultrastructure, we examined expression of their major components such as tubulin, dynein, and chaperonin. Microtubules are formed by protofilaments of α/β-tubulin heterodimers. Among five β-tubulin isotypes, βi- and βiv-tubulin are found in all axoneme structures (43Jensen-Smith H.C. Luduena R.F. Hallworth R. Cell Motil. Cytoskeleton. 2003; 55: 213-220Crossref PubMed Scopus (39) Google Scholar, 44Renthal R. Schneider B.G. Miller M.M. Luduena R.F. Cell Motil. Cytoskeleton. 1993; 25: 19-29Crossref PubMed Scopus (64) Google Scholar), and are required for ciliary function and assembly (45Vent J. Wyatt T.A. Smith D.D. Banerjee A. Luduena R.F. Sisson J.H. Hallworth R. J. Cell Sci. 2005; 118: 4333-4341Crossref PubMed Scopus (33) Google Scholar). We found that the level of βiv-tubulin in the cilia, as well as ciliary α-tubulin, was significantly decreased in the HSF1-null respiratory epithelium (Fig. 3A). Levels of βiv-tubulin were also reduced in cilia of the ventricles, trachea, and oviducts (Fig. 3B). Although the TCP-1 complex, known as eukaryotic cytoplasmic chaperonin, direc
Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based "assisted-diffusion" behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a mechanism of potential importance for physiological cargoes as well.
Recent evidence has implicated dynein and its regulatory factors dynactin and LIS1 in neuronal and non-neuronal cell migration. In the current study we sought to test whether effects on neuronal cell motility might reflect, in part, a role for these proteins in the growth cone. In chick sensory neurons subjected to acute laminin treatment dynein, dynactin, and LIS1 were mobilized strikingly and rapidly to the leading edge of the growth cone, where they were seen to be associated with microtubules converging into the laminin-induced axonal outgrowths. To interfere acutely with LIS1 and dynein function and to minimize secondary phenotypic effects, we injected antibodies to these proteins just before axon initiation. Antibody to both proteins produced an almost complete block of laminin-induced growth cone remodeling and the underlying reorganization of microtubules. Penetration of microtubules into the peripheral zone of differentiating axonal growth cones was decreased dramatically by antibody injection, as judged by live analysis of enhanced green fluorescent protein-tubulin and the microtubule tip-associated EB3 (end-binding protein 3). Dynein and LIS1 inhibition had no detectable effect on microtubule assembly but reduced the ability of microtubules to resist retrograde actin flow. In hippocampal neurons dynein, dynactin, and LIS1 were enriched in axonal growth cones at stage 3, and both growth cone organization and axon elongation were altered by LIS1 RNA interference. Together, our data indicate that dynein and LIS1 play a surprisingly prominent role in microtubule advance during growth cone remodeling associated with axonogenesis. These data may explain, in part, the role of these proteins in brain developmental disease and support an important role in diverse aspects of neuronal differentiation and nervous system development.
Significance Zika virus (ZIKV) infection has been associated with multiple pathologies of the central nervous system (CNS) including microcephaly, Guillain-Barré syndrome, lissencephaly, the loss of white and gray matter volume and acute myelitis. Using organotypic brain slice cultures, we determined that ZIKV replicates across different embryonic developmental stages, and viral infection can disrupt proper brain development leading to congenital CNS complications. These data illustrate that all lineages of ZIKV tested are neurotropic, and that infection may disrupt neuronal migration during brain development. The results expand our understanding of neuropathologies associated with congenital Zika virus syndrome.
Cytoplasmic dynein and its regulatory proteins have been implicated in neuronal and non-neuronal cell migration. A genetic model for analyzing the role of cytoplasmic dynein specifically in these processes has, however, been lacking. The Loa (Legs at odd angles) mouse with a mutation in the dynein heavy chain has been the focus of an increasing number of studies for its role in neuron degeneration. Despite the location of this mutation in the tail domain of the dynein heavy chain, we previously found a striking effect on coordination between the two dynein motor domains, resulting in a defect in dynein run length in vitro and in vivo. We have now tested for effects of the Loa mutation on neuronal migration in the developing neocortex. Loa homozygotes showed clear defects in neocortical lamination and neuronal migration resulting from a reduction in the rate of radial migration of bipolar neurons. These results present a new genetic model for understanding the dynein pathway and its functions during neuronal migration. They also provide the first evidence for a link between dynein processivity and somal movement, which is essential for proper development of the brain.
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