Search

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.08.008 Byline: Daisuke Kobayashi, Norio Iijima, Haruo Hagiwara, Keiichiro Kamura, Hiroyuki Takeda, Takahiko Yokoyama Keywords: Cilia; Primary ciliary dyskinesia; Dynein; Kidney; Cyst; Medaka Abstract: Cilia and flagella are highly conserved organelles that have diverse motility and sensory functions. Motility defects in cilia and flagella result in primary ciliary dyskinesia (PCD). We isolated a novel medaka PCD mutant, jaodori (joi). Positional cloning showed that axonemal dynein intermediate chain 2 (dnai2) is responsible for joi. The joi mutation was caused by genomic insertion of the medaka transposon, Tol1. In the joi mutant, cilia in Kupffer's vesicle (KV), an organ functionally equivalent to the mouse node in terms of left-right (LR) specification, are generated but their motility is disrupted, resulting in a LR defect. Ultrastructural analysis revealed severe reduction in the outer dynein arms in KV cilia of joi mutants. We also found the other dnai2 gene in the medaka genome. These two dnai2 genes function either redundantly or distinctly in tissues possessing motile cilia. Article History: Received 18 March 2010; Revised 9 July 2010; Accepted 5 August 2010

Long-distance transport in cells is driven by kinesin and dynein motors that move along microtubule tracks. These motors must be tightly regulated to ensure the spatial and temporal fidelity of their transport events. Transport motors of the kinesin-1 and kinesin-3 families are regulated by autoinhibition, but little is known about the mechanisms that regulate kinesin-2 motors. We show that the homodimeric kinesin-2 motor KIF17 is kept in an inactive state in the absence of cargo. Autoinhibition is caused by a folded conformation that enables nonmotor regions to directly contact and inhibit the enzymatic activity of the motor domain. We define two molecular mechanisms that contribute to autoinhibition of KIF17. First, the C-terminal tail interferes with microtubule binding; and second, a coiled-coil segment blocks processive motility. The latter is a new mechanism for regulation of kinesin motors. This work supports the model that autoinhibition is a general mechanism for regulation of kinesin motors involved in intracellular trafficking events. doi/10.1083/jcb.201001057

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.12.004 Byline: Heidi N. Fridolfsson, Nina Ly, Marina Meyerzon, Daniel A. Starr Keywords: Nuclear migration; KASH proteins; Nuclear envelope; Kinesin; Dynein; C. elegans Abstract: Nuclei migrate during many events, including fertilization, establishment of polarity, differentiation, and cell division. The Caenorhabditis elegans KASH protein UNC-83 localizes to the outer nuclear membrane where it recruits kinesin-1 to provide the major motor activity required for nuclear migration in embryonic hyp7 cells. Here we show that UNC-83 also recruits two dynein-regulating complexes to the cytoplasmic face of the nucleus that play a regulatory role. One consists of the NudE homolog NUD-2 and the NudF/Lis1/Pac1 homolog LIS-1, and the other includes dynein light chain DLC-1, the BicaudalD homolog BICD-1, and the Egalitarian homologue EGAL-1. Genetic disruption of any member of these two complexes caused nuclear migration defects that were enhanced in some double mutant animals, suggesting that BICD-1 and EGAL-1 function in parallel to NUD-2. Dynein heavy chain mutant animals also had a nuclear migration defect, suggesting these complexes function through dynein. Deletion analysis indicated that independent domains of UNC-83 interact with kinesin and dynein. These data suggest a model where UNC-83 acts as the cargo-specific adaptor between the outer nuclear membrane and the microtubule motors kinesin-1 and dynein. Kinesin-1 functions as the major force generator during nuclear migration, while dynein is involved in regulation of bidirectional transport of the nucleus. Author Affiliation: Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA Article History: Received 16 July 2009; Revised 4 December 2009; Accepted 4 December 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.10.012 Byline: Alu Konno (a), Maiko Kaizu (a), Kohji Hotta (b), Takeo Horie (a), Yasunori Sasakura (a), Kazuho Ikeo (c), Kazuo Inaba (a) Keywords: Primary cilia; Flagellar axoneme; Ciona; Ascidian larva; Tadpole larva; Dynein; Embryogenesis Abstract: Accumulating evidence demonstrates that cilia play important roles in a variety of processes in embryogenesis. For functional survey of larval cilia at the cellular level, we exploited the simple cell organization of tadpole larvae in the ascidian Ciona intestinalis. Immunofluorescent microscopy showed distribution of cilia not only in previously described tissues but also in a subpopulation of ependymal cells in the sensory vesicle, gut primordium, papillae, apical trunk epidermal neurons, and the endodermal strand. Transmission electron microscopy revealed a variety of axonemal structures, including a 9+0 structure similar to vertebrate primary cilia, a 9+0 structure with electron-dense materials in the center, a 9+2 structure with no dynein arms, and an axoneme with a disorganized structure at the distal end. Extensive description of cilia in the present study gives important insights into the evolution of the ciliary structure and provides a basis for analysis of ciliary functions in establishment of chordate body plan. Author Affiliation: (a) Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka 415-0025, Japan (b) Department of Bioscience and Informatics, Faculty of Science and Technology, Keio University, Kouhoku-ku, Yokohama 223-8522, Japan (c) Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan Article History: Received 31 May 2009; Revised 22 September 2009; Accepted 3 October 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.12.030 Byline: Marina Meyerzon (a), Zhizhen Gao (b), Jin Liu (a), Jui-Ching Wu (a), Christian J. Malone (b), Daniel A. Starr (a) Keywords: Nuclear envelope; Centrosome; Male pronucleus; Pronuclear migration; Lamin; LEM; BAF; Microtubule aster; Outer nuclear membrane; Dynein Abstract: A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein. Author Affiliation: (a) Department of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA (b) Department of Biochemistry and Molecular Biology, Penn State University, University Park, PA 16802, USA Article History: Received 30 April 2008; Revised 13 December 2008; Accepted 19 December 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.11.049 Byline: Anthony J. Roberts (1), Naoki Numata (2), Matt L. Walker (3), Yusuke S. Kato (1), Bara Malkova (1), Takahide Kon (2), Reiko Ohkura (2), Fumio Arisaka (4), Peter J. Knight (1), Kazuo Sutoh (2), Stan A. Burgess (1) Keywords: CELLBIO Abstract: Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by [approximately equal to]17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism. Author Affiliation: (1) Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK (2) Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Tokyo 153-8902, Japan (3) MLW Consulting, 11 Race Hill, Launceston, Cornwall PL15 9BB, UK (4) Graduate School and School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Yokohama 226-8501, Japan Article History: Received 5 June 2008; Revised 12 October 2008; Accepted 26 November 2008 Article Note: (miscellaneous) Published: February 5, 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.05.046 Byline: Samara L. Reck-Peterson (2), Ahmet Yildiz (2), Andrew P. Carter (2), Arne Gennerich (2), Nan Zhang (1)(2), Ronald D. Vale (1)(2) Abstract: Cytoplasmic dynein, the 1.2 MDa motor driving minus-end-directed motility, has been reported to move processively along microtubules, but its mechanism of motility remains poorly understood. Here, using S. cerevisiae to produce recombinant dynein with a chemically controlled dimerization switch, we show by structural and single-molecule analysis that processivity requires two dynein motor domains but not dynein's tail domain or any associated subunits. Dynein advances most frequently in 8 nm steps, although longer as well as side and backward steps are observed. Individual motor domains show a different stepping pattern, which is best explained by the two motor domains shuffling in an alternating manner between rear and forward positions. Our results suggest that cytoplasmic dynein moves processively through the coordination of its two motor domains, but its variable step size and direction suggest a considerable diffusional component to its step, which differs from Kinesin-1 and is more akin to myosin VI. Author Affiliation: (1) Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94143 USA (2) Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158 USA Article History: Received 5 March 2006; Revised 4 May 2006; Accepted 17 May 2006 Article Note: (miscellaneous) Published: July 27, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.06.002 Byline: Sadhana A. Samant, Olugbemiga O. Ogunkua, Ling Hui, Jing Lu, Yibing Han, Joanne M. Orth, Stephen H. Pilder Keywords: t complex; Dynein heavy chain subtype; Inner dynein arm; Outer dynein arm; Axoneme; Flagellum; Flagellar waveform; Principal piece; Midpiece; Male sterility; Male TRD Abstract: Heterozygosity for a t haplotype (t) in male mice results in distorted transmission (TRD) of the t-bearing chromosome 17 homolog to their offspring. However, homozygosity for t causes male sterility, thus limiting the spread of t through the population at large. The Ca.sup.2+-dependent sperm tail curvature phenotypes, 'fishhook', where abnormally high levels of sperm exhibit sharp bends in the midpiece, and 'curlicue', where motile sperm exhibit a chronic negative curving of the entire tail, have been tightly linked to t-associated male TRD and sterility traits, respectively. Genetic studies have indicated that homozygosity for the t allele of Dnahc8, an axonemal [gamma]-type dynein heavy chain ([gamma]DHC) gene, is partially responsible for expression of 'curlicue'; however, its involvement in 'fishhook'/TRD, if any, is unknown. Here we report that the major isoform of DNAHC8 is copiously expressed, carries an extended N-terminus and full-length C-terminus, and is stable and equally abundant in both testis and sperm from +/+ and t/t animals. By in silico analysis we also demonstrate that at least three of the seventeen DNAHC8.sup.t mutations at highly conserved positions in wild-type DHCs may be capable of substantially altering normal DNAHC8 function. Interestingly, DNAHC8 is confined to the principal piece of the sperm tail. The combined results of this study suggest possible mechanisms of DNAHC8.sup.t dysfunction and involvement in 'curlicue', and support the hypothesis that 'curlicue' is a multigenic phenomenon. They also demonstrate that the accelerated 'fishhook' phenotype of sperm from +/t males is not directly linked to DNAHC8.sup.t dysfunction. Author Affiliation: Department of Anatomy and Cell Biology, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140, USA Article History: Received 24 November 2004; Revised 26 May 2005; Accepted 3 June 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2004.03.018 Byline: Yannick Marrari, Christian Rouviere, Evelyn Houliston Keywords: Xenopus; Egg; Cortical rotation; Microtubule; Dynein; Dynactin; Dynamitin; Kinesin; Cortex Abstract: Aligned vegetal subcortical microtubules in fertilized Xenopus eggs mediate the 'cortical rotation', a translocation of the vegetal cortex and of dorsalizing factors toward the egg equator. Kinesin-related protein (KRP) function is essential for the cortical rotation, and dynein has been implicated indirectly; however, the role of neither microtubule motor protein family is understood. We examined the consequence of inhibiting dynein-dynactin-based transport by microinjection of excess dynamitin beneath the vegetal egg surface. Dynamitin introduced before the cortical rotation prevented formation of the subcortical array, blocking microtubule incorporation from deeper regions. In contrast, dynamitin injected after the microtubule array was fully established did not block cortical translocation, unlike inhibitory-KRP antibodies. During an early phase of cortical rotation, when microtubules showed a distinctive wavy organization, dynamitin disrupted microtubule alignment and perturbed cortical movement. These findings indicate that dynein is required for formation and early maintenance of the vegetal microtubule array, while KRPs are largely responsible for displacing the cortex once the microtubule tracks are established. Consistent with this model for the cortical rotation, photobleach analysis revealed both microtubules that translocated with the vegetal cytoplasm relative to the cortex, and ones that moved with the cortex relative to the cytoplasm. Article History: Received 20 January 2004; Revised 10 March 2004; Accepted 11 March 2004

A study was conducted to illustrate how genetic mutations can prevent the association of dynein heavy chains with the kinetochores of meiotic and mitotic chromosomes in Drosophila melanogaster. Results showed that the kinetochore localization of dynein was affected by tension across the centromere. In addition, interaction between ZW10 and dynamitin was observed to control the targeting function of ZW10.

Molecular mechanisms that orient left-right (LR) axis in embryos have been unknown. It has been shown recently that the inverted viscerum mutation in mice may come from a mutation in a gene that encodes an axonemal dynein heavy chain. Along with answers, this has brought new questions: what the role of axonemal dynein is as opposed to that of cytoplasmic dynein and how dynein action is moved across fields of cells. Timing of initial LR actions in embryogenesis and evolutionary functions of asymmetry are of interest.

Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.

The dyamitin overexpression phenotype interphase mammalian cells was determined through time-lapsed observation of nocodazole treated HeLa cell lines. Results have shown that residual plus end-directed motor activity associated with late endosomes and lysosomes indicated that dynactin specifically inhibited minus end-directed motor activity. It was indicated that dynamitin regulated organelle assembly at the motor-cargo binding level.

The importance of centrosome in spindle formation and bipolarity was investigated through analysis of the mechanism of spindle assembly in Xenopus egg extracts. Results revealed that in the presence of one centrosome, a dominant site, was generated for microtubule nucleation and focal organization. This monopolized development resulted in monopolar spindle.

Green fluorescent protein (GFP)-tubulin fusion protein was utilized to investigate the role of mitotic spindle in the orientation of microtubules relative to dynein-dependent interactions with the cell cortex of yeast cells. Microtubules from one of the centrosomes interacted with a cortical attachment site on the anterior cortex, resulting in the rotation of the same centrosome toward that site. The absence of dynein resulted in the alteration of the microtubular dynamics.

Rapid DNA amplification and peptide sequence analysis were utilized for the biochemical characterization of the 19,000-Mr protein of the Chlamydomonas flagellum. Molecular analysis of the Chlamydomonas 19,000-Mr protein indicated its homology with the Tctex-2 putative murine t complex distorter. The Chlamydomonas homologue of the putative murine t complex distorter was also characterized as a light chain of outer arm dynein. Furthermore, chemical dissection of the outer arm dynein exhibited the tight association between the 19,000-Mr LC and beta dynein heavy chains.

The cytoplasmic dynein heavy chain (CDHC) encoded in the nudA gene of Aspergillus nidulans is situated at the growing terminal of the fungal mycelium. The introduction of four temperature-dependent mutations in the nudA gene reduces the CDHC protein. For nudA, the deletion/disruption mutant is phenotypically identical to the original temperature-sensitive mutants. The cytoplasmic dynein intervenes in nuclear migration.

The products of the Drosophila Glued gene, which are subunits of a 20S complex, are similar to the vertebrate homologues in dynactin. The Glued complexes are physically bound to the cytoplasmic dynein motor complexes. The Glued polypeptides copurify as microtubule-linked proteins. During oogenesis the spatial and temporal arrangement of the Glued complex are sensitive to the dynein activity and other gene products.

Analysis of phenotypically similar mutants from Neurospora crassa fungus, called ropy, that lack the coding for dynactin complex or cytoplasmic dynein reveals that the mutants have defective nuclear organization, indicating that dynactin complex, actin-related protein Arp1 and cytoplasmic dynein are essential for normal nuclear arrangement. The mutants do not express the genes ro-1 and ro-4 that encode the cytoplasmic dynein and Arp1. Maintenance of the nuclear profiles occurs through antiparallel microtubule sliding.

In vitro biochemical assays and immunofluorescence analyses of actin-related protein Arp1 indicate that Arp1 plays a role in the microtubule-based motility activities of the cytoplasmic dynein protein. Arp1 exists in the 1.2-md molecule dynactin complex of the cytoplasm and attaches dynein to intracellular membranes to enable microtubule-based motility. Arp1 mediates nuclear migration and spindle orientation like cytoplasmic dynein, which also mediates endocytic pathway processes, retrograde transport in axons and apical epithelial transcytosis. Removal of dynein or Arp1 induces defective spindle orientation and nuclear migration.

The microtubule motor molecule, dynein, influences nuclear migration process which is crucial to the development and growth of many organisms such as the multinuclear fungus Aspergillus nidulans. The cytoplasmic dynein heavy chain was observed to have four characteristic ATP-binding sites.

Investigations on the yeast Saccharomyces cerevisiae have established the gene DYN1 which encodes the heavy chain of cytoplasmic dynein. The correct arrangement of the mitotic spindle in yeast may depend on the force along cytoplasmic microtubules, induced by cytoplasmic dynein. Irregular distribution of the dividing nuclei between the mother cells and the bud results from the misalignment of the spindle, relative to the bud neck during cell division, arising from the disruption of the DYN1.

An analysis to establish the exact site of Dhc-Yh3 on the Y chromosome involved the application of several feasible translocation between the X and Y chromosomes. The ordinary genes that function during spermiogenesis are contained in the Y chromosome, and are crucial for male fertility. Dynein heavy-chain sequences are regionalized to Y chromosome kl-5 site.

Identification of and mutational studies conducted on the gene DHC1 reveal that it encodes a dynein heavy chain isoform within the Saccharomyces cerevisiae. Sequence analysis of a 9-kb genomic fragment of the DHC1 gene yielded four polypeptides closely resembling the dynein sequences derived from the sea urchin, Dictyostelium, Drosophila and rat. The dynein microtubule motor protein has an unimportant role in spindle assembly in yeast. However, it has a vital role in regulating spindle orientation in the course of cell division.

Molecular identification of polypeptides consisting of the complex structure of microtubule motor dyneins facilitates analysis of the framework and molecular pathway of dynein enzyme activity. The 70-kDa Chlamydomonas flagellar dynein external arm intermediate chain (IC70) is molecularly cloned during the study of outer dynein arm (oda) mutants. Intracellular mobility of microtubules is attributed to dyneins. These proteins are characteristic microtubule-activated ATpases involved in the transformation of chemical energy to mechanical energy.

The heavy chain of rat brain cytoplasmic dynein, composed of four putative nucleotide-finding consensus sequences GX4GK(T/S) in the middle one third region, and inferred protein sequence are analogous to the sea urchin flagellar beta-dynein by 27.0% and to Dictyostelium cytoplasmic dynein by 53.5%. The N-terminal one third of the heavy chain of rat brain cytoplasmic dynein resembles the Dictyostelium cytoplasmic dynein by 43.8% and has a low resemblance of 19.4% to the sea urchin flagellar dynein. The inference shows that the N-terminal regions are different between sea urchin and Dictyostelium dyneins and that the C-terminal two-thirds of the dyneins function in the microtubule-dependent motility action.

An analysis of conventional protein coding genes in the Drosophila Y chromosome revealed that the ATP-bonding site of a likely functional dynein gene is related to D. melanogaster's kl-5 fertility gene. The existence of a protein coding gene not on the Y chromosome, associated with one of the fertility genes, is conclusively confirmed. A faulty dynein gene leads to male sterility, owing to disorders in development induced by the lack of dynein.

Structural and single-molecule analysis is used to show that processivity requires two dynein motor domains but does not require dynein's tail domain or any associated subunits. The variable step size and direction of the two motor domains suggest a considerable diffusion component to its size, which differs from Kinesin-1 but is similar to myosin VI.

Research shows that misfolded protein-induced stress is managed by the microtubule-associated deacetylase, HDAC6, which is a component of the aggresome. The HDAC6 binds both polyubiquinitated misfolded proteins and dynein motors and recruit misfolded proteins to dynein motors for transport to aggresomes.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.01.026 Byline: Anne Houdusse (1)(2), Andrew P. Carter (3) Abstract: Motor proteins, such as dynein, use chemical energy from ATP hydrolysis to move along the cytoskeleton. now describe the arrangement of subdomains in the motor domain of dynein and propose a model for how these regions function together in force generation. Author Affiliation: (1) Structural Motility Team, Institut Curie, Centre de Recherche, Paris F-75248, France (2) CNRS, UMR144, Paris F-75248, France (3) Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA