Rotokinesis, a novel phenomenon of cell locomotion-assisted cytokinesis in the ciliate Tetrahymena thermophila

The mechanism responsible for final cell separation at the end of cytokinesis is currently unknown. Knockout strains of the ciliate, Tetrahymena thermophila lacking the kinesin-II homologous molecular motors, Kin1p and Kin2p are paralyzed due to their complete loss of cilia and undergo frequent cytokinesis failures. Observations of live dividing cells revealed that cleavage furrow ingression is normal in kinesin-II double knockout cells until the final stage of cell separation (Brown et al., 1999). During closer inspection of dividing cells using video differential interference contrast microscopy, we found that wild-type cells undergo an extremely complex motile behavior near the end of cytokinesis. This process, which we have named rotokinesis, appears to facilitate the physical separation of daughter cells. Here we present recent work onTetrahymena rotokinesis, and review studies in other organisms which suggest that the use of cell locomotion in the completion of cytokinesis is a general phenomenon of motile cell types.     

Hypoxia regulates assembly of cilia in suppressors of Tetrahymena lacking an intraflagellar transport subunit gene

We cloned a Tetrahymena thermophila gene, IFT52, encoding a homolog of the Chlamydomonas intraflagellar transport protein, IFT52. Disruption of IFT52 led to loss of cilia and incomplete cytokinesis, a phenotype indistinguishable from that of mutants lacking kinesin-II, a known ciliary assembly transporter. The cytokinesis failures seem to result from lack of cell movement rather than from direct involvement of ciliary assembly pathway components in cytokinesis. Spontaneous partial suppressors of the IFT52 null mutants occurred, which assembled cilia at high cell density and resorbed cilia at low cell density. The stimulating effect of high cell density on cilia formation is based on the creation of pericellular hypoxia. Thus, at least under certain conditions, ciliary assembly is affected by an extracellular signal and the Ift52p function may be integrated into signaling pathways that regulate ciliogenesis.     

Heterotrimeric Kinesin-II Is Required for the Assembly of Motile 9+2 Ciliary Axonemes on Sea Urchin Embryos

Heterotrimeric kinesin-II is a plus end– directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti–kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti–kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti–kinesin-II–injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding.     

The protein kinase kin1 is required for cellular symmetry in fission yeast

The fission yeast Schizosaccharomyces pombe is a highly polarized unicellular eukaryote with two opposite growing poles in which F-actin cytoskeleton is focused. The KIN1/PAR-1/MARK protein family is composed of conserved eukaryotic serine/threonine kinases which are involved in cell polarity, microtubule stability or cell cycle regulation. Here, we investigate the function of the fission yeast KIN1/PAR-1/MARK member, kin1p. Using a deletion allele (kin1Delta), we show that kin1 mutation promotes a delay in septation. Kin1p regulates the structure of the new cell end after cytokinesis by modulating cell wall remodeling. Abnormal shaped interphase kin1Delta cells misplace F-actin patches and the premitotic nucleus. Thus, mitotic kin1Delta cells misposition the F-actin ring assembly site that is dependent on the position of the interphase nucleus. The resulting asymmetric cell division produces daughter cells with distinct shapes. Overexpressed kin1p accumulates asymmetrically at the cell cortex and affects cell shape, F-actin organization and microtubules. Our results suggest that correct dosage of kin1p at the cortex is required for spatial organization of the fission yeast cell.     

Kin3 protein,a NIMA-related kinase of Saccharomyces cerevisiae,is involved in DNA adduct damage response

Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.     

驱动蛋白Kin11调控嗜热四膜虫生殖系小核的减数分裂

驱动蛋白(kinesin)是分子马达蛋白质超家族成员,主要参与囊泡与细胞器的运输、纺锤体组装、有丝分裂和减数分裂等过程。在减数分裂期,不同驱动蛋白发挥功能的调控机制并不十分清楚。嗜热四膜虫(Tetrahymena thermophila)中含有14个驱动蛋白家族成员。其中,kinesin-6家族的唯一成员Kin11(TTHERM_00637750),在营养生长期低表达,饥饿期不表达,有性生殖期表达上调。Kin11编码1608个氨基酸,包含1个N端保守的马达蛋白结构域,C端卷曲螺旋(coiled-coil)结构域,并在N端和C端分别含有核定位信号NLS1和NLS2。Kin11在营养生长期和有性生殖期,定位在有丝分裂和减数分裂的小核和纺锤体上,并在有性生殖后期alignment阶段定位于小核上。Kin11与微管蛋白共定位于有丝分裂和减数分裂的纺锤体上。将Kin11的N端含有NLS1的1~400位氨基酸序列截短后,截断突变体定位在有性生殖减数分裂期的小核和纺锤体上。而将其C端含有NLS2的1008~1608位氨基酸残基截短后,截断突变体只能定位在有丝分裂和减数分裂后期的小核及有丝分裂的纺锤体上。敲除KIN11导致减数分裂过程中的纺锤体结构发生异常变化,小核染色体不均等分离与丢失,有性生殖发育停滞。结果表明,嗜热四膜虫驱动蛋白Kin11通过影响纺锤体结构,参与调控四膜虫生殖系小核在减数分裂过程中的正常分离。     

Kinesin-13 regulates the quantity and quality of tubulin inside cilia

Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena. One of the paralogues, Kin13Ap, localizes to the nuclei and is essential for nuclear divisions. The remaining two paralogues, Kin13Bp and Kin13Cp, localize to the cell body and inside assembling cilia. Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase. The mutant cilia assembled slowly and contained abnormal tubulin, characterized by altered posttranslational modifications and hypersensitivity to paclitaxel. The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding. Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility.     

KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin–RNA interacting proteins (CRIPs) conserved from fission yeast to man

In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.     

Cak1 Is Required for Kin28 Phosphorylation and Activation In Vivo

Complete activation of most cyclin-dependent protein kinases (CDKs) requires phosphorylation by the CDK-activating kinase (CAK). In the budding yeast, Saccharomyces cerevisiae, the major CAK is a 44-kDa protein kinase known as Cak1. Cak1 is required for the phosphorylation and activation of Cdc28, a major CDK involved in cell cycle control. We addressed the possibility that Cak1 is also required for the activation of other yeast CDKs, such as Kin28, Pho85, and Srb10. We generated three new temperature-sensitive cak1 mutant strains, which arrested at the restrictive temperature with nonuniform budding morphology. All three cak1 mutants displayed significant synthetic interactions with loss-of-function mutations in CDC28 and KIN28. Loss of Cak1 function reduced the phosphorylation and activity of both Cdc28 and Kin28 but did not affect the activity of Pho85 or Srb10. In the presence of the Kin28 regulatory subunits Ccl1 and Tfb3, Kin28 was phosphorylated and activated when coexpressed with Cak1 in insect cells. We conclude that Cak1 is required for the activating phosphorylation of Kin28 as well as that of Cdc28.     

A Conditional Mutant Having Paralyzed Cilia and a Block in Cytokinesis Is Rescued by Cytoplasmic Exchange in Tetrahymena Thermophila

Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.     

Distinct IFT mechanisms contribute to the generation of ciliary structural diversity in C. elegans

Individual cell types can elaborate morphologically diverse cilia. Cilia are assembled via intraflagellar transport (IFT) of ciliary precursors; however, the mechanisms that generate ciliary diversity are unknown. Here, we examine IFT in the structurally distinct cilia of the ASH/ASI and the AWB chemosensory neurons in Caenorhabditis elegans, enabling us to compare IFT in specific cilia types. We show that unlike in the ASH/ASI cilia, the OSM-3 kinesin moves independently of the kinesin-II motor in the AWB cilia. Although OSM-3 is essential to extend the distal segments of the ASH/ASI cilia, it is not required to build the AWB distal segments. Mutations in the fkh-2 forkhead domain gene result in AWB-specific defects in ciliary morphology, and FKH-2 regulates kinesin-II subunit gene expression specifically in AWB. Our results suggest that cell-specific regulation of IFT contributes to the generation of ciliary diversity, and provide insights into the networks coupling the acquisition of ciliary specializations with other aspects of cell fate.     

The yeast par-1 homologs kin1 and kin2 show genetic and physical interactions with components of the exocytic machinery

Kin1 and Kin2 are Saccharomyces cerevisiae counterparts of Par-1, the Caenorhabditis elegans kinase essential for the establishment of polarity in the one cell embryo. Here, we present evidence for a novel link between Kin1, Kin2, and the secretory machinery of the budding yeast. We isolated KIN1 and KIN2 as suppressors of a mutant form of Rho3, a Rho-GTPase acting in polarized trafficking. Genetic analysis suggests that KIN1 and KIN2 act downstream of the Rab-GTPase Sec4, its exchange factor Sec2, and several components of the vesicle tethering complex, the Exocyst. We show that Kin1 and Kin2 physically interact with the t-SNARE Sec9 and the Lgl homologue Sro7, proteins acting at the final stage of exocytosis. Structural analysis of Kin2 reveals that its catalytic activity is essential for its function in the secretory pathway and implicates the conserved 42-amino acid tail at the carboxy terminal of the kinase in autoinhibition. Finally, we find that Kin1 and Kin2 induce phosphorylation of t-SNARE Sec9 in vivo and stimulate its release from the plasma membrane. In summary, we report the finding that yeast Par-1 counterparts are associated with and regulate the function of the exocytic apparatus via phosphorylation of Sec9.     

Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans.

The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 microm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope-based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at approximately 1.1 microm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end-directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.     

The FLA3 KAP subunit is required for localization of kinesin-2 to the site of flagellar assembly and processive anterograde intraflagellar transport

Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21 degrees C, but cannot maintain them at 33 degrees C. Although the Kinesin-2 complex is present at both 21 and 33 degrees C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.     

Effect of superexpression of DNA-binding protein heterochromatin Abp1p on frequency of loss of minichromosomes and growth of Schizosaccharomyces pombe with mutations of the gene coding cofactor D

Mitotic chromosome segregation is partly determined by interaction between microtubules (MTs) and the kinetochores of sister chromatids. The precise mechanism of the interaction between kinetochores and MTs remains unclear. This process has been studied in fission yeast Schizosaccharomyces pombe by analyzing interaction between genes encoding kinetochore components, such as DNA-binding protein Abp1p, and genes whose protein products affect the dynamics of MTs, such as cofactor D of tubulin dimer assembly. Analysis of cell growth and minichromosome loss frequency has demonstrated that mutations in the gene of cofactor D, especially mutation tsm1-512, increase the rate of minichromosome loss and the sensitivity to changes in Abp1p concentration in cells compared to wild-type cells Probably, mutations alp1-1315 and tsm1-512 of the cofactor D gene cause defects in the kinetochore-MT interaction.     

Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules

Tuberous sclerosis complex (TSC) is a multiorgan hamartomatous disease caused by loss of function mutations of either the TSC1 or TSC2 genes. Neurological symptoms of TSC predominate in younger patients, but renal pathologies are a serious aspect of the disease in older children and adults. To study TSC pathogenesis in the kidney, we inactivated the mouse Tsc1 gene in the distal convoluted tubules (DCT). At young ages, Tsc1 conditional knockout (CKO) mice have enlarged kidneys and mild cystogenesis with increased mammalian target of rapamycin complex (mTORC)1 but decreased mTORC2 signaling. Treatment with the mTORC1 inhibitor rapamycin reduces kidney size and cystogenesis. Rapamycin withdrawal led to massive cystogenesis involving both distal as well as proximal tubules. To assess the contribution of decreased mTORC2 signaling in kidney pathogenesis, we also generated Rictor CKO mice. These animals did not have any detectable kidney pathology. Finally, we examined primary cilia in the DCT. Cilia were longer in Tsc1 CKO mice, and rapamycin treatment returned cilia length to normal. Rictor CKO mice had normal cilia in the DCT. Overall, our findings suggest that loss of the Tsc1 gene in the DCT is sufficient for renal cystogenesis. This cytogenesis appears to be mTORC1 but not mTORC2 dependent. Intriguingly, the mechanism may be cell autonomous as well as non-cell autonomous and possibly involves the length and function of primary cilia.     

Cell type‐specific regulation of ciliary transition zone assembly in vertebrates

Ciliopathies are life‐threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans. By performing quantitative immunofluorescence studies in RPGRIP1L^?/? mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type‐specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate‐specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.     

noxin, a novel stress-induced gene involved in cell cycle and apoptosis

We describe a novel stress-induced gene, noxin, and a knockout mouse line with an inactivated noxin gene. The noxin gene does not have sequelogs in the genome and encodes a highly serine-rich protein with predicted phosphorylation sites for ATM, Akt, and DNA-dependent protein kinase kinases; nuclear localization signals; and a Zn finger domain. noxin mRNA and protein levels are under tight control by the cell cycle. noxin, identified as a nitric oxide-inducible gene, is strongly induced by a wide range of stress signals: gamma- and UV irradiation, hydrogen peroxide, adriamycin, and cytokines. This induction is dependent on p53. Noxin accumulates in the nucleus in response to stress and, when ectopically expressed, Noxin arrests the cell cycle at G1; although it also induces p53, the cell cycle arrest function of Noxin is independent of p53 activity. noxin knockout mice are viable and fertile; however, they have an enlarged heart, several altered hematopoietic parameters, and a decreased number of spermatids. Importantly, loss or downregulation of Noxin leads to increased cell death. Our results suggest that Noxin may be a component of the cell defense system: it is activated by various stress stimuli, helps cells to withdraw from cycling, and opposes apoptosis.