Kinesin-like Protein Research Articles

Intercellular protein movement in syncytialDrosophilafollicle cells

Ring canals connecting Drosophila germline, follicle and imaginal disc cells provide direct contact of cytoplasm between cells. To date, little is known about the formation, structure, or function of the somatic ring canals present in follicle and imaginal disc cells. Here, we show by confocal and electron microscopy that Pavarotti kinesin-like protein and Visgun are stable components of somatic ring canals. Using live-cell confocal microscopy, we show that somatic ring canals form from the stabilization of mitotic cleavage furrows. In contrast to germline cells, syncytial follicle cells do not divide synchronously, are not maximally branched and their ring canals do not increase in size during egg chamber development. We show for the first time that somatic ring canals permit exchange of cytoplasmic proteins between follicle cells. These results provide insight into the composition and function of ring canals in somatic cells, implying a broader functional significance for syncytial organization of cells outside the germline.

Whole-Exome Sequencing Identifies Mutations of KIF22 in Spondyloepimetaphyseal Dysplasia with Joint Laxity, Leptodactylic Type

Spondyloepimetaphyseal dysplasia with joint laxity (SEMDJL), leptodactylic (lepto-SEMDJL) or Hall type, is an autosomal-dominant skeletal dysplasia manifesting with short stature, joint laxity with dislocation(s), limb malalignment, and spinal deformity. Its causative gene mutation has not yet been discovered. We captured and sequenced the exomes of eight affected individuals in six unrelated kindreds (three individuals in a family and five simplex individuals). Five novel sequence variants in KIF22, which encodes a member of the kinesin-like protein family, were identified in seven individuals. Sanger sequencing of KIF22 confirmed that c.443C>T (p.Pro148Ser) cosegregated with the phenotype in the affected individuals in the family; c.442C>T (p.Pro148Leu) or c.446G>A (p.Arg149Gln) was present in four of five simplex individuals, but was absent in unaffected individuals in their family and 505 normal cohorts. KIF22 mRNA was detected in human bone, cartilage, joint capsule, ligament, skin, and primary cultured chondrocytes. In silico analysis of KIF22 protein structure indicates that Pro148 and Arg149 are important in maintaining hydrogen bonds in the ATP binding and motor domains of KIF22. We conclude that these mutations in KIF22 cause lepto-SEMDJL.

KIF20A mRNA and Its Product MKlp2 Are Increased During Hepatocyte Proliferation and Hepatocarcinogenesis

Mitotic kinesin-like protein 2 (MKlp2), a microtubule-associated motor, is required during mitosis exit for the final step of cytokinesis. It also contributes to retrograde vesicular trafficking from the Golgi apparatus to the endoplasmic reticulum in interphase. The KIF20A gene encoding MKlp2 is controlled by the E2F-retinoblastoma protein-p16 pathway, and its widely expressed mRNA is found in fetal and proliferating adult tissues. The expression pattern and function of MKlp2 in the adult liver, however, have not been investigated. We report herein that MKlp2 transiently accumulates in vivo during mouse liver regeneration after partial hepatectomy and is strongly overexpressed in preneoplastic and neoplastic mouse liver. In vitro in mitogen-stimulated primary hepatocytes, MKlp2 accumulated in the nucleus during the G2 phase of the cell cycle coincident with the mitotic kinase Aurora B. Human hepatoma cell lines exhibited high levels of MKlp2; however, it was undetectable in normal human hepatocytes. RNAi-mediated MKlp2 knockdown in hepatoma cells induced polyploidization consistent with its essential function in promoting cytokinesis and inhibited cell proliferation without inducing apoptosis. KIF20A mRNA was strongly accumulated in a large series of human hepatocellular carcinomas, with the highest expression observed in tumors with genomic instability. Accumulation of MKlp2 in normal proliferating, preneoplastic, and transformed hepatocytes suggests that MKlp2 contributes to both normal and pathologic hepatocyte proliferation and is linked to tumor aggressiveness in human hepatocellular carcinomas.

Phosphorylation of a mitotic kinesin-like protein and a MAPKKK by cyclin-dependent kinases (CDKs) is involved in the transition to cytokinesis in plants

Cytokinesis in eukaryotes involves specific arrays of microtubules (MTs), which are known as the "central spindle" in animals, the "anaphase spindle" in yeasts, and the "phragmoplast" in plants. Control of these arrays, which are composed mainly of bundled nonkinetochore MTs, is critically important during cytokinesis. In plants, an MAPK cascade stimulates the turnover of phragmoplast MTs, and a crucial aspect of the activation of this cascade is the interaction between the MAPKKK, nucleus- and phragmoplast-localized protein kinase 1 (NPK1) and the NPK1-activating kinesin-like protein 1 (NACK1), a key regulator of plant cytokinesis. However, little is known about the control of this interaction at the molecular level during progression through the M phase. We demonstrated that cyclin-dependent kinases (CDKs) phosphorylate both NPK1 and NACK1 before metaphase in tobacco cells, thereby inhibiting the interaction between these proteins, suggesting that such phosphorylation prevents the transition to cytokinesis. Failure to inactivate CDKs after metaphase prevents dephosphorylation of these two proteins, causing incomplete mitosis. Experiments with Arabidopsis NACK1 (AtNACK1/HINKEL) revealed that phosphorylated NACK1 fails to mediate cytokinesis. Thus, timely and coordinated phosphorylation by CDKs and dephosphorylation of cytokinetic regulators from prophase to anaphase appear to be critical for the appropriate onset and/or progression of cytokinesis.

Elk-1 interacts with dynein upon serum stimulation but independent of serine 383 phosphorylation.

Elk-1 belongs to the Ternary Complex Factor (TCF) subfamily of the ETS (from E26 viral oncogene) domain superfamily of transcription factors, and has been known as a regulator of mitogen-induced immediate early gene transcription upon Mitogen Activated Protein Kinase (MAPK) activation. Elk-1 has been previously shown to interact with neuronal microtubules, and here we show that P-S383-Elk-1, in addition to co-localizing with motor proteins kinesin, Eg5 and Mitotik Kinesin-Like Protein (MKLP) at the mitotic spindles, it physically interacts with dynein in a serum induction-dependent but Ser383 phosphorylation-independent manner.

Structural Basis of Specific Binding between Aurora A and TPX2 by Molecular Dynamics Simulations

In the present study, the impacts of G198N and W128F mutations on the recognition between Aurora A and targeting protein of Xenopus kinesin-like protein 2 (TPX2) were investigated using molecular dynamics (MD) simulations, free energy calculations, and free energy decomposition analysis. The predicted binding free energy of the wild-type complex is more favorable than those of three mutants, indicating that both single and double mutations are unfavorable for the Aurora A and TPX2 binding. It is also observed that the mutations alternate the binding pattern between Aurora A and TPX2, especially the downstream of TPX2. An intramolecular hydrogen bond between the atom OD of Asp11(TPX2) and the atom HE1 of Trp34(TPX2) disappear in three mutants and thus lead to the instability of the secondary structure of TPX2. The combination of different molecular modeling techniques is an efficient way to understand how mutation has impacts on the protein-protein binding and our work gives valuable information for the future design of specific peptide inhibitors for Aurora A.

Role of Survivin in cytokinesis revealed by a separation-of-function allele

The chromosomal passenger complex (CPC), containing Aurora B kinase, Inner Centromere Protein, Survivin, and Borealin, regulates chromosome condensation and interaction between kinetochores and microtubules at metaphase, then relocalizes to midzone microtubules at anaphase and regulates central spindle organization and cytokinesis. However, the precise role(s) played by the CPC in anaphase have been obscured by its prior functions in metaphase. Here we identify a missense allele of Drosophila Survivin that allows CPC localization and function during metaphase but not cytokinesis. Analysis of mutant cells showed that Survivin is essential to target the CPC and the mitotic kinesin-like protein 1 orthologue Pavarotti (Pav) to the central spindle and equatorial cell cortex during anaphase in both larval neuroblasts and spermatocytes. Survivin also enabled localization of Polo kinase and Rho at the equatorial cortex in spermatocytes, critical for contractile ring assembly. In neuroblasts, in contrast, Survivin function was not required for localization of Rho, Polo, or Myosin II to a broad equatorial cortical band but was required for Myosin II to transition to a compact, fully constricted ring. Analysis of this "separation-of-function" allele demonstrates the direct role of Survivin and the CPC in cytokinesis and highlights striking differences in regulation of cytokinesis in different cell systems.

Lack of Association of KIF6 Genotype With Vascular Disease and Statin Response

### Study Hypothesis A common polymorphism in the kinesin-like protein 6 ( KIF6 ) gene, the Trp719Arg variant, was reported in multiple exploratory studies to be associated with both vascular risk and response to statin therapy. The clinical implication of these findings was that noncarriers of the Trp719Arg variant receive little benefit from statin therapy, suggesting clinical utility for a KIF6 pharmacogenetic test to predict the suitability of statin therapy for individual patients. More recent publications have disputed the reported associations. The authors of both studies described here (Hopewell et al1 and Ridker et al2) sought to definitively confirm or reject the hypothesis that the Trp719Arg variant affects the response to statin therapy with respect to incident vascular disease. ### How Was the Hypothesis Tested? Hopewell et al1 performed a retrospective analysis of data from the Heart Protection Study (HPS), in which more than 20 000 patients with established vascular disease or risk factors for vascular disease were randomly assigned to the use of 40 mg simvastatin daily versus placebo. Genotyping of the KIF6 Trp719Arg polymorphism (also designated rs20455) was successfully performed in 18 348 HPS participants of self-reported white ethnicity, with almost equal numbers from the simvastatin arm (n=9167) and the placebo arm (n=9181). The primary outcome was incident major vascular events (defined as …

KLP6: a newly identified kinesin that regulates the morphology and transport of mitochondria in neuronal cells

Mitochondria utilize diverse cytoskeleton-based mechanisms to control their functions and morphology. Here, we report a role for kinesin-like protein KLP6, a newly identified member of the kinesin family, in mitochondrial morphology and dynamics. An RNA interference screen using Caenorhabditis elegans led us to identify a C. elegans KLP-6 involved in maintaining mitochondrial morphology. We cloned a cDNA coding for a rat homolog of C. elegans KLP-6, which is an uncharacterized kinesin in vertebrates. A rat KLP6 mutant protein lacking the motor domain induced changes in mitochondrial morphology and significantly decreased mitochondrial motility in HeLa cells, but did not affect the morphology of other organelles. In addition, the KLP6 mutant inhibited transport of mitochondria during anterograde movement in differentiated neuro 2a cells. To date, two kinesins, KIF1Bα and kinesin heavy chain (KHC; also known as KIF5) have been shown to be involved in the distribution of mitochondria in neurons. Expression of the kinesin heavy chain/KIF5 mutant prevented mitochondria from entering into neurites, whereas both the KLP6 and KIF1Bα mutants decreased mitochondrial transport in axonal neurites. Furthermore, both KLP6 and KIF1Bα bind to KBP, a KIF1-binding protein required for axonal outgrowth and mitochondrial distribution. Thus, KLP6 is a newly identified kinesin family member that regulates mitochondrial morphology and transport.

Arabidopsis thaliana MAP65-1 and MAP65-2 function redundantly with MAP65-3/PLEIADE in cytokinesis downstream of MPK4

Plant cytokinesis occurs by the growth of cell plates from the interior to the periphery of the cell. These dynamic events in cytokinesis are mediated by a plant-specific microtubule (MT) array called the phragmoplast, which consists of bundled antiparallel MTs between the two daughter nuclei. The NACK-PQR pathway, a NACK1 kinesin-like protein and mitogen activated protein kinase (MAPK) cascade, is a key regulator of plant cytokinesis through the regulation of phragmoplast MTs. The MT-associated protein MAP65 has been identified as one of the structural components of MT assays involved in cell division, and we recently showed that Arabidopsis AtMAP65-3/PLEIADE (PLE) is a substrate of MPK4 that is a component of the NACK-PQR pathway in Arabidopsis. Here we show that AtMAP65-1 and AtMAP65-2 are also phosphorylated by MPK4. AtMAP65-1 and AtMAP65-2 that localize to the phragmoplast were phosphorylated by MPK4 in vitro. Although mutants of the Arabidopsis AtMAP65-1 and AtMAP65-2 genes exhibited a wild-type phenotype, double mutations of AtMAP65-3 and AtMAP65-1 or AtMAP65-2 caused more severe growth and cytokinetic defects than the single atmap65-3/ple mutation. These results suggest that AtMAP65-1 and AtMAP65-2 also function in cytokinesis downstream of MPK4.

Kinesin-Like Protein 6 (KIF6) Polymorphism and the Efficacy of Rosuvastatin in Primary Prevention

Hypothesis-generating data raise the possibility that carriers of the kinesin-like protein 6 (KIF6) 719 arginine (Arg) allele preferentially benefit from statin therapy, and, on this basis, a commercial assay for KIF6 has been developed. In the recently completed JUPITER trial, men and women without prior cardiovascular disease or diabetes who had baseline low-density lipoprotein cholesterol <130 mg/dL and high-sensitivity C-reactive protein ≥ 2 mg/L were randomly allocated to rosuvastatin 20 mg daily or to placebo and followed for first major vascular events (nonfatal myocardial infarction, nonfatal stroke, hospitalization for unstable angina, arterial revascularization, or vascular death) and for all-cause mortality. We evaluated the effect of polymorphism at rs20455 encoding the KIF6 719Arg allele on outcomes in this primary prevention trial, both among Caucasian participants and in the trial as a whole. Among 8781 Caucasian trial participants, we observed no increase in vascular event rates among carriers of the KIF6 719Arg allele as compared with noncarriers (hazard ratio, 0.91; 95% confidence interval, 0.66 to 1.26) nor any difference in percent low-density lipoprotein cholesterol reduction with rosuvastatin according to genotype (-52 versus -52 mg/dL, P = 0.11). Rosuvastatin allocation was associated with an almost identical reduction in the trial primary end point among carriers (hazard ratio, 0.61; 95% confidence interval, 0.43 to 0.87) as among noncarriers (hazard ratio, 0.59; 95% confidence interval, 0.39 to 0.88) (P-interaction = 0.90). Genotype had no impact on rosuvastatin efficacy in further analyses that included all-cause mortality, in analyses conducted in the total trial cohort that adjusted for race, or in analyses using generalized models of inheritance rather than recessive models. In the large primary prevention JUPITER trial, rosuvastatin was equally effective at reducing cardiovascular event rates among carriers and noncarriers of the KIF6 719Arg allele. Thus, at least for rosuvastatin, there appears to be no clinical utility to screening for KIF6 genotype as a method to determine vascular risk or to predict statin efficacy. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00239681.

Survival bias and drug interaction can attenuate cross-sectional case-control comparisons of genes with health outcomes. An example of the kinesin-like protein 6 (KIF6) Trp719Arg polymorphism and coronary heart disease.

BackgroundCase-control studies typically exclude fatal endpoints from the case set, which we hypothesize will substantially underestimate risk if survival is genotype-dependent. The loss of fatal cases is particularly nontrivial for studies of coronary heart disease (CHD) because of significantly reduced survival (34% one-year fatality following a coronary attack). A case in point is the KIF6 Trp719Arg polymorphism (rs20455). Whereas six prospective studies have shown that carriers of the KIF6 Trp719Arg risk allele have 20% to 50% greater CHD risk than non-carriers, several cross-sectional case-control studies failed to show that carrier status is related to CHD. Computer simulations were therefore employed to assess the impact of the loss of fatal events on gene associations in cross-sectional case-control studies, using KIF6 Trp719Arg as an example.ResultsTen replicates of 1,000,000 observations each were generated reflecting Canadian demographics. Cardiovascular disease (CVD) risks were assigned by the Framingham equation and events distributed among KIF6 Trp719Arg genotypes according to published prospective studies. Logistic regression analysis was used to estimate odds ratios between KIF6 genotypes. Results were examined for 33%, 41.5%, and 50% fatality rates for incident CVD.In the absence of any difference in percent fatalities between genotypes, the odds ratios (carriers vs. noncarriers) were unaffected by survival bias, otherwise the odds ratios were increasingly attenuated as the disparity between fatality rates increased between genotypes. Additional simulations demonstrated that statin usage, shown in four clinical trials to substantially reduce the excess CHD risk in the KIF6 719Arg variant, should also attenuate the KIF6 719Arg odds ratio in case-control studies.ConclusionsThese computer simulations show that exclusions of prior CHD fatalities attenuate odds ratios of case-control studies in proportion to the difference in the percent fatalities between genotypes. Disproportionate CHD survival for KIF6 Trip719Arg carriers is suggested by their 50% greater risk for recurrent myocardial infarction. This, and the attenuation of KIF6 719Arg carrier risk with statin use, may explain the genotype's weak association with CHD in cross-sectional case-control studies. The results may be relevant to the underestimation of risk in cross-sectional case-control studies of other genetic CHD-risk factors affecting survival.

ArabidopsisKinesin KP1 Specifically Interacts with VDAC3, a Mitochondrial Protein, and Regulates Respiration during Seed Germination at Low Temperature

The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochondrial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3 (VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 4° C, KP1 dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plant-specific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3 regulate aerobic respiration during seed germination at low temperature.

Bidirectional regulation of thermotaxis by glutamate transmissions inCaenorhabditis elegans

In complex neural circuits of the brain, massive information is processed with neuronal communication through synaptic transmissions. It is thus fundamental to delineate information flows encoded by various kinds of transmissions. Here, we show that glutamate signals from two distinct sensory neurons bidirectionally affect the same postsynaptic interneuron, thereby producing the opposite behaviours. EAT-4/VGLUT (vesicular glutamate transporter)-dependent glutamate signals from AFD thermosensory neurons inhibit the postsynaptic AIY interneurons through activation of GLC-3/GluCl inhibitory glutamate receptor and behaviourally drive migration towards colder temperature. By contrast, EAT-4-dependent glutamate signals from AWC thermosensory neurons stimulate the AIY neurons to induce migration towards warmer temperature. Alteration of the strength of AFD and AWC signals led to significant changes of AIY activity, resulting in drastic modulation of behaviour. We thus provide an important insight on information processing, in which two glutamate transmissions encoding opposite information flows regulate neural activities to produce a large spectrum of behavioural outputs.

Mutation of RiceBC12/GDD1, Which Encodes a Kinesin-Like Protein That Binds to a GA Biosynthesis Gene Promoter, Leads to Dwarfism with Impaired Cell Elongation

The kinesins are a family of microtubule-based motor proteins that move directionally along microtubules and are involved in many crucial cellular processes, including cell elongation in plants. Less is known about kinesins directly regulating gene transcription to affect cellular physiological processes. Here, we describe a rice (Oryza sativa) mutant, gibberellin-deficient dwarf1 (gdd1), that has a phenotype of greatly reduced length of root, stems, spikes, and seeds. This reduced length is due to decreased cell elongation and can be rescued by exogenous gibberellic acid (GA₃) treatment. GDD1 was cloned by a map-based approach, was expressed constitutively, and was found to encode the kinesin-like protein BRITTLE CULM12 (BC12). Microtubule cosedimentation assays revealed that BC12/GDD1 bound to microtubules in an ATP-dependent manner. Whole-genome microarray analysis revealed the expression of ent-kaurene oxidase (KO2), which encodes an enzyme involved in GA biosynthesis, was downregulated in gdd1. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that GDD1 bound to the element ACCAACTTGAA in the KO2 promoter. In addition, GDD1 was shown to have transactivation activity. The level of endogenous GAs was reduced in gdd1, and the reorganization of cortical microtubules was altered. Therefore, BC12/GDD1, a kinesin-like protein with transcription regulation activity, mediates cell elongation by regulating the GA biosynthesis pathway in rice.

Structures of the Motor Subunits from a Novel Asymmetric Kinesin Provide Insight Into its Motile Mechanism

The spatial arrangement of a cell's contents, and its ability to redistribute them, is fundamental to its survival. In all animal and plant cells, kinesins fill a large part of this need, hauling chromosomes and vital cargo-containing sacks to their required destinations. Many kinesins operate as a homodimer in which the two subunits coordinate their movement along microtubules and perform a single cellular function. However, a few kinesins in certain species mix-and-match different molecules in ways that allow the motor to perform multiple functions. Certain types of pathogenic fungi harbor an unusual group of kinesins that form heterodimeric complexes with one or more non-catalytic kinesin-like proteins that regulate the function and cellular localization of their catalytic partner. A challenge in the motor protein field is to provide a mechanistic view of how these asymmetric motors move using this unconventional form of motor subunit arrangement. Our recent determination of the X-ray crystal structures of the motor domain region of both the catalytic and non-catalytic subunits of a heterodimeric kinesin from Candida glabrata revealed that certain regions of the latter subunit are highly dynamic. Specifically, our crystals of CgVik1 (the non-catalytic subunit) exhibit three very different conformations of an alpha-helical segment that is analogous to the force-producing ‘neck’ element of kinesins. The intramolecular interactions of the CgVik1 neck and its motor core differ in each conformation and are accompanied by movements in elements that are analogous to the nucleotide binding ‘P-loop’ and part of the microtubule binding surface of catalytic kinesins. This suggests a functional link between CgVik1 neck orientation and microtubule interactions and motility of the motor complex, and sheds light on how this kinesin works at the atomic level as an asymmetric motor complex.

Klp Navigation of Spindle Assembly from Poles

Spindle assembly is the crucial initiating step in the mitotic mechanism. Its formation utilizes coordinated actions of Kinesin-like proteins (Klps) at poles, on microtubules and at chromosomes. We previously identified a novel conserved Kinesin-14 pole-based mechanism for spindle assembly by Klp binding to the γ-tubulin small complex (γ-TuSC) microtubule organizing center, (MTOC). Changes to the γ-TuSC MTOC in fission yeast alter microtubule dynamics to regulate bipolarity and both human and fission yeast Kinesin-14 Klps can regulate this mechanism. The Kinesin-14 Pkl1 motor domain associates with residues in helix 11 of γ-tubulin at a novel Klp binding site. The Kinesin-14 Pkl1 Tail domain is distinct from that of the well-characterized Drosophila Ncd, replacing microtubule association sites with specialized elements for spindle pole targeting and regulation of the γ-TuSC. Thus unlike Drosophila Ncd that crosslinks microtubules to stabilize mitotic spindle assembly, Kinesin-14 Pkl1 regulates assembly through the γ-TuSC. Klps can be classified into 14 families. Members of two ubiquitous families, Kinesin-14 and opposing Kinesin-5 Klps, each exhibit distinctions at multiple levels amongst members, including domain elements, functional mechanisms and spindle localization. In fission yeast, Kinesin-5 also localizes to spindle poles, consistent with the hypothesis that similar localization is needed to optimally counterbalance forces by these two Klp families. Although the stoichiometry of proteins within γ-TuSC is known and conserved in eukaryotes little is understood of the γ-TuSC mechanism. Cross-species analysis is defining shared and distinct functions and novel insights into the γ-TuSC interactome and the role of associating Klps in regulating its mechanism for spindle assembly. Our studies apply site directed mutagenesis, genetics, yeast two hybrid, biochemistry, timelapse microscopy and bioinformatic/structural approaches with novel reagents we have developed for Klp and γ-TuSC analysis.

Coiled-Coil Stalk of Active Kinesin-Like Protein CENP-E is Stably Folded

CENP-E is a plus-end directed kinesin-like motor, which resides at the kinetochores of mitotic chromosomes. Its activity contributes to chromosome congression in metaphase, and to the capture and stabilization of kinetochore microtubules. Full length CENP-E from Xenopus laevis has a flexible 230nm-long coiled coil stalk, whose function is not known. The stalk is thought to be extended when the motor is active, but it becomes folded in a phosphorylation-dependent manner when the motor domain is inhibited via intramolecular binding to the tail-domain. Here, we investigate the role of this stalk in vitro by studying the motility of microbeads coated with purified recombinant full length CENP-E and a truncated construct, which lacks the stalk and the tail. We show that when conjugated to the beads, both constructs exhibit similar velocities and run lengths even in the absence of in vitro phosphorylation, indicating that the full length CENP-E is entirely active. However, when we used tethered particle motion analysis of the beads carried along microtubules by a single full length motor, we found that CENP-E was not fully extended and behaved as a 40-70nm tether. Phosphorylation of CENP-E with CDK1, Aurora A or MPS1 kinases did not change this length, so this conformation does not depend on phosphorylation. To probe the elastic properties of this compact conformation we applied periodic sideways force to a bead moving along a microtubule. Our measurements demonstrate that the full length CENP-E can be stretched up to 100nm, but it cannot be fully unfolded even by the forces up to 5pN. Together, these data suggest that the compact conformation of CENP-E is stable and phosphorylation-independent, and it does not interfere with normal motor activity.

Detection of the quantity of kinesin and microgravity-sensitive kinesin genes in rat bone marrow stromal cells grown in a simulated microgravity environment

Kinesin and kinesin-like proteins (KLPs) constitute a superfamily of microtubule motor proteins found in all eukaryotic organisms. Members of the kinesin superfamily are known to play important roles in many fundamental cellular and developmental processes. To date, few published studies have reported on the effects of microgravity on kinesin expression. In this paper, we describe the expression pattern and microgravity-sensitive genes of kinesin in rat bone marrow stromal cells cultured in a ground-based rotating bioreactor. The quantity of kinesin under the clinorotation condition was examined by immunoblot analysis with anti-kinesin. Furthermore, the distribution of kinesin at various times during clinorotation was determined by dual immunostaining, using anti-kinesin monoclonal antibody or anti-β-tubulin monoclonal antibody. In terms of kinesin quantity, we found that the ratios of the amounts of clinorotated/stationary KLPs decreased from clinorotation day 5 to day 10, although it increased on days 2 and 3. Immunofluorescence analysis revealed that kinesin in the nucleus was the first to be affected by simulated microgravity, following the kinesin at the periphery that was affected at various times during clinorotation. Real-time RT-PCR analysis of kinesin mRNA expression was performed and led to the identification of 3 microgravity-sensitive kinesin genes: KIF9, KIFC1, and KIF21A. Our results suggest that kinesin has a distinct expression pattern, and the identification of microgravity-sensitive kinesin genes offers insight into fundamental cell biology.

250 The polymorphism Trp719Arg in the kinesin-like protein 6 is associated with the presence of late outgrowth endothelial progenitor cells in acute myocardial infarction

Much attention is focused on genetic polymorphisms associated with coronary artery disease. A candidate gene controversially associated with risk of coronary events is the kinesin-like protein 6 (KIF6), which is correlated with Trp719Arg polymorphism. In acute myocardial infarction (AMI), endothelial progenitor cells, particularly endothelial colony forming cells (ECFC) are mobilized from bone marrow and correlated with infarct size reduction. We investigated whether there was a relationship between presence of ECFC in AMI at admission and genetic status with regard to the KIF6 Trp719Arg polymorphism (rs20455). Forty five patients aged <75 years old referred for a first STEMI or non STEMI AMI. Peripheral blood samples were drawn on admission. Isolated peripheral blood mononuclear cells were obtained by Hypaque-Ficoll density gradient centrifugation and cultured for 4 weeks. Cultured cells were phenotyped to assess the endothelial origin of ECFC. Genomic DNA was extracted in all patients and genotyping for allelic variations of KIF6 was performed. Subjects were divided into two groups comparing the (Arg/Arg) homozygote variants with patients having a Trp allele. The genotype frequencies were 55%, 31% and 13% for Arg/Arg, Arg/Trp and Trp/Trp respectively. Between groups, higher levels of TnI, CK and CK-MB wer observed in the Arg/Arg group (respectively p = 0.026, p = 0.001 and p = 0.031). ECFC were observed in 33% of patients with AMI. The percentage of patients without ECFC was significantly higher in the Arg/Arg group (p = 0.033). No other significant differences were observed between groups. In this report the Arg/Arg group showed a high number of ECFC-negative patients. A possible explanation might be the low mobilization of ECFC from bone marrow in this genotype since KIF6 is involved in cytokinesis. The altered amino acid Trp719Arg could decrease the ECFC released from the bone marrow in response to chemokines released at the onset of AMI.