Kinesin Gene Research Articles

A tomato kinesin-like protein is associated with Tobacco mosaic virus infection

Tobacco mosaic virus (TMV) causes severe damage and economic losses of tomato in crop yield production and quality worldwide. Tomato plants infected with TMV usually show a mosaic pattern and chlorosis symptoms that result from affected chloroplasts. To elucidate tomato–TMV interactions, the transcriptional profiles of tomato tissues at different time intervals were analysed using a differential display-polymerase chain reaction technique. Compared to mock-inoculated plants, and based on gene expression changes in tomato plants, the representative down- and up-regulated genes were selected for further analysis. The Basic Local Alignment Search Tool at the National Center for Biotechnology Information (NCBI-BLAST) analysis revealed that the three up-regulated gene transcripts at 15 days post-inoculation were coding for chloroplast (MG565980 and MG565979) and kinesin-like proteins (MG565981). Additionally, the down-regulated gene was a chloroplast-related gene (MG565978). Interestingly, the two chloroplast genes, MG565978 and MG565980, shared more than 96% sequence identity, and their transmembrane profiles were nearly identical. On the other hand, many protein kinase C phosphorylation, casein kinase II phosphorylation and N-myristoylation sites were detected within the MG565981 gene. Moreover, the high similarity between the kinesin gene and many Arabidopsis defensin genes indicated that it might play an important role in the plant defence system against TMV infection. It should be stressed that studies of the pathways in which chloroplast and kinesin-like protein genes are involved may elucidate the mechanisms of tomato tolerance to viral infection and can lead us to a more comprehensive understanding of tomato–TMV interactions.

Expression profiling of kinesins, involved in the development of autophagy in Arabidopsis thaliana, and the role of tubulin acetylation in the interaction of Atg8 protein with microtubules

Aim. To investigate the interrelation between changes in the expression levels of kinesin genes that are potentially involved in the development of stress-induced autophagy in Arabidopsis thaliana by means of microtubules, and the structural biology analysis of the role of α-tubulin acetylation in the regulation of interaction of α-tubulin with Atg8. Methods. The simulation of the influence of abiotic stresses. PCR analysis of changes in expression levels of kinesin genes. The molecular dynamics simulations of α-tubulin and Atg8 complexes were performed using the GROMACS 4.5.5 program. Results. It was shown that the changes in expression levels were caused by the influence of stressful stimuli. A significant increase in the transcriptional activity of the KIN5B, KIN12B, KIN12F genes after UV-B irradiation, the KIN6, KIN7O, KIN7D, KIN12B genes under osmotic-, and KIN6, KIN12B under salt stress was detected. By means of bioinformatics it was demonstrated that α-tubulin acetylation provides an enhanced interaction of α-tubulin and Atg8 protein. Conclusions. Obtained data point out the important role of kinesins and α-tubulin acetylation in realization of microtubules’ partaking in the development of stress-induced autophagy in plants.
 Keywords: microtubules, α-tubulin, kinesins, Atg8 protein, stress-induced autophagy.

An improved method for vitrification of in vitro matured ovine oocytes; beneficial effects of Ethylene Glycol Tetraacetic acid, an intracellular calcium chelator

Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.

Axonal transport proteins and depressive like behavior, following Chronic Unpredictable Mild Stress in male rat

BackgroundA common mood disorder, depression has long been considered a leading cause of disability worldwide. Chronic stress is involved in the development of various psychiatric diseases including major depressive disorder. Stress can induce depressive-like symptoms and initiate neurodegenerative processes in the brain. The neurodegenerative theory of depression holds impaired axonal transport as a negative factor in neural survival. Axonal transport is a critical mechanism for normal neuronal function, playing crucial roles in axon growth, neurotransmitter secretion, normal mitochondrial function and neural survival. Methods and materialsTo investigate the effects of stress-induced depression, in the present study, we evaluated behavior by forced swimming test (FST), corticosterone plasma level by ELISA assay, hippocampal mRNA expression of three genes (NGF, kinesin and dynein) via real-time PCR and hippocamp count by Nissl staining in male Wistar rats. ResultsOur data demonstrated a significant decrease in the expression of NGF, kinesin and dynein genes in CUMS groups compared to the control group (non-stressed) (p < 0.05). CUMS also caused an elevation in immobility time and corticosterone plasma level in the stressed group compared to the controls (p < 0.01 and p < 0.05, respectively). ConclusionThe results suggested that the possibility of stress-induced depressive behavior associated with hippocampal neurodegeneration process is correlated with a low expression of kinesin and dynein, the two most important proteins in axonal transport.

Theoretical Investigations of the Role of Mutations in Dynamics of Kinesin Motor Proteins.

Motor proteins are active enzymatic molecules that are critically important for a variety of biological phenomena. It is known that some neurodegenerative diseases are caused by specific mutations in motor proteins that lead to their malfunctioning. Hereditary spastic paraplegia is one of such diseases, and it is associated with the mutations in the neuronal conventional kinesin gene, producing the decreased speed and processivity of this motor protein. Despite the importance of this problem, there is no clear understanding on the role of mutations in modifying dynamic properties of motor proteins. In this work, we investigate theoretically the molecular basis for negative effects of two specific mutations, N256S and R280S, on the dynamics of kinesin motor proteins. We hypothesize that these mutations might accelerate the adenosine triphosphate (ATP) release by increasing the probability of open conformations for the ATP-binding pocket. Our approach is based on the use of coarse-grained structure-based molecular dynamics simulations to analyze the conformational changes and chemical transitions in the kinesin molecule, which is also supplemented by investigation of a mesoscopic discrete-state stochastic model. Computer simulations suggest that mutations N256S and R280S can decrease the free energy difference between open and closed biochemical states, making the open conformation more stable and the ATP release faster, which is in agreement with our hypothesis. Furthermore, we show that in the case of N256S mutation, this effect is caused by disruption of interactions between α helix and switch I and loop L11 structural elements. Our computational results are qualitatively supported by the explicit analysis of the discrete-state stochastic model.

Tumor suppressor protein p53-mediated repression of human mitotic centromere-associated kinesin gene expression is exerted via down-regulation of Sp1 level.

The repressive role of p53 on the human mitotic centromere-associated kinesin (MCAK) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53‒/‒) than in HCT116 (p53+/+) cells. Enforced down-regulation of p53 levels either in HCT116 (p53+/+) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at −93/−84 and GC2 at −119/−110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53+/+) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53−/−) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants (p53V143A, p53R248W, and p53R273H) failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation.

Unexpected Roles for Ciliary Kinesins and Intraflagellar Transport Proteins.

Transport of proteins in the ciliary shaft is driven by microtubule-dependent motors, kinesins. Prior studies suggested that the heterotrimeric ciliary kinesin may be dispensable for certain aspects of transport in specialized cilia of vertebrate photoreceptor cells. To test this possibility further, we analyzed the mutant phenotype of the zebrafish kif3a gene, which encodes the common motor subunit of heterotrimeric ciliary kinesins. Cilia are absent in all organs examined, leading to the conclusion that kif3a is indispensable for ciliogenesis in all cells, including photoreceptors. Unexpectedly, kif3a function precedes ciliogenesis as ciliary basal bodies are mispositioned in mutant photoreceptors. This phenotype is much less pronounced in intraflagellar transport (IFT) mutants and reveals that kif3a has a much broader role than previously assumed. Despite the severity of their basal body phenotype, kif3a mutant photoreceptors survive longer compared to those in IFT mutants, which display much weaker basal body mispositioning. This effect is absent in kif3a;IFT double mutants, indicating that IFT proteins have ciliary transport-independent roles, which add to the severity of their photoreceptor phenotype. kif3a is dispensable for basal body docking in otic vesicle sensory epithelia and, surprisingly, short cilia form in mechanosensory cristae even in the absence of kif3a In contrast to Kif3a, the functions of the Kif3c-related protein, encoded by the kif3c-like (kif3cl) gene, and the homodimeric ciliary kinesin, kif17, are dispensable for photoreceptor morphogenesis. These studies demonstrate unexpected new roles for both ciliary heterotrimeric kinesins and IFT particle genes and clarify the function of kif17, the homodimeric ciliary kinesin gene.

XAF1 promotes neuroblastoma tumor suppression and is required for KIF1Bβ-mediated apoptosis.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bβ, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bβ deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bβ. XAF1 silencing protects from NGF withdrawal and from KIF1Bβ-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions.

Aberrantly activated Gli2-KIF20A axis is crucial for growth of hepatocellular carcinoma and predicts poor prognosis.

Glioma-associated oncogene 2 (Gli2), a primary transcriptional regulator of Hedgehog (Hh) signaling, is essential for hepatocellular carcinoma (HCC) growth and survival. However, the underlying molecular mechanism and crucial downstream targets of Gli2 in human HCC are not fully understood. Here, we report the identification of kinesin family member 20A (KIF20A) as a novel downstream target of Gli2, which is important for HCC proliferation and tumor growth. Inhibition of Hh signaling leads to a remarkable decrease of KIF20A expression in HCC cells, whereas overexpression of Gli2 elevates KIF20A expression by activating Forkhead Box M1 (FoxM1)-MMB complex-mediated transcription of this kinesin gene. Gli2-induced HCC cell growth requires enhanced expression of KIF20A, and knockdown of Gli2 or KIF20A represses the proliferation of HCC cells in vitro and in vivo. Correlated with these results, analyses of clinical HCC samples show that Gli2, FoxM1 and KIF20A are highly elevated in primary HCC samples and represent significant risk factors for HCC recurrence and survival. Conclusion: KIF20A is an important downstream target gene of Hh signaling. And, the Gli2-KIF20A axis is essential for the proliferation and growth of human HCC cells. Our study also suggests Gli2-KIF20A axis as a potential target for future therapeutic intervention and as an independent prognostic biomarker for HCC.

Abstract 1413: Significance of KIFC1 in spheroid formation of gastric cancer cells

Abstract Spheroid colony formation is a typically method to identify cancer stem cells (CSC). The growth of spherical colonies is considered indicative of self-renewal ability, and is consistent with a CSC phenotype. However, mechanism of spheroid formation remains unclear in gastric cancer (GC). In the present study, we analyzed spheroid formation associated genes in GC. Comprehensive gene expression profile of MKN-45 and MKN-74 GC cell lines that were grown as monolayers or spheroids was obtained by Affymetrix GeneChip, and we found several kinds of genes whose expression was up-regulated or down-regulated in cells that were grown as spheroids. Among these, 10 kinds of kinesin genes were up-regulated in both MKN-45 and MKN-74 cells that were grown as spheroids. Kinesins are categorized into 14 subfamilies and classified as mitotic kinesins, which are involved in cell division, and non-mitotic kinesins, which are principally involved in intracellular transport. In spheroids, mitotic kinesins were up-regulated. In the present study, we focused on KIFC1, because antibody against KIFC1 is available. qRT-PCR analysis revealed that up-regulation of KIFC1 in both MKN-45 and MKN-74 cells that were grown as spheroids. Immunohistochemical analysis of KIFC1 was performed in 20 GC cases. Expression of KIFC1 was up-regulated in GC tissues in comparison with corresponding non-neoplastic mucosa. These results suggest that KIFC1 plays an important role in gastric carcinogenesis and may be a therapeutic target of GC. Citation Format: Naohide Oue, Shoichiro Mukai, Trang Pham T.B., Kazuhiro Sentani, Reiko Ito, Wataru Yasui. Significance of KIFC1 in spheroid formation of gastric cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2015-1413

De novo mutations in KIF1A cause progressive encephalopathy and brain atrophy.

ObjectiveTo determine the cause and course of a novel syndrome with progressive encephalopathy and brain atrophy in children.MethodsClinical whole-exome sequencing was performed for global developmental delay and intellectual disability; some patients also had spastic paraparesis and evidence of clinical regression. Six patients were identified with de novo missense mutations in the kinesin gene KIF1A. The predicted functional disruption of these mutations was assessed in silico to compare the calculated conformational flexibility and estimated efficiency of ATP binding to kinesin motor domains of wild-type (WT) versus mutant alleles. Additionally, an in vitro microtubule gliding assay was performed to assess the effects of de novo dominant, inherited recessive, and polymorphic variants on KIF1A motor function.ResultsAll six subjects had severe developmental delay, hypotonia, and varying degrees of hyperreflexia and spastic paraparesis. Microcephaly, cortical visual impairment, optic neuropathy, peripheral neuropathy, ataxia, epilepsy, and movement disorders were also observed. All six patients had a degenerative neurologic course with progressive cerebral and cerebellar atrophy seen on sequential magnetic resonance imaging scans. Computational modeling of mutant protein structures when compared to WT kinesin showed substantial differences in conformational flexibility and ATP-binding efficiency. The de novo KIF1A mutants were nonmotile in the microtubule gliding assay.InterpretationDe novo mutations in KIF1A cause a degenerative neurologic syndrome with brain atrophy. Computational and in vitro assays differentiate the severity of dominant de novo heterozygous versus inherited recessive KIF1A mutations. The profound effect de novo mutations have on axonal transport is likely related to the cause of progressive neurologic impairment in these patients.

Somatic Mutations in Cerebral Cortical Malformations

BackgroundAlthough there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated.MethodsUsing a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing.ResultsValidated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria.ConclusionsTargeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.)

Motor protein mutations cause a new form of hereditary spastic paraplegia.

To identify a novel disease gene in 2 families with autosomal recessive hereditary spastic paraplegia (HSP). We used whole-exome sequencing to identify the underlying genetic disease cause in 2 families with apparently autosomal recessive spastic paraplegia. Endogenous expression as well as subcellular localization of wild-type and mutant protein were studied to support the pathogenicity of the identified mutations. In 2 families, we identified compound heterozygous or homozygous mutations in the kinesin gene KIF1C to cause hereditary spastic paraplegia type 58 (SPG58). SPG58 can be complicated by cervical dystonia and cerebellar ataxia. The same mutations in a heterozygous state result in a mild or subclinical phenotype. KIF1C mutations in SPG58 affect the domains involved in adenosine triphosphate hydrolysis and microtubule binding, key functions for this microtubule-based motor protein. KIF1C is the third kinesin gene involved in the pathogenesis of HSPs and is characterized by a mild dominant and a more severe recessive disease phenotype. The identification of KIF1C as an HSP disease gene further supports the key role of intracellular trafficking processes in the pathogenesis of hereditary axonopathies.

Kinesin Family Deregulation Coordinated by Bromodomain Protein ANCCA and Histone Methyltransferase MLL for Breast Cancer Cell Growth, Survival, and Tamoxifen Resistance

Kinesins are a superfamily of motor proteins and often deregulated in different cancers. However, the mechanism of their deregulation has been poorly understood. Through examining kinesin gene family expression in estrogen receptor (ER)-positive breast cancer cells, we found that estrogen stimulation of cancer cell proliferation involves a concerted regulation of specific kinesins. Estrogen strongly induces expression of 19 kinesin genes such as Kif4A/4B, Kif5A/5B, Kif10, Kif11, Kif15, Kif18A/18B, Kif20A/20B, Kif21, Kif23, Kif24, Kif25, and KifC1, whereas suppresses the expression of seven others, including Kif1A, Kif1C, Kif7, and KifC3. Interestingly, the bromodomain protein ANCCA/ATAD2, previously shown to be an estrogen-induced chromatin regulator, plays a crucial role in the up- and downregulation of kinesins by estrogen. Its overexpression drives estrogen-independent upregulation of specific kinesins. Mechanistically, ANCCA (AAA nuclear coregulator cancer associated) mediates E2-dependent recruitment of E2F and MLL1 histone methyltransferase at kinesin gene promoters for gene activation-associated H3K4me3 methylation. Importantly, elevated levels of Kif4A, Kif15, Kif20A, and Kif23 correlate with that of ANCCA in the tumors and with poor relapse-free survival of patients with ER-positive breast cancer. Their knockdown strongly impeded proliferation and induced apoptosis of both tamoxifen-sensitive and resistant cancer cells. Together, the study reveals ANCCA as a key mediator of kinesin family deregulation in breast cancer and the crucial role of multiple kinesins in growth and survival of the tumor cells. These findings support the development of novel inhibitors of cancer-associated kinesins and their regulator ANCCA for effective treatment of cancers including tamoxifen-resistant breast cancers.

The Arabidopsis kinesin gene AtKin-1 plays a role in the nuclear division process during megagametogenesis

Atkin - 1 , the only Kinesin-1 member of Arabidopsis thaliana , plays a role during female gametogenesis through regulation of nuclear division cycles. Kinesins are microtubule-dependent motor proteins found in eukaryotic organisms. They constitute a superfamily that can be further classified into at least 14 families. In the Kinesin-1 family, members from animal and fungi play roles in long-distance transport of organelles and vesicles. Although Kinesin-1-like sequences have been identified in higher plants, little is known about their function in plant cells, other than in a recently identified Kinesin-1-like protein in a rice pollen semi-sterile mutant. In this study, the gene encoding the only Kinesin-1 member in Arabidopsis, AtKin-1 was found to be specifically expressed in ovules and anthers. AtKin-1 loss-of-function mutants showed substantially aborted ovules in siliques, and this finding was supported by complementation testing. Reciprocal crossing between mutant and wild-type plants indicated that a defect in AtKin-1 results in partially aborted megagametophytes, with no observable effects on pollen fertility. Further observation of ovule development in the mutant pistils indicated that the enlargement of the megaspore was blocked and nuclear division arrested at the one-nucleate stage during embryo sac formation. Our data suggest that AtKin-1 plays a role in the nuclear division cycles during megagametogenesis.

The Genome of the Foraminiferan Reticulomyxa filosa

Rhizaria are a major branch of eukaryote evolution with an extensive microfossil record, but only scarce molecular data are available. The rhizarian species Reticulomyxa filosa, belonging to the Foraminifera, is free-living in freshwater environments. In culture, it thrives only as a plasmodium with thousands of haploid nuclei in one cell. The R. filosa genome is the first foraminiferal genome to be deciphered. The genome is extremely repetitive, and the large amounts of identical sequences hint at frequent amplifications and homologous recombination events. Presumably, these mechanisms are employed to provide more gene copies for higher transcriptional activity and to build up a reservoir of gene diversification in certain gene families, such as the kinesin family. The gene repertoire indicates that it is able to switch to a single-celled, flagellated sexual state never observed in culture. Comparison to another rhizarian, the chlorarachniophyte alga Bigelowiella natans, reveals that proteins involved in signaling were likely drivers in establishing the Rhizaria lineage. Compared to some other protists, horizontal gene transfer is limited, but we found evidence of bacterial-to-eukaryote and eukaryote-to-eukaryote transfer events. The R. filosa genome exhibits a unique architecture with extensive repeat homogenization and gene amplification, which highlights its potential for diverse life-cycle stages. The ability of R. filosa to rapidly transport matter from the pseudopodia to the cell body may be supported by the high diversification of actin and kinesin gene family members.

Whole Exome Sequencing Detecting Kinesin Family Gene Defects In Myeloid Neoplasm

Clinical and pathomorphologic diversity in MDS is a reflection of heterogeneity of molecular lesions. Somatic mutations and chromosomal deletions/amplifications affect various pathways in a convergent and divergent fashion, generate phenocopy and can occur in a variety of combinations. Recent technological advances, including high density arrays and the new generation sequencing (NGS) led to the discovery of novel pathway mutations or gene families affected by somatic defects, e.g., cohesin or spliceosomal mutations.We have performed whole exome NGS of paired (tumor/germ line) samples in 222 patients with myeloid neoplasms from the Cleveland Clinic and University of Tokyo. Clinical parameters were studied including age, gender, overall survival (OS), bone marrow blast count, and metaphase cytogenetics. Additionally, we also used in our analysis data sets from 197 AML included in the Cancer Genome Atlas (TCGA). We found 1.4% (6/419) of non-canonical somatic mutations of KIF2Bwhich is a member of kinesin13 family located on the long arm of chromosome 17; 3 cases from our cohort (p.V32M (c.G94A), p.T113M (c.C338T), p.R163C (c.C487T)) and 3 cases from TCGA database (p.T47M (c.C140T), p.T310M (c.C929T), p.H551N (c.C1651A)).By analyzing clonal architecture and intra-tumor heterogeneity in 2 cases (RCMD and RAEB) by targeted deep sequencing, allelic frequencies of KIF2B mutations were more than 45% and larger than for any other concomitant mutations, suggesting that KIF2B mutations might consequently constitute ancestral events followed by subclonal acquisitions of the other mutations. Of note is that 6 non-sense mutations were also reported in lung cancer. Based on SNP-array mapping of chromosomal abnormalities, deletions of 17q involving the KIF2B locus (17q22) was present about 3% (6/215) of myeloid neoplasm. KIF2B defects were frequently detected in higher-risk MDS and AML phenotypes (9%). KIF2B performed an important role in regulation of kinetochore-microtubule attachment. Previous studies showed that the velocity of chromosomes' movement in KIF2B-deficient cells is reduced 80% comparing to control and fail to perform cytokinesis. In our series, 56% of myeloid neoplasms with KI2B defects had complex cytogenetics and 67% cases of them were also UPD, suggesting that KIF2B defects might lead to inducing abnormal chromosomal movements and segregations. We then, expanded our study to the whole kinesin gene family: 17 somatic mutations and 57 deletions were identified in KIF1A (n=6), KIF23 (n=1), KIF26A (n=1), KIF27 (n=7), KIF1C (n=9), KIF21B (n=2), KIF13A (n=10), KIF14 (n=2), KIF17 (n=15), KIF25 (n=1), KIF3C (n=8), KIF6 (n=2) and CENPE (n=10). All mutations were heterozygous and mutually exclusive. By survival analysis of such mutated cases, a tendency towards worse prognosis was observed (HR; 1.72, 95%CI 0.86-3.37). Analysis of concomitant mutations associated with whole kinesin family mutations or deletions showed that most frequently affected genes are TET2 (n=14), DNMT3A (n=8), IDH1/2 (n=8) and MLL (n=5), all involved in epigenetic regulation.In conclusion, somatic mutations in kinesin family genes are found in myeloid malignancies and might be responsible for another pathogenesis of the disease. KIF2B is most frequently found in myeloid malignancies and associated with aggressive type of MDS. Since knockout mice of multiple kinesin family genes (KIF5A, KIF16B and EG5) were lethal in embryo and all the mutations occur in a heterozygous configuration, it is likely synthetic lethal approach might create therapeutic window between defective malignant cells and healthy controls. Kinesin family of motor proteins may be an emerging novel therapeutic target. In fact some kinesins have been already successfully targeted in solid tumors. Disclosures:Polprasert:MDS foundation: Research Funding. Makishima:AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Molecular characterization and expression analysis of a KIFC1-like kinesin gene in the testis of Eumeces chinensis.

The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339bp that contained a 216bp 5'-untranslated region (5'UTR), a 194bp 3'-untranslated region (3'UTR) and a 1,929bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116aa, a stalk domain from 117 to 291aa and a conserved carboxyl motor domain from 292 to 642aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8% identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.

The expression pattern of the C-terminal kinesin gene kifc1 during the spermatogenesis of Sepiella maindroni

In this study, we investigated the gene sequence and characteristic of kifc1 in Sepiella maindroni through PCR and RACE technology. Our research aimed particularly at the spatio-temporal expression pattern of kifc1 in the developmental testis through in situ hybridization. The particular role of kifc1 in the spermatogenesis of S. maindroni was our particular interest. Based on multiple protein sequence alignments of KIFC1 homologues, kifc1 gene from the testis of S. maindroni was identified, which consisted of 2432bp including a 2109 in-frame ORF corresponding to 703 continuous amino acids. The encoded polypeptide shared highest similarity with Octopus tankahkeei. Through the prediction of the secondary and tertiary structures, the motor domain of KIFC1 was conserved at the C-terminal, having putative ATP-binding and microtubule-binding motifs, while the N-terminal was more specific to bind various cargoes for cellular events. The stalk domain connecting between the C-terminal and N-terminal determined the direction of movement. According to RT-PCR results, the kifc1 gene is not tissue-specific, commonly detected in different tissues, for example, the testis, liver, stomach, muscle, caecum and gills. Through an in situ hybridization method, the expression pattern of KIFC1 protein mimics in the spermatogenesis of S. maindroni. During the primary stage of the spermatogenesis, the kifc1 mRNA signal was barely detectable. At the early spermatids, the signal started to be present. With the elongation of spermatids, the signals increased substantially. It peaked and gathered around the acrosome area when the spermatids began to transform to spindle shape. As the spermatids developed into mature sperm, the signal vanished. In summary, the expression of kfic1 at specific stages during spermiogenesis and its distribution shed light on the potential functions of this motor in major cytological transformations. The KIFC1 homologue may provide a direct shaping force to the nucleus or influence the shaping process through indirect regulation.

Characterization and expression profiling of cucumber kinesin genes during early fruit development: revealing the roles of kinesins in exponential cell production and enlargement in cucumber fruit

Rapid cell division and expansion in early fruit development are important phases for cucumber fruit yield and quality. Kinesin proteins are microtubule-based motors responsible for modulating cell division and enlargement. In this work, the candidate kinesin genes involved in rapid cell division and expansion during cucumber fruit development were investigated. The morphological and cellular changes during early fruit development were compared in four cucumber genotypes with varied fruit size. The correlation between the expression profiles of cucumber kinesin genes and cellular changes in fruit was investigated. Finally, the biochemical characteristics and subcellular localizations of three candidate kinesins were studied. The results clarified the morphological and cellular changes during early cucumber fruit development. This study found that CsKF2–CsKF6 were positively correlated with rapid cell production; CsKF1 and CsKF7 showed a strongly positive correlation with rapid cell expansion. The results also indicated that CsKF1 localized to the plasma membrane of fast-expanding fruit cells, that CsKF2 might play a role in fruit chloroplast division, and that CsKF3 is involved in the function or formation of phragmoplasts in fruit telophase cells. The results strongly suggest that specific fruit-enriched kinesins are specialized in their functions in rapid cell division and expansion during cucumber fruit development.