Mechanism of spindle pole organization and instability in human oocytes.

Human oocytes are prone to assembling meiotic spindles with unstable poles, which can favor aneuploidy in human eggs. The underlying causes of spindle instability are unknown. We found that NUMA (nuclear mitotic apparatus protein)-mediated clustering of microtubule minus ends focused the spindle poles in human, bovine, and porcine oocytes and in mouse oocytes depleted of acentriolar microtubule-organizing centers (aMTOCs). However, unlike human oocytes, bovine, porcine, and aMTOC-free mouse oocytes have stable spindles. We identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing protein that is deficient in human oocytes. Depletion of KIFC1 recapitulated spindle instability in bovine and aMTOC-free mouse oocytes, and the introduction of exogenous KIFC1 rescued spindle instability in human oocytes. Thus, the deficiency of KIFC1 contributes to spindle instability in human oocytes.

Deletion of kif3a in CK19 positive cells leads to primary cilia loss, biliary cell proliferation and cystic liver lesions in TAA-treated mice.

BACKGROUND & AIMS: Loss of primary cilia in epithelial cells is known to cause cystic diseases of the liver and kidney. We have previously shown that during experimental and human cirrhosis that primary cilia were predominantly expressed on biliary cells in the ductular reaction. However, the role of primary cilia in the pathogenesis of the ductular reaction is not fully understood. METHODS: Primary cilia were specifically removed in biliary epithelial cells (BECs) by the administration of tamoxifen to Kif3af/f;CK19CreERT mice at week 2 of a 20-week course of TAA treatment. Biliary progenitor cells were isolated and grown as organoids from gallbladders. Cells and tissue were analysed using histology, immunohistochemistry and Western blot assays. RESULTS: At the end of 20 weeks TAA administration, primary cilia loss in liver BECs resulted in multiple microscopic cystic lesions within an unaltered ductular reaction. These were not seen in control mice who did not receive TAA. There was no effect of biliary primary cilia loss on the development of cirrhosis. Increased cellular proliferation was seen within the cystic structures associated with a decrease in hepatocyte lobular proliferation. Loss of primary cilia within biliary organoids was initially associated with reduced cell passage survival but this inhibitory effect was diminished in later passages. ERK but not WNT signalling was enhanced in primary cilia loss-induced cystic lesions in vivo and its inhibition reduced the expansion of primary cilia deficient biliary progenitor cells in vitro. CONCLUSIONS: TAA-treated kif3a BEC-specific knockout mice had an unaltered progression to cirrhosis, but developed cystic lesions that showed increased proliferation.

Betaine ameliorates schizophrenic traits by functionally compensating for KIF3-based CRMP2 transport.

In schizophrenia (SCZ), neurons in the brain tend to undergo gross morphological changes, but the related molecular mechanism remains largely elusive. Using Kif3b+/- mice as a model with SCZ-like behaviors, we found that a high-betaine diet can significantly alleviate schizophrenic traits related to neuronal morphogenesis and behaviors. According to a deficiency in the transport of collapsin response mediator protein 2 (CRMP2) by the KIF3 motor, we identified a significant reduction in lamellipodial dynamics in developing Kif3b+/- neurons as a cause of neurite hyperbranching. Betaine administration significantly decreases CRMP2 carbonylation, which enhances the F-actin bundling needed for proper lamellipodial dynamics and microtubule exclusion and may thus functionally compensate for KIF3 deficiency. Because the KIF3 expression levels tend to be downregulated in the human prefrontal cortex of the postmortem brains of SCZ patients, this mechanism may partly participate in human SCZ pathogenesis, which we hypothesize could be alleviated by betaine administration.

Genetic background-dependent abnormalities of the enteric nervous system and intestinal function in Kif26a-deficient mice.

The Kif26a protein-coding gene has been identified as a negative regulator of the GDNF-Ret signaling pathway in enteric neurons. The aim of this study was to investigate the influence of genetic background on the phenotype of Kif26a-deficient (KO, -/-) mice. KO mice with both C57BL/6 and BALB/c genetic backgrounds were established. Survival rates and megacolon development were compared between these two strains of KO mice. Functional bowel assessments and enteric neuron histopathology were performed in the deficient mice. KO mice with the BALB/c genetic background survived more than 400 days without evidence of megacolon, while all C57BL/6 KO mice developed megacolon and died within 30 days. Local enteric neuron hyperplasia in the colon and functional bowel abnormalities were observed in BALB/c KO mice. These results indicated that megacolon and enteric neuron hyperplasia in KO mice are influenced by the genetic background. BALB/c KO mice may represent a viable model for functional gastrointestinal diseases such as chronic constipation, facilitating studies on the underlying mechanisms and providing a foundation for the development of treatments.

Whole-genome doubling confers unique genetic vulnerabilities on tumour cells.

Whole-genome doubling (WGD) is common in human cancers, occurring early in tumorigenesis and generating genetically unstable tetraploid cells that fuel tumour development1,2. Cells that undergo WGD (WGD+ cells) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can be exploited therapeutically, is unclear. Here, using sequencing data from roughly 10,000 primary human cancer samples and essentiality data from approximately 600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent than WGD- cells on signalling from the spindle-assembly checkpoint, DNA-replication factors and proteasome function. We also identify KIF18A, which encodes a mitotic kinesin protein, as being specifically required for the viability of WGD+ cells. Although KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD- cells, its loss induces notable mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results suggest new strategies for specifically targeting WGD+ cancer cells while sparing the normal, non-transformed WGD- cells that comprise human tissue.

Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition.

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.

KIF5A-dependent axonal transport deficiency disrupts autophagic flux in trimethyltin chloride-induced neurotoxicity.

Trimethyltin chloride (TMT) is widely used as a constituent of fungicides and plastic stabilizers in the industrial and agricultural fields, and is generally acknowledged to have potent neurotoxicity, especially in the hippocampus; however, the mechanism of induction of neurotoxicity by TMT remains elusive. Herein, we exposed Neuro-2a cells to different concentrations of TMT (2, 4, and 8 µM) for 24 h. Proteomic analysis, coupled with bioinformatics analysis, revealed the important role of macroautophagy/autophagy-lysosome machinery in TMT-induced neurotoxicity. Further analysis indicated significant impairment of autophagic flux by TMT via suppressed lysosomal function, such as by inhibiting lysosomal proteolysis and changing the lysosomal pH, thereby contributing to defects in autophagic clearance and subsequently leading to nerve cell death. Mechanistically, molecular interaction networks of Ingenuity Pathway Analysis identified a downregulated molecule, KIF5A (kinesin family member 5A), as a key target in TMT-impaired autophagic flux. TMT decreased KIF5A protein expression, disrupted the interaction between KIF5A and lysosome, and impaired lysosomal axonal transport. Moreover, Kif5a overexpression restored axonal transport, increased lysosomal dysfunction, and antagonized TMT-induced neurotoxicity in vitro. Importantly, in TMT-administered mice with seizure symptoms and histomorphological injury in the hippocampus, TMT inhibited KIF5A expression in the hippocampus. Gene transfer of Kif5a enhanced autophagic clearance in the hippocampus and alleviated TMT-induced neurotoxicity in vivo. Our results are the first to demonstrate KIF5A-dependent axonal transport deficiency to cause autophagic flux impairment via disturbance of lysosomal function in TMT-induced neurotoxicity; manipulation of KIF5A may be a therapeutic approach for antagonizing TMT-induced neurotoxicity.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTB: actin beta; AGC: automatic gain control; ATG: autophagy-related; ATP6V0D1: ATPase H+ transporting lysosomal V0 subunit D1; ATP6V1E1: ATPase H+ transporting lysosomal V1 subunit E1; CA: cornu ammonis; CQ: chloroquine; CTSB: cathepsin B; CTSD: cathepsin D; DCTN1: dynactin subunit 1; DG: dentate gyrus; DYNLL1: dynein light chain LC8-type 1; FBS: fetal bovine serum; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IPA: Ingenuity Pathway Analysis; KEGG: Kyoto Encyclopedia of Genes and Genomes; KIF5A: kinesin family member 5A; LAMP: lysosomal-associated membrane protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PBS: phosphate-buffered saline; PFA: paraformaldehyde; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PRM: parallel reaction monitoring; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; SYP: synaptophysin; TAX1BP1: Tax1 binding protein 1; TMT: trimethyltin chloride; TUB: tubulin.

Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells.

Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1's role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.

Retinal ganglion cell loss in kinesin-1 cargo Alcadein α deficient mice.

Maintenance of retinal ganglion cells (RGCs) activity is relied on axonal transport conveying materials required for their survival such as neurotrophic factors. Kinesin-1 undergoes anterograde transport in axons, and Alcadein α (Alcα; also called calsyntenin-1) is a major cargo adaptor protein that can drive kinesin-1 to transport vesicles containing Alcα. The long-term effects of Alcα-deficiency on retinal morphology and survival of RGCs during postnatal development were examined in Alcα knockout mice. At 1.5, 3, 6, and 15 months postnatal, the number of retrogradely labeled RGCs was determined in flat-mounted retinas of Alcα-deficient and wild-type mice. Retinal damage was assessed histologically by determining the retinal thickness. Intraocular pressure (IOP) was measured with a Tonolab tonometer. At 1.5 months postnatal, the number of retrogradely labeled RGCs was not different between wild-type and Alcα-deficient mice. However, at 3, 6, and 15 months postnatal, the number of RGCs was significantly lower in Alcα deficient mice than those of wild-type mice (143 ± 41.1 cells/mm2 vs. 208 ± 28.4 cells/mm2, respectively, at 3 months; P < 0.01). No differences were seen in retinal thickness or IOP between the two types of mice at any postnatal age. Alcα-deficient mice showed spontaneous loss of RGCs but no elevation in IOP. These mice mimic normal-tension glaucoma and will be useful for investigating the mechanism of neurodegeneration in this disorder and for developing treatments for RGC loss that does not involve changes in IOP.

Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal.

Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity.IMPORTANCECandida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14-canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

Kinesin-14 motor protein KIFC1 participates in DNA synthesis and chromatin maintenance.

The nuclear localization signal (NLS) in kinesin-14 KIFC1 is associated with nuclear importins and Ran gradient, but detailed mechanism remains unknown. In this study, we found that KIFC1 proteins have specific transport characteristics during cell cycle. In the absence of KIFC1, cell cycle kinetics decrease significantly with a prolonged S phase. After KIFC1 overexpression, the duration of S phase becomes shorten. KIFC1 may transport the recombinant/replicate-related proteins into the nucleus, meanwhile avoiding excessive KIFC1 in the cytoplasm, which results in aberrant microtubule bundling. Interestingly, the deletion of kifc1 in human cells results in a higher ratio of aberrant nuclear membrane, and the degradation of lamin B and lamin A/C. We also found that kifc1 deletion leads to defects in metaphase mitotic spindle assembly, and then results in chromosome structural abnormality. The kifc1-/- cells finally form micronuclei in daughter cells, and results in aneuploidy and chromosome loss in cell cycle. In this study, we demonstrate that kinesin-14 KIFC1 proteins involve in regulating DNA synthesis in S phase, and chromatin maintenance in mitosis, and maintain cell growth in a nuclear transport-independent way.

Lysosome Positioning Influences mTORC2 and AKT Signaling.

Growth factor signaling is initiated at the plasma membrane and propagated through the cytoplasm for eventual relay to intracellular organelles such as lysosomes. The serine/threonine kinase mTOR participates in growth factor signaling as a component of two multi-subunit complexes, mTORC1 and mTORC2. mTORC1 associates with lysosomes, and its activity depends on the positioning of lysosomes within the cytoplasm, although there is no consensus regarding the exact effect of perinuclear versus peripheral distribution. mTORC2 and its substrate kinase AKT have a widespread distribution, but they are thought to act mainly at the plasma membrane. Using cell lines with knockout of components of the lysosome-positioning machinery, we show that perinuclear clustering of lysosomes delays reactivation of not only mTORC1, but also mTORC2 and AKT upon serum replenishment. These experiments demonstrate the existence of pools of mTORC2 and AKT that are sensitive to lysosome positioning.

Fibroblast Primary Cilia Are Required for Cardiac Fibrosis.

BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

Mutations in Kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development.

Cerebrospinal fluid flow is crucial for neurodevelopment and homeostasis of the ventricular system of the brain, with localized flow being established by the polarized beating of the ependymal cell (EC) cilia. Here, we report a homozygous one base-pair deletion, c.1193delT (p.Leu398Glnfs*2), in the Kinesin Family Member 6 (KIF6) gene in a child displaying neurodevelopmental defects and intellectual disability. To test the pathogenicity of this novel human KIF6 mutation we engineered an analogous C-terminal truncating mutation in mouse. These mutant mice display severe, postnatal-onset hydrocephalus. We generated a Kif6-LacZ transgenic mouse strain and report expression specifically and uniquely within the ependymal cells (ECs) of the brain, without labeling other multiciliated mouse tissues. Analysis of Kif6 mutant mice with scanning electron microscopy (SEM) and immunofluorescence (IF) revealed specific defects in the formation of EC cilia, without obvious effect of cilia of other multiciliated tissues. Dilation of the ventricular system and defects in the formation of EC cilia were also observed in adult kif6 mutant zebrafish. Finally, we report Kif6-GFP localization at the axoneme and basal bodies of multi-ciliated cells (MCCs) of the mucociliary Xenopus epidermis. Overall, this work describes the first clinically-defined KIF6 homozygous null mutation in human and defines KIF6 as a conserved mediator of neurological development with a specific role for EC ciliogenesis in vertebrates.

Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia.

Primary cilia are solitary organelles that emanate from the plasma membrane during growth arrest in almost all mammalian cells. The canonical Hedgehog (HH) pathway requires trafficking of the G protein-coupled receptor SMOOTHENED (SMO) and the GLI transcription factors to the primary cilium upon binding of a HH ligand to PATCHED1. However, it is unknown if activation of the small GTPase RHOA by SMO coupling to heterotrimeric Gi proteins, a form of non-canonical HH signaling, requires localization of SMO in the primary cilium. In this study, we compared RHOA and Gi protein stimulation by activation of SMO or sphingosine 1-phosphate receptor (S1P) receptors in WT and KIF3A-deficient mouse embryonic fibroblasts that lack primary cilia. We found that activation of SMO in response to Sonic HH (SHH) or purmorphamine (PUR), a small molecule agonist of SMO, stimulates Gi proteins and RHOA independently of the presence of primary cilia, similar to the effects of S1P. However, while S1P induced a fast activation of AKT that is sensitive to the Gi inhibitor pertussis toxin, HH pathway activators did not significantly activate AKT, suggesting that RHOA activation is not downstream of AKT. Our findings demonstrate that early events in some forms of non-canonical HH signaling occur in extraciliary membranes, which might be particularly relevant for actively-cycling cells, for some cancers characterized by loss of primary cilia, and in ciliopathies.

KIF20A/MKLP2 regulates the division modes of neural progenitor cells during cortical development.

Balanced symmetric and asymmetric divisions of neural progenitor cells (NPCs) are crucial for brain development, but the underlying mechanisms are not fully understood. Here we report that mitotic kinesin KIF20A/MKLP2 interacts with RGS3 and plays a crucial role in controlling the division modes of NPCs during cortical neurogenesis. Knockdown of KIF20A in NPCs causes dislocation of RGS3 from the intercellular bridge (ICB), impairs the function of Ephrin-B-RGS cell fate signaling complex, and leads to a transition from proliferative to differentiative divisions. Germline and inducible knockout of KIF20A causes a loss of progenitor cells and neurons and results in thinner cortex and ventriculomegaly. Interestingly, loss of function of KIF20A induces early cell cycle exit and precocious neuronal differentiation without causing substantial cytokinesis defect or apoptosis. Our results identify a RGS-KIF20A axis in the regulation of cell division and suggest a potential link of the ICB to regulation of cell fate determination.

The Molecular Motor KIF21B Mediates Synaptic Plasticity and Fear Extinction by Terminating Rac1 Activation.

Fear extinction is a component of cognitive flexibility that is relevant for important psychiatric diseases, but its molecular mechanism is still largely elusive. We established mice lacking the kinesin-4 motor KIF21B as a model for fear extinction defects. Postsynaptic NMDAR-dependent long-term depression (LTD) is specifically impaired in knockouts. NMDAR-mediated LTD-causing stimuli induce dynamic association of KIF21B with the Rac1GEF subunit engulfment and cell motility protein 1 (ELMO1), leading to ELMO1 translocation out of dendritic spines and its sequestration in endosomes. This process may essentially terminate transient activation of Rac1, shrink spines, facilitate AMPAR endocytosis, and reduce postsynaptic strength, thereby forming a mechanistic link to LTD expression. Antagonizing ELMO1/Dock Rac1GEF activity by the administration of 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) significantly reverses the knockout phenotype. Therefore, we propose that KIF21B-mediated Rac1 inactivation is a key molecular event in NMDAR-dependent LTD expression underlying cognitive flexibility in fear extinction.

Down-regulation of KIF2A inhibits gastric cancer cell invasion via suppressing MT1-MMP.

Gastric cancer accounts for a sizeable proportion of global cancer mortality with high morbidity and poor prognosis. Kinesin superfamily proteins (KIFs) are microtubule-dependent motor proteins that function as oncogenes in cancer cells, it has been discovered in recent years. Kinesin family member 2a (KIF2A), a member of the KIFs, has received attention for its role in carcinogenesis and its prognostic value in several human cancers such as breast cancer, colorectal cancer, and squamous cell carcinoma. However, the role of KIF2A in human gastric cancer remains unknown. In this study we aimed to explore the expression and biological functions of KIF2A in human gastric cancer cells, as well as to reveal its potential action mechanism. First, we found that KIF2A was markedly increased in gastric cancer cells (MKN-28, MKN-45, NCI-N87 and SGC-7901) compared to normal gastric mucosa epithelial cells (GES-1). Then KIF2A was successfully silenced in MKN-45 and SGC-7901 cells to facilitate further research into its function. We discovered that KIF2A silencing can significantly inhibit the growth and invasion of MKN-45 and SGC-7901 cells in a time-independent manner, accompanying a decreased expression of Membrane type 1-matrix metalloproteinase (MT1-MMP). When MT1-MMP was reintroduced into MKN-45 and SGC-7901 cells in the KIF2A-siRNA group, only invasion inhibition effects on MKN-45 and SGC-7901 cells induced by KIF2A silencing can be reversed. In conclusion, our study reveals that down-regulation of KIF2A can inhibit gastric cancer cell invasion by suppressing MT1-MMP.

Kinesin-1 Is a New Actor Involved in Platelet Secretion and Thrombus Stability.

OBJECTIVE: Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized. APPROACH AND RESULTS: By studying a mouse model (cKO [conditional knockout]Kif5b) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKOKif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKOKif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKOKif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 (Ras-related protein Rab-27) protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein. CONCLUSIONS: Our results indicate that a kinesin-1-dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

Regulation of Mitoflash Biogenesis and Signaling by Mitochondrial Dynamics.

Mitochondria are highly dynamic organelles undergoing constant network reorganization and exhibiting stochastic signaling events in the form of mitochondrial flashes (mitoflashes). Here we investigate whether and how mitochondrial network dynamics regulate mitoflash biogenesis and signaling. We found that mitoflash frequency was largely invariant when network fragmentized or redistributed in the absence of mitofusin (Mfn) 1, Mfn2, or Kif5b. However, Opa1 deficiency decreased spontaneous mitoflash frequency due to superimposing changes in respiratory function, whereas mitoflash response to non-metabolic stimulation was unchanged despite network fragmentation. In Drp1- or Mff-deficient cells whose mitochondria hyperfused into a single whole-cell reticulum, the frequency of mitoflashes of regular amplitude and duration was again unaltered, although brief and low-amplitude "miniflashes" emerged because of improved detection ability. As the network reorganized, however, the signal mass of mitoflash signaling was dynamically regulated in accordance with the degree of network connectivity. These findings demonstrate a novel functional role of mitochondrial network dynamics and uncover a magnitude- rather than frequency-modulatory mechanism in the regulation of mitoflash signaling. In addition, our data support a stochastic trigger model for the ignition of mitoflashes.