RESUMO
RAB11 small GTPases and associated recycling endosome have been localized to mitotic spindles and implicated in regulating mitosis. However, the physiological significance of such regulation has not been observed in mammalian tissues. We have used newly engineered mouse models to investigate intestinal epithelial renewal in the absence of single or double isoforms of RAB11 family members: Rab11a and Rab11b. Comparing with single knockouts, mice with compound ablation demonstrate a defective cell cycle entry and robust mitotic arrest followed by apoptosis, leading to a total penetrance of lethality within 3 days of gene ablation. Upon Rab11 deletion ex vivo, enteroids show abnormal mitotic spindle formation and cell death. Untargeted proteomic profiling of Rab11a and Rab11b immunoprecipitates has uncovered a shared interactome containing mitotic spindle microtubule regulators. Disrupting Rab11 alters kinesin motor KIF11 function and impairs bipolar spindle formation and cell division. These data demonstrate that RAB11A and RAB11B redundantly control mitotic spindle function and intestinal progenitor cell division, a mechanism that may be utilized to govern the homeostasis and renewal of other mammalian tissues.
Assuntos
Proteômica , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Mamíferos/metabolismo , Mitose , Proteínas rab de Ligação ao GTP/metabolismo , Fuso Acromático/metabolismo , Células-Tronco/metabolismoRESUMO
Kinesin motors play a fundamental role in development by controlling intracellular transport, spindle assembly, and microtubule organization. In humans, patients carrying mutations in KIF11 suffer from an autosomal dominant inheritable disease called microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR). While mitotic functions of KIF11 proteins have been well documented in centrosome separation and spindle assembly, cellular mechanisms underlying KIF11 dysfunction and MCLMR remain unclear. In this study, we generate KIF11-inhibition chick and zebrafish models and find that KIF11 inhibition results in microcephaly, chorioretinopathy, and severe developmental defects in vivo. Notably, loss-of-function of KIF11 causes the formation of monopolar spindle and chromosome misalignment, which finally contribute to cell cycle arrest, chromosome instability, and cell death. Our results demonstrate that KIF11 is crucial for spindle assembly, chromosome alignment, and cell cycle progression of progenitor stem cells, indicating a potential link between polyploidy and MCLMR. Our data have revealed that KIF11 inhibition cause microcephaly, chorioretinopathy, and development disorders through the formation of monopolar spindle, polyploid, and cell cycle arrest.
Assuntos
Fácies , Linfedema , Microcefalia , Doenças Retinianas , Displasia Retiniana , Animais , Pontos de Checagem do Ciclo Celular/genética , Instabilidade Cromossômica , Deficiências do Desenvolvimento , Cinesinas/genética , Cinesinas/metabolismo , Microcefalia/genética , Fenótipo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Chromosomal instability (CIN), the process of increased chromosomal alterations, compromises genomic integrity and has profound consequences on human health. Yet, our understanding of the molecular and mechanistic basis of CIN initiation remains limited. We developed a high-throughput, single-cell, image-based pipeline employing deep-learning and spot-counting models to detect CIN by automatically counting chromosomes and micronuclei. To identify CIN-initiating conditions, we used CRISPR activation in human diploid cells to upregulate, at physiologically relevant levels, 14 genes that are functionally important in cancer. We found that upregulation of CCND1, FOXA1 and NEK2 resulted in pronounced changes in chromosome counts, and KIF11 upregulation resulted in micronuclei formation. We identified KIF11-dependent fragilities within the mitotic spindle; increased levels of KIF11 caused centrosome fragmentation, higher microtubule stability, lagging chromosomes or mitotic catastrophe. Our findings demonstrate that even modest changes in the average expression of single genes in a karyotypically stable background are sufficient for initiating CIN by exposing fragilities of the mitotic spindle, which can lead to a genomically diverse cell population.
Assuntos
Instabilidade Cromossômica , Cinesinas , Fuso Acromático , Aneuploidia , Centrossomo/metabolismo , Instabilidade Cromossômica/genética , Aberrações Cromossômicas , Humanos , Cinesinas/genética , Microtúbulos/genética , Mitose/genética , Quinases Relacionadas a NIMA/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
Few efficacious treatment options are available for patients with small cell lung carcinoma (SCLC), indicating the need to develop novel therapeutic approaches. In this study, we explored kinesin family member 11 (KIF11), a potential therapeutic target in SCLC. An analysis of publicly available data suggested that KIF11 mRNA expression levels are significantly higher in SCLC tissues than in normal lung tissues. When KIF11 was targeted by RNA interference or a small-molecule inhibitor (SB743921) in two SCLC cell lines, Lu-135 and NCI-H69, cell cycle progression was arrested at the G2/M phase with complete growth suppression. Further work suggested that the two cell lines were more significantly affected when both KIF11 and BCL2L1, an anti-apoptotic BCL2 family member, were inhibited. This dual inhibition resulted in markedly decreased cell viability. These findings collectively indicate that SCLC cells are critically dependent on KIF11 activity for survival and/or proliferation, as well as that KIF11 inhibition could be a new strategy for SCLC treatment.
Assuntos
Sobrevivência Celular , Cinesinas , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Cinesinas/metabolismo , Cinesinas/genética , Cinesinas/antagonistas & inibidores , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Linhagem Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proliferação de Células , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Regulação Neoplásica da Expressão Gênica , Apoptose/genética , Benzamidas , QuinazolinasRESUMO
Hepatocellular carcinoma (HCC) is the tumor with the second highest mortality rate worldwide. Recent research data show that KIF11, a member of the kinesin family (KIF), plays an important role in the progression of various tumors. However, its expression and molecular mechanism in HCC remain elusive. Here, we evaluated the potential role of KIF11 in HCC. The effect of KIF11 was evaluated using the hepatocellular carcinoma cell lines, LM3 and Huh7, after genetic or pharmacological treatment. Evaluating the role of KIF11 in the xenograft animal models using its specific inhibitor. The role of KIF11 was systematically evaluated using specimens obtained from the aforementioned animal and cell models after various in vivo and in vitro experiments. The clinicopathological analysis showed that KIF11 was expressed at high levels in patients with hepatocellular carcinoma. Cell experiments in vitro showed that KIF11 deficiency significantly slowed the proliferation of liver tumor cells. And in the experiment using liver cancer cells overexpressing OCT4, overexpression of OCT4 substantially increased the proliferation of tumor cells compared with tumor cells with KIF11 knockdown alone. Both in vitro cell experiment and in vivo xenotransplantation tumor experiment showed that monastrol, an inhibitor of KIF11, could effectively delay the proliferation and migration of tumor cells. Based on these results, KIF11 is expressed at high levels in hepatocellular carcinoma and promotes tumor proliferation in an OCT4-dependent manner. KIF11 may become a therapeutic target for hepatocellular carcinoma, and its inhibitor monastrol may become a clinical antitumor drug.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animais , Carcinoma Hepatocelular/genética , Cinesinas/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , FamíliaRESUMO
EGFR-mutant lung adenocarcinoma (LUAD) mostly depends on EGFR for survival and consequently responds well to EGFR inhibitors. However, resistance to the drugs develops almost universally during treatment. We previously demonstrated that EGFR-mutant LUAD cell lines, HCC827 and H1975, have subpopulations of cells, which we termed HCC827 GR2 and H1975 WR7 cells, that can thrive independently of EGFR signaling. These EGFR-independent EGFR-mutant cancer cells are difficult to treat because they lack sensitivity to EGFR inhibitors. Therefore, the development of novel strategies to target EGFR-independent EGFR-mutant LUAD is particularly important. We found that high expression of kinesin family member 11 (KIF11) correlated with poor survival in patients with LUAD. We also observed that KIF11 silencing caused cell cycle arrest at G2/M in HCC827 GR2 and H1975 WR7 cells. Furthermore, dual silencing of KIF11 plus BCL2L1, an anti-apoptotic BCL2 family member, in these two EGFR-independent sublines resulted in marked apoptosis levels. Dual inhibition of KIF11 plus BCL2L1 also induced apoptosis in HCC827 and H1975 parental cells and a KRAS-mutant LUAD cell line, H441. These findings collectively suggest that dual inhibition of KIF11 plus BCL2L1 may be a new approach for the treatment of LUAD.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Cinesinas/genética , Apoptose , Adenocarcinoma de Pulmão/genética , Proteína bcl-X , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Receptores ErbB/genéticaRESUMO
Abnormal spindle-like microcephaly-associated (ASPM) protein is crucial to the mitotic spindle function during cell replication and tumor progression in multiple tumor types. However, the effect of ASPM in anaplastic thyroid carcinoma (ATC) has not yet been understood. The present study is to elucidate the function of ASPM in the migration and invasion of ATC. ASPM expression is incrementally upregulated in ATC tissues and cell lines. Knockout (KO) of ASPM pronouncedly attenuates the migration and invasion of ATC cells. ASPM KO significantly reduces the transcript levels of Vimentin, N-cadherin, and Snail and increases E-cadherin and Occludin, thereby inhibiting epithelial-to-mesenchymal transition (EMT). Mechanistically, ASPM regulates the movement of ATC cells by inhibiting the ubiquitin degradation of KIF11 and thus stabilizing it via direct binding to it. Moreover, xenograft tumors in nude mice proved that KO of ASPM could ameliorate tumorigenesis and tumor growth accompanied by a decreased protein expression of KIF11 and an inhibition of EMT. In conclusion, ASPM is a potentially useful therapeutic target for ATC. Our results also reveal a novel mechanism by which ASPM inhibits the ubiquitin process in KIF11.
Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Camundongos , Humanos , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Camundongos Nus , Linhagem Celular Tumoral , Camundongos Knockout , Proteínas do Tecido Nervoso , Ubiquitinas/farmacologia , Movimento Celular , Proliferação de Células , Cinesinas/genéticaRESUMO
Our group recently demonstrated that K858, an inhibitor of motor kinesin Eg5, has important antiproliferative and apoptotic effects on breast cancer, prostatic cancer, melanoma and glioblastoma cells. Since high levels of kinesin Eg5 expression have been correlated with a poor prognosis in laryngeal carcinoma, we decided to test the anticancer activity of K858 toward this tumor, which belongs to the group of head and neck squamous cell carcinomas (HNSCCs). These cancers are characterized by low responsiveness to therapy. The effects of K858 on the proliferation and assembly of mitotic spindles of three human HNSCC cell lines were studied using cytotoxicity assays and immunofluorescence for tubulin. The effect of K858 on the cell cycle was analyzed by FACS. The expression levels of cyclin B1 and several markers of apoptosis and invasion were studied by Western blot. Finally, the negative regulation of the malignant phenotype by K858 was evaluated by an invasion assay. K858 inhibited cell replication by rendering cells incapable of developing normal bipolar mitotic spindles. At the same time, K858 blocked the cell cycle in the G2 phase and induced the accumulation of cytoplasmic cyclin B and, eventually, apoptosis. Additionally, K858 inhibited cell migration and attenuated the malignant phenotype. The data described confirm that kinesin Eg5 is an interesting target for new anticancer strategies and suggest that this compound may be a powerful tool for an alternative therapeutic approach to HNSCCs.
Assuntos
Antineoplásicos , Neoplasias de Cabeça e Pescoço , Cinesinas , Tiadiazóis , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Cinesinas/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Tiadiazóis/farmacologiaRESUMO
Glioma is the most common type of primary brain malignancy with high morbidity and mortality, but little is known about its pathological mechanisms. Kinesin family member 11 (KIF11) is a key driver of malignancy in glioblastoma, a grade IV glioma, but its involvement in glioma chemoresistance remains to be determined. We accessed the TCGA open datasets, collected glioma tumor tissue samples, and analyzed the expression of KIF11 in glioma patients. Meanwhile, the correlation between KIF11 and survival outcomes was determined by the Kaplan-Meier analysis. The role of KIF11 in glioma tumor cell function was assessed in an in vitro knockdown and overexpressing system. Here, we found that KIF11 was upregulated in glioma tumors and negatively correlated with overall survival outcomes via analyzing the open datasets. KIF11 was negatively correlated with TP53 expression. Furthermore, KIF11 promoted the stemness in glioma cells, accompanied by increased cell proliferation and chemoresistance. Mechanistically, we found that KIF11 promoted cell cycle progression via upregulating cyclin expression.
Assuntos
Neoplasias Encefálicas , Glioma , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Resistência a Medicamentos , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , Humanos , Cinesinas/genética , Células-Tronco Neoplásicas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/genéticaRESUMO
While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug's target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resistência a Medicamentos/genética , Endonucleases/metabolismo , Testes Genéticos/métodos , Mutação , Sítios de Ligação , Ligação ProteicaRESUMO
Emerging evidence suggests that KIF11 could play a pivotal role in cancer cell proliferation; however, its biological functions and molecular mechanisms in Wilms tumor (WT) cells are largely unknown. The aim of this study was to evaluate the clinical significance and therapeutic potential of KIF11 proteins in WT. KIF11 expression in WT tissues and adjacent nontumor tissues was determined using qRT-PCR, Western blotting, immunohistochemistry (IHC) and bioinformatics. The function of KIF11 protein was determined by its correlation with tumor cell growth, angiogenesis, and apoptosis using IHC and lentiviral vector-mediated KIF11 depletion. KIF11 expression was upregulated in WT tissues and was associated with WT clinical outcomes. Tumor KIF11 expression was significantly associated with the Ki67 proliferation index. CCK-8, flow-cytometric analysis, and Western blotting revealed that KIF11 knockdown significantly inhibited WT cell growth. Functional studies have indicated that increased KIF11 expression is significantly correlated with vascular endothelial growth factor (VEGF) expression and intratumoral microvessel density. We further confirmed that downregulated expression of KIF11 promoted cell apoptosis and significantly increased Bcl-2 and Bax expression. Our findings demonstrate that KIF11 plays a role in promoting the development of human WT and can serve as a potential molecular marker for the treatment of WT.
Assuntos
Neoplasias Renais , Tumor de Wilms , Apoptose/fisiologia , Carcinogênese , Proliferação de Células/genética , Criança , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Cinesinas/genética , Prognóstico , Fator A de Crescimento do Endotélio Vascular/genética , Tumor de Wilms/genética , Tumor de Wilms/patologiaRESUMO
INTRODUCTION: Unresectable renal cell carcinoma continues to be a great challenge due to our limited understanding of its underlying pathophysiology. We explored the relationship between KIF11 protein expression and the clinical courses of clear cell renal cell carcinoma (ccRCC) using a tissue microarray. MATERIAL AND METHODS: The tissue microarray contained specimens derived from 90 patients, cancer and matched adjacent non-cancerous tissue (2 cores per case), followed up for 7 years. Tumour samples were evaluated for KIF11 expression using the H-score, and their correlations with clinicopathological data and survival data were analysed. RESULTS: 72.7% of ccRCC tissues presented KIF11 cytoplasmic expression with a median value of 20 (interquartile range 0-200). The nuclear staining was positive in 36.36% of ccRCC tissues. Among controls, nuclear KIF11 expression was absent, but cytoplasmic expression was identified in all cases, with a median value of 230 (interquartile range 45-290). Cytoplasmic KIF11 expression in ccRCC tissues was lower than in the control tissues and was positively correlated with tumour grade and mortality (p < 0.05). KIF11 nuclear expression did not correlate with overall survival. CONCLUSIONS: Elevated expression of KIF11 predicts poor clinical outcome in ccRCC patients. Downregulation of KIF11 may provide a new therapeutic strategy for ccRCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Prognóstico , Biomarcadores Tumorais/análise , CinesinasRESUMO
Genomic instability (GIN) has an important contribution to the pathology of colorectal cancer (CRC). Therefore, we selected mitosis and cytokinesis kinesins, KIF11 and KIF14, as factors of potential clinical and functional value in CRC, as their aberrant expression has been suspected to underlie GIN. We examined the expression and the prognostic and biological significance of KIF11 and KIF14 in CRC via in-house immunohistochemistry on tissue microarrays, public mRNA expression datasets, as well as bioinformatics tools. We found that KIF11 and KIF14 expression, at both the protein and mRNA level, was markedly altered in cancer tissues compared to respective controls, which was reflected in the clinical outcome of CRC patients. Specifically, we provide the first evidence that KIF11 protein and mRNA, KIF14 mRNA, as well as both proteins together, can significantly discriminate between CRC patients with better and worse overall survival independently of other relevant clinical risk factors. The negative prognostic factors for OS were high KIF11 protein, high KIF11 protein + low KIF14 protein, low KIF11 mRNA and low KIF14 mRNA. Functional enrichment analysis revealed that the gene sets related to the cell cycle, DNA replication, DNA repair and recombination, among others, were positively associated with KIF11 or KIF14 expression in CRC tissues. In TCGA cohort, the positive correlations between several measures related to GIN and the expression of KIFs were also demonstrated. In conclusion, our results suggest that CRC patients can be stratified into distinct risk categories by biological and molecular determinants, such as KIF11 and KIF14 expression and, mechanistically, this is likely attributable to their role in maintaining genome integrity.
Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Cinesinas/genética , Proteínas Oncogênicas/genética , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Biologia Computacional , Feminino , Perfilação da Expressão Gênica , Instabilidade Genômica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Cinesinas/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas Oncogênicas/metabolismo , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
ZBP1-modulated localization of ß-actin mRNA enables a cell to establish polarity and structural asymmetry. Although the mechanism of ß-actin mRNA localization has been well established, the underlying mechanism of how a specific molecular motor contributes to the transport of the ZBP1 (also known as IGF2BP1) complex in non-neuronal cells remains elusive. In this study, we report the isolation and identification of KIF11, a microtubule motor, which physically interacts with ZBP1 and is a component of ß-actin messenger ribonucleoprotein particles (mRNPs). We show that KIF11 colocalizes with the ß-actin mRNA, and the ability of KIF11 to transport ß-actin mRNA is dependent on ZBP1. We characterize the corresponding regions of ZBP1 and KIF11 that mediate the interaction of the two proteins in vitro and in vivo. Disruption of the in vivo interaction of KIF11 with ZBP1 delocalizes ß-actin mRNA and affects cell migration. Our study reveals a molecular mechanism by which a particular microtubule motor mediates the transport of an mRNP through direct interaction with an mRNA-binding protein.
Assuntos
Actinas/metabolismo , Movimento Celular/fisiologia , Glicoproteínas/metabolismo , Cinesinas/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Actinas/genética , Animais , Transporte Biológico Ativo/fisiologia , Linhagem Celular , Glicoproteínas/genética , Cinesinas/genética , Camundongos , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Ribonucleoproteínas/genéticaRESUMO
Although in vitro maturation (IVM) of oocytes has been used for a relatively long time, during which the culture conditions have improved remarkably, the resulting germ cells are still not fully comparable to the cells obtained from the ovary in many important aspects, namely in fertilization rate and subsequent embryonic development. Some of the differences between IVM and in vivo maturation (IVV) oocytes were already discovered, including variability in spindle assembly and morphology. In this study we focused on a role of molecular motor Kif11 (hereafter referred to as Eg5) in maintaining bipolar spindle structure in IVM and IVV oocytes. Our experiments revealed that in IVM oocytes, Eg5 is abundant on meiosis II spindle, which makes these cells more sensitive to Eg5 inhibition than IVV oocytes. We further demonstrate that this sensitivity is acquired gradually with exposure to the in vitro conditions. This is a remarkable difference in function of spindle apparatus between IVM and IVV oocytes, and we believe our results are important not only for understanding of the chromosome segregation in mammalian oocytes but also because they indicate cells are using alternative pathways to achieve the same function when exposed to different conditions.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Cinesinas/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Fuso Acromático/metabolismo , Animais , Feminino , CamundongosRESUMO
Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of compensatory regulation may exist to maintain overall proportions in the neural tube. We propose a model in which Kif11 normally functions during mitotic spindle formation to facilitate the progression of radial glia through mitosis, which leads to the maturation of progeny into specific secondary neuronal and glial lineages in the developing neural tube.
Assuntos
Células Ependimogliais/metabolismo , Cinesinas/metabolismo , Tubo Neural/embriologia , Neurogênese/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Apoptose/genética , Caspase 3/biossíntese , Contagem de Células , Proliferação de Células , Cisteína/análogos & derivados , Cisteína/farmacologia , Células Ependimogliais/citologia , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Mitose/efeitos dos fármacos , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Tubo Neural/citologia , Tubo Neural/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
Establishment of microtubule polarity is critical for directional cell migration involved in morphogenesis, differentiation, cell division, and metastasis. Current models, involving iterative microtubule capture and inactivation of microtubule depolymerizing mechanisms at the leading edge, cannot account for the biased migration exhibited by cells in culture in the absence of directional cues, suggesting central mechanisms governing microtubule polarity remain unknown. We engineered two human MDA-MB-231/IMP1 breast carcinoma cell lines, denoted kdKIF11-1 and kdKIF11-2, in which the kinesin KIF11 (also known as Eg5) was stably knocked down by two different shRNAs. Western blot analysis showed knockdown by each shRNA decreased KIF11 expression by 58% and 79% for kdKIF11-1 and kdKIF11-2, respectively, whereas Rac1 expression was unaffected. All cell lines retained a well-defined microtubule structure. Compared to cells infected with the control viral vector, both KIF11 knockdown cell lines displayed a 14-45% increase in cell motility in a scratch wound healing assay. In contrast, KIF11 knockdown decreased invasion by 70%, compared to the control, as measured by invasion through Matrigel-coated transwells. To determine whether the reduction in invasion was due to reduced chemotaxis, we substituted collagen for Matrigel in the transwell assay and similarly observed a 44-54% reduction in migration, using EGF as the chemoattractant. However, when including EGF in both the upper and lower chambers of the transwell to stimulate migration but eliminate chemotaxis, transwell migration decreased for the control cell line only, indicating that KIF11 knockdown did not impair migration, but severely impaired chemotaxis. We conclude KIF11 is a key downstream molecule that responds to directional cues in chemotaxis to govern the direction of migration.
Assuntos
Quimiotaxia/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Cinesinas/antagonistas & inibidores , Bioensaio , Engenharia Celular , Linhagem Celular Tumoral , Colágeno/química , Cultura em Câmaras de Difusão , Combinação de Medicamentos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Laminina/química , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Proteoglicanas/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Cicatrização/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The microcephaly-lymphedema-chorioretinal dysplasia (MLCRD) syndrome is a distinct microcephaly syndrome. The hallmark features, microcephaly, chorioretinopathy, and lymphedema are frequently recognized at birth. Another clinical entity, the chorioretinal dysplasia, microcephaly and mental retardation syndrome (CDMMR) is a highly overlapping syndrome characterized by more variable lymphedema. Recently, heterozygous mutations in KIF11, a gene encoding a critical spindle motor protein of the Kinesin family, have been reported in individuals with MLCRD, and in individuals with CDMMR. This finding is suggestive of a single clinically variable spectrum. Here, we report on de novo novel mutations of KIF11 in five individuals with severe microcephaly, marked simplification of the gyral pattern on neuroimaging, bilateral chorioretinopathy, and developmental delay. Three patients had congenital lymphedema, and one had congenital bilateral sensorineural hearing loss. This report, therefore, further expands the clinical and molecular spectrum of KIF11-associated microcephaly.
Assuntos
Heterozigoto , Cinesinas/genética , Microcefalia/genética , Mutação , Displasia Retiniana/genética , Adolescente , Encéfalo/patologia , Criança , Pré-Escolar , Mapeamento Cromossômico , Análise Mutacional de DNA , Éxons , Fácies , Feminino , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Microcefalia/diagnóstico , Fenótipo , Displasia Retiniana/diagnóstico , SíndromeRESUMO
The photoreceptor cilium is vital for maintaining the structure and function of the retina. However, the molecular mechanisms underlying the photoreceptor cilium integrity and retinal homeostasis are largely unknown. Herein, it is shown that kinesin family member 11 (KIF11) localizes at the transition zone (connecting cilium) of the photoreceptor and plays a crucial role in orchestrating the cilium integrity. KIF11 depletion causes malformations of both the photoreceptor ciliary axoneme and membranous discs, resulting in photoreceptor degeneration and the accumulation of drusen-like deposits throughout the retina. Mechanistic studies show that the stability of KIF11 is regulated by an interplay between its UFMylation and ubiquitination; UFMylation of KIF11 at lysine 953 inhibits its ubiquitination by synoviolin 1 and thereby prevents its proteasomal degradation. The lysine 953-to-arginine mutant of KIF11 is more stable than wild-type KIF11 and also more effective in reversing the ciliary and retinal defects induced by KIF11 depletion. These findings identify a critical role for KIF11 UFMylation in the maintenance of photoreceptor cilium integrity and retinal homeostasis.
Assuntos
Cílios , Homeostase , Cinesinas , Retina , Cinesinas/metabolismo , Cinesinas/genética , Animais , Camundongos , Homeostase/fisiologia , Cílios/metabolismo , Cílios/genética , Retina/metabolismo , Modelos Animais de Doenças , Ubiquitinação , Humanos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/genéticaRESUMO
Lung adenocarcinoma (LUAD) is a malignant tumor with the characteristics of progressive advancement and high mortality rate worldwide. We aimed to explore the role and mechanism of helicase Lymphoid-Specific (HELLS) in LUAD. Bioinformatics databases were applied to predict HELLS and kinesin family member (KIF)11 expression in LUAD tissues. The expressions of HELLS and KIF11 before and after HELLS knockdown were detected by RT-qPCR and western blot. After HELLS was knocked down, the proliferative, migratory, and invasive capabilities of A549 cells were evaluated. Cell apoptotic level was assessed using TUNEL. Western blot was employed to evaluate the expressions of Akt/CREB pathway-related proteins. The interaction between HELLS and KIF11 was analyzed using bioinformatics databases, and testified by Co-IP assay. Results revealed that HELLS and KIF11 expressions were significantly upregulated in LUAD cells and tissues. High HELLS and KIF11 expression was correlated with the poor prognosis of patients with LUAD. Additionally, HELLS knockdown suppressed the capabilities of LUAD cells to proliferate, migrate, and invade whereas promoted the cell apoptotic level. Moreover, HELLS could interact with KIF11 and had positive correlation with KIF11. Furthermore, KIF11 overexpression partially counteracted the impacts of HELLS knockdown on cell proliferative, migratory, invasive capabilities, and apoptotic level in LUAD cells. Besides, Akt/CREB pathway was blocked by HELLS silencing, which was restored by KIF11 overexpression. Collectively, HELLS knockdown blocked Akt/CREB pathway by downregulating KIF11 expression, thereby inhibiting LUAD cell proliferation, invasion, migration, and promoting apoptosis.