Journal of Molecular Cell Biology最新文献

Cells round up when they enter mitosis and maintain this rounded morphology until they pass the spindle assembly checkpoint during anaphase. However, the mechanisms that regulate and maintain this transient spherical state remain unclear. In this study, we demonstrate that both astral microtubules and Aurora B kinase are required to maintain cortex stability during prometaphase. Simultaneous inhibition of astral microtubules and Aurora B leads to severe and continuous deformation of mitotic cells, resulting in micronuclei containing chromosomes after the cells exit mitosis. Mechanistically, active Aurora B kinase reduces the activity of myosin light chain kinase through phosphorylation, which in turn decreases the motor activity of myosin II. Additionally, Aurora B kinase regulates the distribution of actin at the cortex by phosphorylating the intermediate filament protein vimentin. Blocking these phosphorylation events disrupts the para-cortex localization of vimentin around the cortex and leads to the dislocalization of actin at the cortex. These regulatory effects occur in highly mobile cells expressing vimentin. In summary, we show that during mitosis, Aurora B kinase coordinates the interactions between microtubules, actin, and intermediate filaments to stabilize the cortex of rounded mitotic cells, ensuring the successful completion of mitosis.

Metabolic adaptability, controlled by transcription factors or oncogenes, is critical for the survival of cancer cells. However, the mechanism by which the transcription factor forkhead box protein O1 (FOXO1) regulates the proliferation and survival of malignant tumor cells under high levels of reactive oxygen species (ROS) remains poorly understood. Here, we found that FOXO1 endows cancer cells with the strong antioxidative capacity and rapid proliferation. By upregulating the expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, FOXO1 promotes the synthesis of nicotinamide adenine dinucleotide phosphate and ribose 5-phosphate and thus enhances the antioxidative and proliferative capabilities of cancer cells. Induction of G6PD expression in FOXO1-deficient cells mitigates tumor growth inhibition and alleviates ROS level elevation. These results establish a critical role of FOXO1 in the regulation of G6PD during the antioxidative and proliferative processes of cancer cells.

The differentiation of a round spermatid into a streamlined sperm cell involves a series of remarkable morphological changes, such as sperm head shaping and flagellum formation. However, the underlying mechanism of spermatid shaping remains unclear. In this study, we find that CCDC92 deficiency in mice leads to severe abnormalities of the sperm head and flagellum and causes male infertility. Ultrastructural analyses of testicular elongating Ccdc92 knockout spermatids reveal severely deformed manchette structures. The manchette defects impair the subsequent sperm nucleus elongation and acrosome anchoring, resulting in misshapen rod-like nuclei and detached acrosomes. Molecularly, CCDC92 interacts with intraflagellar transport (IFT) complex components and colocalizes with IFT proteins at the manchette in developing spermatids. Quantitative proteomics further reveals the requirement of CCDC92 for proper flagellar distribution of axonemal microtubule inner proteins. Our findings demonstrate an essential role of CCDC92 in regulating spermatid shaping and provide novel insights into the pathology of male infertility.

Cilia are microtubule-based organelles that protrude from the surface of various eukaryotic cell types. Microtubules are assembled by α/β-tubulin heterodimers, all of which comprise multiple isotypes encoded by distinct genes. However, the composition and function of tubulin isotypes in cilia are largely unclear. Here, we successfully labeled the endogenous α-tubulin and β-tubulin isotypes with HA or GFP tag in cultured mouse ependymal cells (EPCs) via the CRISPR/Cas9 system. TUBA1A, TUBA1B, TUBA1C, TUBB2A, TUBB2B, TUBB4B, and TUBB5 were identified to be incorporated in ependymal cilia, with TUBB4B showing the highest expression. Overexpression assay revealed that the ependymal cilia did not display a preference for the entrance of specific tubulin isotypes. Furthermore, luciferase reporter assay showed that the expression of TUBB4B in EPCs was specifically regulated by the ciliogenesis factor FOXJ1. TUBB4B deficiency disrupted planar polarity of EPCs and impaired cerebrospinal fluid flow, resulting in hydrocephalus. This study reveals the composition of tubulin isotypes in ependymal cilia and the specific role of FOXJ1-promoted TUBB4B in ciliary motility.

Centromeres are defined by the histone H3 variant CENP-A, which serve as the foundation for kinetochore assembly and ensure faithful chromosome segregation. CENP-A nucleosomes possess distinctive dynamic features, including flexible DNA ends at the entry/exit sites and a mobile N-terminal region, which are properties proposed to facilitate kinetochore assembly, yet the underlying molecular mechanisms remain elusive. Here, we present cryo-electron microscopy structures of Cnp1, the Schizosaccharomyces pombe (S. pombe) ortholog of CENP-A, alone and in complex with Mis15, the fission yeast ortholog of CENP-N. By integrating structural, biochemical, and molecular dynamics analyses, we demonstrate that the N-terminal region of Cnp1 regulates both DNA-end breathing and the conformational mobility of the L1 loop, a critical structural element for Mis15 recognition. Either enhanced dynamics caused by N-terminal deletion or reduced dynamics from targeted residue substitution disrupt Mis15 binding in vitro and impair its centromeric localization in vivo, thereby compromising the earliest steps of constitutive centromere-associated network assembly. Our findings establish the Cnp1 N-terminus as a dynamic allosteric modulator of chromatin architecture and reveal an L1 loop modulation mechanism that links nucleosome flexibility to kinetochore specification and chromosome segregation fidelity in fission yeast.

Hepatitis B virus (HBV) infection remains a major global health challenge. While sodium taurocholate co-transporting polypeptide (NTCP) is the primary receptor for HBV entry, the molecular mechanisms regulating NTCP-mediated viral entry remain incompletely understood. Here, we identified CD46 as a crucial regulatory factor for NTCP membrane expression. We found that CD46 interacted with NTCP in cis at the plasma membrane through proximity-based labeling screening. The depletion of CD46 significantly reduced cell-surface NTCP levels and HBV infection in hepatocytes. Anti-CD46 monoclonal antibodies, particularly clone E4.3, inhibited HBV infection by triggering NTCP internalization from the plasma membrane to intracellular vesicles. The antiviral effect of CD46 antibodies was also confirmed in primary human hepatocytes. Our study reveals a previously unknown mechanism regulating NTCP-mediated HBV entry and suggests CD46 as a potential therapeutic target for HBV infection.

Fused in sarcoma (FUS), a DNA-RNA binding protein, affects gene transcription while its role in non-alcoholic steatohepatitis (NASH)-fibrosis is not well understood. In this study, Immunohistochemistry and western blot analysis were used to detect the expression of FUS in liver samples from patients with NASH and in LX-2 cells. Immunofluorescence staining showed that FUS co-localized with growth arrest and DNA damage 45β (GADD45B) in hepatic stellate cells (HSCs). Chromatin immunoprecipitation combined with quantitative PCR and luciferase assays were performed to validate the binding sites and transcriptional activity of FUS to the TGFB1 and COL1A1 promoters. Gadd45b knockout (Gadd45b KO) and wild-type mice with NASH-fibrosis model validated the role of GADD45B in NASH-fibrosis. Downregulation of GADD45B reduced HSC activation triggered by transforming growth factor beta-1 (TGFB1) stimulation or FUS overexpression. Ameliorated collagen deposition and decreased nuclear FUS content in HSCs were detected in Gadd45b KO mice. Overall, this study suggests that FUS and GADD45B could be potential treatment targets for NASH-fibrosis.

Mutations in the gene encoding cytoskeleton-associated protein 2-like (CKAP2L) have been identified as a causative factor for Filippi syndrome, a rare developmental disorder characterized by facial dysmorphism, syndactyly, and microcephaly. However, the cellular and molecular mechanisms by which CKAP2L contributes to the pathogenesis of this syndrome remain largely unknown. Here, we generate a Ckap2l knockout mouse model to investigate the in vivo and cellular roles of CKAP2L. Interestingly, Ckap2l knockout mice show no overt developmental abnormalities, with the exception of reduced male fertility, evidenced by decreased sperm count, impaired motility, and abnormally elongated flagella. At the cellular level, CKAP2L is a bona fide microtubule-associated protein that localizes to microtubule-based organelles, including the centrosome, mitotic spindle, and ciliary basal body. Depletion of CKAP2L leads to shortened mitotic spindles and cytokinesis failure, resulting in multinucleation. Furthermore, we uncover a conserved function for CKAP2L as a negative regulator of primary cilium length; its loss markedly increases ciliary length in both human and mouse cells. Collectively, these findings position CKAP2L as a multifunctional regulator of microtubule-based organelles and propose that Filippi syndrome can be classified as a 'centrosomopathy' arising from concurrent defects in cell proliferation and ciliary function.

The Werner syndrome (WS) is characterized with both premature aging and tumorigenic phenotypes. In this study, we introduced a tumorigenic mutation p53N236S (referred as p53S later), which is found in immortalized WS mouse embryo fibroblasts, back into WS mice to investigate its impact on the telomere dysfunction-induced aging process. Intriguingly, the introduction of p53S rescued the aging phenotypes of WS mice, showing the extension of the lifespan and the delay in organ degeneration. Further studies revealed that the introduction of p53S transcriptionally upregulated the DREAM/MMB pathway and downstream DNA helicases and telomere maintenance proteins, facilitated the recruitment of these proteins to G-quadruplex (G4) DNA structures proximal to DNA replication forks, and promoted the unwinding of G4. By comparing the cellular responses to pyridostatin and hydroxyurea, respectively, we confirmed that p53S specifically regulates G4-related DNA replication stress. Thus, p53S compensates the loss of Wrn and telomerase function, solves the DNA replication, telomere lengthening, and cell proliferation problems in WS cells, and ultimately, rescues the aging phenotypes of WS. Together, our data indicate that certain tumorigenic features can be applied to balance with premature aging, rescuing the aging phenotype without tumor risk. This study suggests a new mechanism in aging regulation and provides the possibility of developing a tumor-free longevity strategy and targeting G4 and DNA replication in aging-related tumor therapy.