Journal of cell science最新文献

A highly curved membrane region connecting the inner and the outer nuclear membrane serves as a platform where nucleoporins with one or more transmembrane domains promote anchoring of the nuclear pore complex to the nuclear envelope. In mammalian cells, three transmembrane nucleoporins, Nup210, POM121 and NDC1, are inserted at this site. Here, we characterize TMEM209, which had initially been identified as a protein concentrated at the nuclear envelope, as a fourth transmembrane nucleoporin. Proximity labeling revealed that TMEM209 occurs close to proteins of the inner nuclear membrane and to other nucleoporins. TMEM209 localized to the nuclear pore complex in immunofluorescence microscopy and biochemically interacted with Nup210 via a region containing its two transmembrane domains. TMEM209 depletion impaired cell growth and delayed entry into S, G2 and M phases of the cell cycle. Conversely, its overexpression specifically dissociated Nup210 from the nuclear envelope. Together, these findings establish TMEM209 as a novel transmembrane nucleoporin that cooperates with Nup210 in cell cycle progression and cell proliferation.

Epithelial tissues form protective barriers while supporting critical functions such as absorption and secretion. Their structural and functional integrity relies on adherens junctions, which coordinate migration and transmit forces between adjacent cells by connecting their actin cytoskeleton. In this study, we report the presence of an apical supracellular actin network in squamous epithelial cells. Using squamous carcinoma A431 cells as a model, we characterized this network composed of star-shaped actin structures interconnected by linear actin bundles that span multiple cells. We demonstrate that the network's formation and maintenance require actomyosin contractility and intact adherens junctions, while tight junctions seem dispensable. Furthermore, this network dynamically reorganizes as cells migrate and preferentially aligns with the direction of movement. This contractile structure generates mechanical tension that extends across the apical surface of multiple cells. Our findings suggest that this supracellular actin network functions as a long-range force transmission device in squamous cells, advancing our understanding of the biomechanical properties of epithelia.

Mitotic chromosome dimensions differ between species, and they differ between developmental stages within an organism. The physiological determinants of chromosome size remain poorly understood. Here, we investigate chromosome size determinants in the fission yeast Schizosaccharomyces pombe. Super-resolution microscopy and semi-automated measurements reveal that cell or nuclear volume in interphase, or the time spent in mitosis (both previously proposed chromosome size determinants), have little influence on resultant chromosome dimensions. Instead, levels of the chromosomal condensin complex affect chromosome size, with increasing condensin levels resulting in more compact, thinner and shorter, chromosomes. Our observations inform the understanding of how chromosome dimensions are controlled in an organism. They suggest that a chromosome-intrinsic mechanism sets chromosome size, more so than the environment in which chromosomes find themselves in.

The structural integrity of the nucleus is dependent on nuclear mechanical elements of chromatin and lamins to resist antagonistic actin cytoskeleton forces. Force imbalance results in nuclear blebbing, rupture, and cellular dysfunction found in many human diseases. We used Fluorescent Ubiquitin Cell Cycle Indicator (FUCCI) cells to determine how cell cycle changes affect the nucleus and actin force balance. While nuclear blebs are present equally throughout interphase, nuclear blebs form predominantly in G1 and then persist into G2. Actin-based nuclear confinement and focal adhesion density is greater in G1 vs. G2 cells. Removal of focal adhesions via an inhibitor resulted in decreased nuclear confinement and blebbing, supporting this as the underlying mechanism. Upon artificial confinement, G2 nuclei ruptured more than G1 nuclei. Single nucleus micromanipulation force measurements confirmed that G1 nuclei are stiffer than G2 nuclei in both the chromatin-based and lamin-based nuclear stiffness regimes. Decreased nuclear stiffness can be explained by loss of peripheral H3K9me3 from G1 to G2, recapitulated by H3K9me3 inhibition via Chaetocin. Cell cycle-based changes in nuclear and actin mechanics impact nuclear integrity and shape.

The genome folds inside the cell nucleus into hierarchical architectural features, such as chromatin loops and domains. If and how this genome organization influences the regulation of gene expression remains only partially understood. The structure-function relationship of genomes has traditionally been probed by population-wide measurements after mutation of critical DNA elements or by perturbation of chromatin-associated proteins. To circumvent possible pleiotropic effects of such approaches, we have developed OptoLoop, an optogenetic system that allows direct manipulation of chromatin contacts by light in a controlled fashion. OptoLoop is based on the fusion between a nuclease-dead SpCas9 protein and the light-inducible oligomerizing protein CRY2. We demonstrate that OptoLoop can bring together genomically distant, repetitive DNA loci. As a proof-of-principle application of OptoLoop, we probed the functional role of DNA looping in the regulation of the human telomerase gene TERT by long-range contacts with the telomere. By analyzing the extent of chromatin looping and nascent RNA production at individual alleles, we find evidence for looping-mediated repression of TERT. In sum, OptoLoop represents a novel means for the interrogation of structure-function relationships in the genome.

Complex transcriptional programs and signaling pathways control early hematopoietic lineage specification. Many key regulators have been identified; however, a substantial portion of the genome remains functionally uncharacterized. Here, we investigated six uncharacterized 'Riken' genes identified through transcriptomic profiling of Flk-1+ (also known as Kdr)/Pdgfrα- (hematoendothelial-enriched) and Flk-1+/Pdgfrα+ (cardiac mesoderm-enriched) populations at day 4 of embryoid body (EB) differentiation. We generated knockouts in mouse embryonic stem cells and performed bulk RNA-sequencing at day 4. Three of these genes (C130074G19Rik, I830077J02Rik and A530016L24Rik) were selected for further investigation by single-cell RNA-sequencing at day 7 of differentiation, which provided novel insight for two of these genes. Knockout of C130074G19Rik (hereafter G19Rik) increased the abundance of megakaryocyte progenitors and reduced endothelial populations, with differentially expressed genes enriched for hemostasis and membrane trafficking pathways. I830077J02Rik (hereafter J02Rik)-knockout cells showed subtle changes in extracellular matrix and cell adhesion genes, with a shift toward hematoendothelial lineages. Both G19Rik and J02Rik genes encode (predicted) transmembrane proteins that modulate membrane-associated processes in early hematopoietic development. This work establishes a framework for the study of uncharacterized genes with potential roles in cell fate determination.

Paraspeckles are stress-induced nuclear RNA-protein condensates that assemble on the long non-coding RNA NEAT1. Their increased formation under certain cellular circumstances has gained growing interest due to their association with serious human diseases, such as neurodegenerative disorders and cancer. The biological functions of paraspeckles still appear obscure, but increasing evidence suggests that they contribute to regulation of gene expression by recruiting specific proteins and RNA molecules. Here, we have characterized and compared two stress-enriched interactomes of the essential paraspeckle protein NONO in both wild-type and paraspeckle-deficient NEAT1 knockout cells. We identified Hsp70 as part of stress-enriched NONO complexes in wild-type but not in NEAT1-depleted cells. We show that proteotoxic stress-induced paraspeckle formation and NEAT1 expression are strictly dependent on Hsp70 chaperone activity. Our data demonstrate that both NONO and Hsp70 transiently translocate to the nucleolus during heat shock and that paraspeckle formation during recovery follows Hsp70-dependent relocation of NONO from the nucleolus to the nucleoplasm. Taken together, we demonstrate an important role of Hsp70 in paraspeckle assembly and identify a possible link between the nuclear protein quality control system and paraspeckles.

LAMP1 and LAMP2A are abundant proteins of late endosomal/lysosomal compartments that are often used interchangeably to label what is assumed to be the same organelle population, potentially obscuring distinct physiological roles. Here, we characterised the axonal transport dynamics of LAMP1- and LAMP2A-positive compartments in human iPSC-derived cortical neurons. We found that LAMP1-positive organelles move slower in the retrograde direction, pause more frequently, and display a broader anterograde velocity distribution than LAMP2A-positive vesicles, indicating distinct trafficking behaviours. Co-transport analysis revealed that approximately 65% of motile LAMP-positive organelles carry both markers, with higher co-transport in the retrograde direction. To explore molecular differences underlying these behaviours, we performed proximity labelling using full-length LAMP1 or LAMP2A fused to the light-activated biotin ligase, LOV-Turbo. This approach revealed largely overlapping interactomes, with LAMP2A-associated proteins forming a subset of the LAMP1 interactome and showing an enrichment for synaptic vesicle-related proteins. We further validated ZFYVE16 as a novel interactor of both compartments. Together, our findings indicate that LAMP1- and LAMP2A- positive organelles share overlapping molecular identities but represent functionally distinct axonal populations with divergent transport dynamics.

Models of membrane fusion generally assume that strong repulsion between membranes hinders fusion and use macroscopic properties to describe how proteins induce fusion of membranes. Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are commonly believed to mediate intracellular membrane fusion by forming continuous α-helices that assemble into stable complexes, acting as semi-rigid rods that overcome the repulsion between the membranes, bending and squeezing them to cause fusion. However, some evidence indicates that membranes can readily be brought into contact and that helix continuity is not necessary for SNARE function. This Hypothesis article reviews recent molecular dynamics simulations that suggested a fundamentally different model, which postulates that the key obstacle to initiating fusion is the difficulty of mixing the hydrophobic interiors of the two membranes at the polar membrane-membrane interface and that SNAREs act as local detergents to catalyze such mixing. The SNAREs also facilitate evolution of the resulting hydrophobic core into a series of asymmetric stalk-like structures and formation of the fusion pore. The structural properties of cell-cell and virus-cell fusion proteins indicate that they all can act as local detergents; therefore, this central aspect of SNARE function could be universal for all types of biological membrane fusion.