Journal of cell science最新文献

Vesicle-associated membrane protein-associated protein A (VAPA) is a protein of the endoplasmic reticulum (ER) and a component of several membrane contact sites (MCSs). We show here that VAPA also localizes to the inner nuclear membrane (INM), in close proximity to nuclear lamins, INM proteins and nucleoporins. Using our proteomics approach 'rapamycin- and APEX-dependent identification of proteins by SILAC' (RAPIDS), we identified several nuclear proximity partners of VAPA, including emerin, different LAP2 isoforms, lamin A/C and Nup153. Depletion of VAPA in various cellular systems resulted in reduced nuclear lamin levels and aberrant nuclear morphology, including the formation of membrane invaginations and tunnels. Furthermore, histone acetylation levels were altered. Our data suggest that VAPA has distinct nuclear functions, in addition to its established role as an ER organizer.

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 is present 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.

Polycomb repressor complex 2 (PRC2) tri-methylates histone 3 at lysine 27 (H3K27me3), a post translational modification that induces heterochromatin formation and transcriptional repression. Survivin (also known as BIRC5) is a nucleocytoplasmic shuttling protein that is kept out of the nucleus in clement conditions, but that accumulates there in times of stress and in certain specialised cells. Although the cytoplasmic functions of survivin are well documented, there is comparatively less understanding of its roles within the nucleus. Here, we investigated whether nuclear survivin can affect transcriptional programming. Using interaction analyses and qPCR, we report that it binds to the enzymatic subunit of PRC2 (EZH2) and H3K27me3, and causes depression of its target genes in a variety of human cells.

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 and nuclear volume in interphase, and 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.

Microtubule organization plays a central role in cell differentiation, orchestrating essential processes such as cell polarization, mechanotransduction, organelle positioning and intracellular transport. A hallmark of many differentiated cells is the transition from a centrosomal to a non-centrosomal microtubule-organizing center (MTOC). Here, we demonstrate that both centrosomal and nuclear envelope (NE)-associated MTOCs coexist in osteoclasts. We show that the key players for NE-MTOC formation, the AKAP6 and nesprin-1 (SYNE1) isoforms AKAP6β and nesprin-1α, previously considered muscle specific, are upregulated during osteoclast differentiation, suggesting a conserved role in NE-MTOC assembly across cell types. Targeted depletion of AKAP6 in RAW264.7-derived osteoclasts led to the displacement of the Golgi and MTOC-associated proteins PCM1, pericentrin and CDK5RAP2 from the NE, while their centrosomal localization remained intact. This selectively impaired microtubule nucleation from the NE without disrupting centrosomal microtubule activity, enabling a functional dissection of the two MTOCs. Loss of NE-MTOC activity, through AKAP6 depletion, impaired podosome formation and significantly reduced bone resorption capacity, highlighting the distinct and essential role of NE-derived microtubules in osteoclast function.

Regulation of lamin A/C levels and distribution is crucial for nuclear integrity and mechanotransduction via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Dysregulation of lamin A/C correlates with poor cancer prognosis, and its levels determine sensitivity to the microtubule-stabilising drug paclitaxel. Paclitaxel is well-known for disrupting mitosis, yet it also reduces tumour size in slow-dividing tumours, indicating an additional, poorly characterised interphase mechanism. Here, we reveal that paclitaxel induces nuclear aberrations in interphase through SUN2-dependent lamin A/C disruption. Using advanced optical imaging and electron cryo-tomography, we show the formation of aberrant microtubule-vimentin bundles during paclitaxel treatment, which coincides with nuclear deformation and altered lamin A/C protein levels and organisation at the nuclear envelope. SUN2 is required for lamin A/C reduction upon paclitaxel treatment and is in turn regulated by polyubiquitylation. Furthermore, lamin A/C expression levels determine not only cell survival during treatment but also recovery after drug removal. Our findings support a model in which paclitaxel acts through both defective mitosis and interphase nuclear-cytoskeletal disruption, providing additional mechanistic insights into a widely used anticancer drug.

The structural integrity of the nucleus is dependent on nuclear mechanical elements of chromatin and lamins resisting antagonistic actin cytoskeleton forces. Force imbalance results in nuclear blebbing, rupture and cellular dysfunction found in many human diseases. Here, we used the fluorescent ubiquitin cell cycle indicator (FUCCI) cells to determine how cell cycle changes affect the nucleus and actin force balance. Whereas nuclear blebs were present equally throughout interphase, nuclear blebs formed predominantly in G1 and then persisted into G2. Actin-based nuclear confinement and focal adhesion density was greater in G1 versus G2 cells. Removal of focal adhesions through treatment with 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 were 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 through treatment with 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 crucial 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 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.

Within the nucleus of each human cell, ∼2 m of linear DNA is compacted and organized. The structures and principles of genome organization are developmentally regulated and broadly evolutionarily conserved. However, conclusive links between genome structure and function have been difficult to find. In this Review, we provide an overview of mammalian genome organization, highlight recent studies demonstrating how it interacts with evolutionary diversity, and explore its contributions to development. We propose an innovative perspective - that variability in genome organization supports plastic cell fates in multicellular organisms - and draw analogies to show how evolutionary variation can inform study of the function of genome organization.

As the mechanosensitive sensory cells in the inner ear, hair cells are characterized by their apical F-actin-filled stereocilia. The stereocilia are organized into a staircase-like pattern with rows of increasing height. The development and maintenance of stereocilia are tightly regulated, and deficits in this process usually lead to hearing loss. Recently, our group reported that RNA-binding proteins (RBPs) such as RBM24 and ESRP1 play essential roles in the inner ear hair cells. In the present work, we showed that Nucleolin (NCL), a highly conserved RBP, is required for stereocilia maintenance in inner ear hair cells. Ncl knockout leads to progressive stereocilia degeneration in the outer hair cells in a basal-to-apical gradient. Meanwhile, Ncl knockout results in progressive stereocilia fusion in the inner hair cells in an apical-to-basal gradient. As a result, these stereocilia deficits lead to hair cell loss and eventually cause hearing loss in Ncl conditional knockout mice. RNA-seq analysis identified several genes whose mRNA level is affected by Ncl knockout. Among them are Espnl and Ptprq, which have been shown to play essential roles in stereocilia development and/or maintenance. Further investigations confirmed that NCL could directly bind to Espnl and Ptprq mRNAs, and that NCL could increase the stability of Espnl and Ptprq mRNAs. Together, our data demonstrate that NCL plays essential roles in stereocilia maintenance through regulating the stability of its target mRNAs.