Journal of Cell Biology最新文献

Despite the well-established role of condensin II in mitotic chromosome assembly, its function in interphase chromosome organization remains poorly understood. Here, we applied multiscale FISH techniques to human cell lines engineered for single or double depletion of condensin II and cohesin and examined their functional collaboration at two distinct stages of the cell cycle. Our results demonstrate that a functional interplay between condensin II and cohesin during the mitosis-to-G1 transition is critical for establishing chromosome territories (CTs) in the newly assembling nucleus. During the G2 phase, condensin II and cohesin cooperate to maintain global CT morphology, although they act at different genomic scales. Strikingly, double depletion of both complexes causes CTs to collapse and accumulate abnormally at the nucleolar periphery. Based on these findings, we will discuss how the condensin and cohesin complexes act in an orderly and cooperative manner to orchestrate chromatin dynamics across genomic scales, thereby supporting higher-order chromosome organization throughout the cell cycle.

Platelet α-granules are lysosome-related organelles produced in megakaryocytes, the platelet precursor cells. The biogenesis of α-granules is incompletely understood but depends on common endosomal pathways. Here, we discovered GRIPAP1, a partially characterized ubiquitous protein, as a new component of the α-granule biogenesis machinery. GRIPAP1-deficient megakaryocytes showed a significant decrease of α-granule numbers and overall cargo levels. In WT megakaryocytes, fibrinogen taken up by endocytosis and newly synthesized PF4 trafficked through GRIPAP1-labeled compartments en route to α-granules. GRIPAP1 localized to endosome subdomains decorated by Rab4a and Stx12, known players in α-granule biogenesis. GRIPAP1 bound GTP-loaded Rab4a, a key interaction for GRIPAP1 recruitment to membranes. Biochemically, GRIPAP1 behaved as an elongated homodimer akin to membrane tethering factors. Consistently, artificial mislocalization of GRIPAP1 to the mitochondria was sufficient to recruit Rab4a compartments containing internalized transferrin and newly synthesized PF4 to mitochondria. Together, the data advance understanding of endosomal transport, the biogenesis of α-granules, and likely other endo-lysosomal organelles.

Landscape expansion microscopy (land-ExM) is a light microscopy technique that visualizes both the lipid and protein ultrastructural context of cells. Achieving this level of detail requires both superresolution and a high signal-to-noise ratio. Although expansion microscopy (ExM) provides superresolution, obtaining high signal-to-noise images of both proteins and lipids remains challenging. land-ExM overcomes this limitation by using self-retention trifunctional anchors to significantly enhance protein and lipid signals in expanded samples. This improvement enables the accurate visualization of diverse membrane organelles and phase separations, as well as the 3D visualization of their contact sites. As a demonstration, we revealed triple-organellar contact sites among the stress granule, the nuclear tunnel, and the nucleolus. Overall, land-ExM offers a high-contrast superresolution platform that advances our understanding of how cells spatially coordinate interactions between membrane organelles and phase separations.

Homeostatic pathways maintain the lipid composition of organelle membranes, and mechanistic links between lipid sensing, synthesis, and trafficking are lacking. Acute depletion of cell cholesterol elicits an increase in the rate of very-long-chain (VLC) sphingomyelin synthesis in the Golgi apparatus, thereby promoting cholesterol retention in the plasma membrane. Stable isotope metabolic analyses and lipid trafficking assays showed that the increase in VLC-sphingomyelin results from an increase in the rate of coatomer II-dependent trafficking of VLC-ceramide from the endoplasmic reticulum to the Golgi apparatus. An integral membrane protein of the coatomer II network, cTAGE5, is required for endoplasmic reticulum-to-Golgi trafficking of ceramide and cTAGE5 overexpression caused herniations of the endoplasmic reticulum network that entrapped a synthetic ceramide analog to which cTAGE5 could be photochemically cross-linked. We propose that cTAGE5 is a ceramide sensor for export of VLC-ceramide from the endoplasmic reticulum exit site.

Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.

Loss-of-function studies are a central approach to understanding gene/protein function. In mice, this often relies upon heritable recombination at the DNA level. This approach is slow and nonreversible, which limits both spatial and temporal resolution of analysis. Recently, degron techniques that directly target proteins for degradation have been successfully used to quickly and reversibly knock down proteins. Currently, these systems have been limited by lack of tissue/cell type specificity. Here, we generated mice that allow spatial and temporal control of GFP-tagged protein degradation. This DegronGFP line leads to degradation of GFP-tagged proteins in different cellular compartments and in distinct cell types. Further, it is rapid and reversible. We used DegronGFP to probe the function of the glucocorticoid receptor in the epidermis and demonstrate that it has distinct functions in proliferative and differentiated cells-an analysis that would not have been possible with traditional recombination approaches. We propose that the ability to use GFP knock-in lines for loss-of-function analysis will provide additional motivation for generation of these useful tools.

Segregating a complete set of chromosomes into the gametes relies on exchanges of genetic material that occur during meiosis. It is only exchanges that form between the homologous chromosomes (homologs), rather than between the identical sister chromatids, that enable correct chromosome segregation. Understanding how the homologs and the sisters are distinguished requires knowledge of how they are organized relative to each other. Here, we use selective labeling of a single sister in Caenorhabditis elegans to define the organization of the sister chromatids when meiotic exchanges form. We find that pairs of sisters are well separated (resolved) throughout pachytene, despite being tethered to each other along their length. Depleting the cohesin loader NIPBLSCC-2 impairs sister resolution, suggesting that an active process-likely loop extrusion-demixes the sisters. Our work shows that meiotic exchanges form in C. elegans when the sisters and homolog are roughly the same distance from one another, suggesting that repair template choice is unlikely to rely on relative proximity.

SQSTM1/p62 is a master regulator of the autophagic and ubiquitination pathways of protein degradation and the antioxidant response. p62 functions in these pathways via reversible assembly and sequestration of additional factors into cytoplasmic phase-separated structures termed p62 bodies. The physiological roles of p62 in these various pathways depend on numerous mechanisms for regulating p62 body formation and dynamics that are incompletely understood. Here, we identify a new mechanism for regulation of p62 oligomerization and incorporation into p62 bodies by SHKBP1, a cullin-3 E3 ubiquitin ligase adaptor, that is independent of its potential functions in ubiquitination. We map an SHKBP1-p62 protein-protein interaction outside of p62 bodies that limits p62 assembly into p62 bodies and affects the antioxidant response involving sequestration of Keap1 and nuclear translocation of Nrf2. These studies provide a non-ubiquitination-based mechanism for an E3 ligase adaptor in regulating p62 body formation and cellular responses to oxidative stress.

Micronuclei (MN), a hallmark of chromosome instability, frequently rupture, leading to protumorigenic consequences. MN rupture requires nuclear lamina defects, yet their underlying causes remain unclear. Here, we demonstrate that MN lamina gaps are linked to excessive MN growth resulting from impaired protein export. This export defect arises from reduced levels of the transport protein RCC1 in MN. Overexpressing RCC1 increases protein export and protects MN from rupture. Differences in RCC1 levels linked to chromatin state also explain why high euchromatin content increases the stability of small MN. Additional RCC1 loss in euchromatic MN results in impaired protein import. For these MN, increasing RCC1, directly or through increasing histone methylation, accelerates rupture. Our findings define a new model of MN rupture, where defects in protein export drives continuous MN growth causing nuclear lamina gaps that predispose MN to membrane rupture and where chromatin-specific features can alter rupture of small MN by further impairing nuclear transport.

Hundreds of mitochondrial proteins rely on N-terminal presequences for organellar targeting and import. While generally described as positively charged amphiphilic helices, presequences lack a consensus motif and thus likely promote protein import into mitochondria with variable efficiencies. Indeed, the concept of presequence strength underlies biological models such as stress sensing, yet a quantitative analysis of what dictates strong versus weak presequences is lacking. Furthermore, the extent to which presequence strength affects mitochondrial function and cellular fitness remains unclear. Here, we capitalize on the MitoLuc protein import assay to define multiple aspects of presequence strength. We find that select presequences, including those that regulate the mitochondrial unfolded protein response (UPRmt), impart differential import efficiencies during mitochondrial uncoupling. Surprisingly, we find that presequences beyond those associated with stress signaling promote highly variable import efficiency in vitro, suggesting presequence strength may influence a broader array of processes than currently appreciated. We exploit this variability to demonstrate that only presequences that promote robust in vitro import can fully rescue defects in respiratory growth in complex IV-deficient yeast, suggesting that presequence strength dictates metabolic potential. Collectively, our findings demonstrate that presequence strength can describe numerous metrics, such as total imported protein, maximal import velocity, or sensitivity to uncoupling, suggesting that the annotation of presequences as weak or strong requires more nuanced characterization than typically performed. Importantly, we find that such variability in presequence strength meaningfully affects cellular fitness beyond stress signaling, suggesting that organisms may broadly exploit presequence strength to fine-tune mitochondrial import and thus organellar homeostasis.