Journal of cell science最新文献

Actin microfilaments (F-actin) serve as tracks for myosin-driven organelle movement in plants. To understand how the F-actin network supports organelle movement, we examined the motility of peroxisomes as a common proxy for overall organelle motility in Arabidopsis thaliana. Using mutants of three villin (VLN) genes encoding major actin-bundling proteins that are actively expressed in vegetative tissues, we found that the vln4 mutation exacerbated the growth and subcellular F-actin defects in the vln2 vln3 double mutant. Compared to wild-type cells, the double and triple vln mutants exhibited progressive reduction of stable F-actin bundles and rapid remodeling of the fine filaments into a dynamic mesh. The defective F-actin organization caused significantly reduced mean speed and displacement distance of peroxisomes, although both rapid and slow movements were observed. Correlation analysis grouped complex heterogeneous peroxisome movement patterns into clusters reflecting distinct movement patterns. The vln2 vln3 vln4 triple mutant had significantly fewer peroxisomes with long-range and linear movement but produced an actin mesh network sufficient to retain basal peroxisome function. Our results provide insights into how VLN-dependent F-actin organization is coupled with the complex patterns of actomyosin-mediated organelle movement.

The first step to probing any potential interaction between two biomolecules is to determine their spatial association. In other words, if two biomolecules localize similarly within a cell, then it is plausible they could interact. Traditionally, this is quantified through various colocalization metrics. These measures infer this association by estimating the degree to which fluorescent signals from each biomolecule overlap or correlate. However, these metrics are, at best, proxies, and they depend strongly on various experimental choices. Here, we define a new strategy that leverages multispectral imaging and phasor analysis, termed the phasor mixing coefficient (PMC). The PMC measures the precise mixing of fluorescent signals in each pixel. We demonstrate how the PMC captures complex biological subtlety by offering two distinct values, a global measure of overall color mixing and the homogeneity thereof. We additionally show that the PMC exhibits less sensitivity to signal-to-noise ratio, intensity threshold and background signal compared to canonical methods. Moreover, this method provides a means to visualize color mixing at each pixel. We show that the PMC offers users a nuanced and robust metric to quantify biological association.

Chromera velia is a photosynthetic, free-living alga closely related to the apicomplexan parasites, a phylum of intracellular pathogens responsible for many devastating diseases, including malaria, cryptosporidiosis and toxoplasmosis. With molecular and cellular landmarks clearly related to but distinguishable from those found in apicomplexan parasites, Chromera provides an opportunity to investigate the evolutionary origin of the structures and processes needed for intracellular parasitism. However, tools for defining localization and functions of gene products do not exist for Chromera, which creates a major bottleneck for exploring its biology. Here we report two major advances in exploring the cell biology of this free-living relative of a large group of intracellular parasites: (1) successful cell transformation and (2) the implementation of expansion microscopy. The initial analysis enabled by these tools generated new insights into subcellular organization in different life stages of Chromera. These new developments boost the potential of Chromera as a model system for understanding the evolution of parasitism in apicomplexans.

Tumor protein p53 inducible nuclear protein 2 (TP53INP2; also known as DOR) is a multifunctional protein involved in transcriptional coactivation, ribosomal RNA synthesis and autophagy, regulated by subcellular localization. Using CRISPR/Cas9-generated TP53INP2-knockout HeLa cells reconstituted with EGFP-TP53INP2, we show that TP53INP2 is predominantly degraded by nuclear proteasomes under basal conditions. Under stress, including starvation and various chemical stress inducers, TP53INP2 accumulates in the cytoplasm independently of ATG5, CRM1-mediated export, phosphorylation, ubiquitylation or acetylation. We identify a nuclear localization signal (NLS) overlapping a nucleolar localization signal (NoLS) in the C-terminus, which mediates nuclear import and nucleolar enrichment. Deletion of this region redirects TP53INP2 to LC3B-positive puncta. A conserved nine-amino-acid cytoplasmic retention motif (CRM) in the C-terminus prevents nuclear re-entry under stress. This motif and regulation of subcellular localization is conserved in the related TP53INP1 protein. Fluorescence recovery after photobleaching (FRAP) and importin-binding assays show that nutrient starvation disrupts nuclear import of TP53INP2. Finally, we show that starvation enhances TP53INP2 translation via the m6A demethylase FTO, without altering mRNA stability. These findings uncover coordinated regulation of TP53INP2 localization and turnover by cellular stress.

Evolutionary cell biology is emerging as a vibrant discipline, integrating comparative cell biology, evolutionary theory and modern molecular approaches to understand how cells evolve and diversify. With roots dating back to the foundational work of Darwin and Haeckel in the 1800s, the field was historically eclipsed by a focus on a handful of genetically tractable model organisms. Yet, breakthroughs in genomics, imaging, experimental evolution and phylogenetics are driving the rapid growth of the field. Modern evolutionary cell biology faces four central challenges: integrating cell biology with evolutionary theory and experimental evolution to understand both adaptive and non-adaptive processes, bridging the genotype-phenotype gap, identifying and developing new model systems beyond traditional organisms to capture the full diversity of cellular mechanisms, and integrating ecological context with evolutionary processes to understand how environmental forces shape cellular phenotypes. In this Perspective, we discuss how meeting these challenges will illuminate fundamental evolutionary rules governing cellular complexity, innovation and adaptation across the tree of life, with potential applications for predicting cellular responses to future environmental challenges.

Ubiquilins are a family of extrinsic ubiquitin receptors that are thought to facilitate protein degradation by shuttling proteins to the proteasome. However, the defining characteristics of ubiquilin clients, and the steps of ubiquilin-mediated degradation, have been elusive. Previously, we showed that ubiquilin 2 (UBQLN2) regulates the proteasomal degradation of PEG10, a unique virus-like protein that comes in two forms: a gag protein, which is not regulated by UBQLN2, and a gag-pol protein, which is dependent on UBQLN2. Here, we refine the model of ubiquilin activity through further investigation of the UBQLN2-mediated degradation of PEG10. Gag-pol and gag proteins undergo distinct degradation processes; both forms bind to UBQLN2 independently of their ubiquitylation status, but only gag-pol protein is degraded in a UBQLN2-, ubiquitin- and proteasome-dependent fashion. Cellular gag-pol is ubiquitylated, and mutation of key lysine residues in the pol region rendered gag-pol insensitive to UBQLN2. Degradation of gag-pol was also dependent on the E3 ubiquitin ligase UBE3A, which requires UBQLN2 to regulate gag-pol levels. Together, these data clarify our understanding of UBQLN2-mediated degradation and highlight the importance of UBE3A in regulating PEG10.

In the nematode Caenorhabditis elegans, sperm-derived mitochondria and membranous organelles (MOs) are selectively degraded by autophagy in embryos in a process termed allophagy. For this process, ALLO-1 functions as an autophagy adaptor. The allo-1 gene encodes two splice isoforms, ALLO-1a and ALLO-1b, which have different C-terminal sequences and are predominantly targeted to MOs and paternal mitochondria, respectively. However, the mechanism by which ALLO-1 targets the paternal organelles remains unknown. In this study, X-ray crystallography analysis reveals that the C-terminal region of ALLO-1a forms a parallel coiled-coil structure. In addition, AlphaFold2-Multimer predicts that this region directly interacts with ubiquitin. We showed that ALLO-1a interacts with K48- and K63-linked polyubiquitin in vitro and found that the D355 residue of ALLO-1a at the predicted interface with ubiquitin is important for its ubiquitin binding in vitro and also for its MO targeting and MO degradation in embryos. These results suggest that ubiquitin is a marker for the recognition of MOs by the autophagy machinery in C. elegans embryos.

MYO1F, a long-tailed myosin of class I, is selectively expressed in immune cells and upregulated in microglia associated with neurodegenerative pathogenesis. Myosin motor functions are regulated by adaptor proteins that mediate cargo attachment and motor recruitment. To define the MYO1F interactome, we used in situ proximity labelling and proteomics in human myeloid cells. We identified a distinct SH3-domain-dependent adaptor module comprising CD2AP, ASAP1, SH3BP2 and SH3KBP1 (herein termed the CASS group of proteins). Interestingly, CD2AP is an Alzheimer's disease (AD) risk gene upregulated in the microglia of individuals with AD, which are implicated in phagocytic responses to amyloid-β. Structural modelling and mutagenesis confirmed multivalent proline-rich motif interactions between the CASS group of proteins and the MYO1F SH3 domain. Additional binding partners associate with the MYO1F pleckstrin homology (PH) domain. Immunofluorescence revealed colocalisation of MYO1F and the CASS group of proteins at actin-rich podosomes and phagocytic cups in macrophages and microglia. Functional assays demonstrated that MYO1F recruitment to the phagocytic cup requires motor activity and intact PH and SH3 domains. We provide the first MYO1F interactome identifying adaptor proteins for MYO1F in podosomes and during phagocytosis, offering new insights into its function in disease-associated microglia during neurodegeneration.

Serum response factor (SRF) and its cofactors, myocardin-related transcription factors A and B (MRTF-A and MRTF-B, respectively), regulate transcription of numerous cytoskeletal structural and regulatory genes, and most MRTF/SRF inactivation phenotypes reflect deficits in cytoskeletal dynamics. We show that MRTF-SRF activity is required for effective proliferation of both primary and immortalised fibroblast and epithelial cells. Cells lacking the MRTFs or SRF proliferate very slowly, express elevated levels of senescence-associated secretory phenotype (SASP) factors and senescence-associated β-galactosidase activity, and inhibit proliferation of co-cultured primary wild-type cells. They exhibit decreased levels of CDK1 and CKS2 proteins, and elevated levels of CDK inhibitors, usually p27 (also known as CDKN1B). These phenotypes, which can be fully reversed by re-expression of MRTF-A, are also seen in wild-type cells arrested by serum deprivation. Moreover, in wild-type cells direct interference with cytoskeletal dynamics through inhibition of Rho kinases (ROCKs) or myosin ATPase induces a similar proliferative defect to that seen in MRTF-null cells. MRTF-null cells exhibit multiple cytoskeletal defects and markedly reduced contractility. We propose that MRTF-SRF signalling will be required for cell proliferation in cell types and environments where physical progression through cell cycle transitions requires high contractility.