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 that encode 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 vln2 vln3. 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 vln triple mutant had significantly fewer peroxisomes with long-range and linear movement but produced 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. Alternatively, here we define a new strategy which leverages multispectral imaging and phasor analysis, termed the Phasor Mixing Coefficient (PMC). PMC measures the precise mixing of fluorescent signals in each pixel. We demonstrate how 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 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 PMC offers users a nuanced and robust metric to quantify biological association.

The endoplasmic reticulum (ER) and mitochondria are known to affect myriad cellular mechanisms processes. More recently, dynamic association between them has been identified in different eukaryotes; these interactions vary in their composition and involvement in regulation of intracellular machineries. FAM134B (also known as RETREG1), originally identified as an oncogene, regulates ER membrane shape and curvature. It is a key ER-phagy or reticulophagy receptor, which promotes autophagy of not only the ER but also simultaneous dual autophagy of ER and mitochondria. Although it is known that FAM134B can potentiate contact with mitochondria, its direct involvement in affecting mitochondrial dynamics remains unexplored. Here, we show that FAM134B can interact with the canonical fission-promoting protein DRP1 (also known as DNM1L). Functional depletion of FAM134B leads to local actin rearrangement and reduced DRP1 recruitment onto mitochondria, resulting in hyperfusion. A decrease in FAM134B levels is observed with aging in rat brains, cell and mouse models of Parkinson's disease and samples derived from individuals with disease. Our study establishes FAM134B as the ER partner that helps in maintaining mitochondrial morphology and dynamics.

CYRI proteins promote lamellipodial dynamics by opposing Rac1-mediated activation of the Scar/WAVE complex. This activity also supports resolution of macropinocytic cups, promoting internalisation of surface proteins, including integrins. Here, we show that CYRI-B also promotes focal adhesion maturation and dynamics. Focal adhesions in CYRI-B-depleted cells show accelerated maturation and become excessively large. We probed the composition of these enlarged focal adhesions, using a Bio-ID screen, with paxillin as bait. Our screen revealed changes in adhesion proteins proximal to paxillin suggesting early activation of stress fibre contraction and depletion of the integrin internalisation mediator ERC1. Lack of CYRI-B leads to more stable lamellipodia and accumulation of polymerised actin in stress fibres. This actin acts as a barrier to microtubule targeting for adhesion turnover. Thus, our studies reveal an important connection between lamellipodia dynamics controlled by CYRI-B and microtubule targeting of ERC1 to modulate adhesion maturation and turnover.

The meiotic spindle forms only around the chromosomes in oocytes, despite the exceptionally large volume of the cytoplasm. This spatial restriction is likely to be governed by local activation of key microtubule regulators around the chromosomes in oocytes, but the identities of these microtubule regulators and the mechanisms remain unclear. To address this, we developed a novel assay to visualise spatial regulation of spindle-associated proteins in Drosophila oocytes by inducing ectopic microtubule clusters. This assay identified several proteins including the TPX2 homologue Mei-38, which localised more strongly to microtubules near the chromosomes than away from them. In Mei-38, we identified a microtubule-binding domain containing a region that was also highly conserved in humans. The domain itself is regulated spatially, and contains a conserved serine and a nearby PP2A-B56-docking motif. A non-phosphorylatable mutation of this serine residue allowed the domain to localise to ectopic microtubules as well as spindle microtubules, whereas mutations in the PP2A-B56-docking motif greatly reduced the spindle localisation. As this phosphatase is concentrated at the kinetochores, it might act as a novel chromosomal signal spatially regulating spindle proteins within oocytes.

Clathrin-mediated endocytosis (CME) is an essential, highly conserved process in eukaryotic cells that facilitates the internalization of plasma membrane components, transmembrane proteins and extracellular nutrients. This complex pathway involves the concerted assembly and disassembly of many different proteins at the plasma membrane. Budding yeast has served as a powerful model for dissecting CME through combined genetic, biochemical, quantitative imaging and mathematical approaches. In this Cell Science at a Glance article, we integrate decades of quantitative data to generate a three-dimensional molecular animation depicting the full progression of CME in budding yeast (Movie 1). The animation and accompanying poster capture the spatial and temporal dynamics of key protein players. In addition, we highlight recent advances in understanding of the condensation of endocytic proteins into distinct sites and the organization of actin networks that generate the forces necessary to deform and internalize the membrane against the high internal turgor pressure of the budding yeast cell.

Long-distance transport in neurons relies on motor proteins that can move towards either the plus- or minus-end of microtubules. In axons, microtubules uniformly have a plus-end-out orientation, whereas dendrites of vertebrate neurons contain mixed polarity bundles: stable microtubules are typically minus-end-out, and dynamic microtubules are plus-end-out. This organization supports selective transport, yet how this dedicated microtubule organization is established is unclear. Here, we use single-molecule localization microscopy, expansion microscopy and live-cell imaging to examine how the microtubule cytoskeleton is reorganized during neuronal development in cultured rat hippocampal neurons. We find that early neurites contain mixed polarity microtubules, with stable microtubules initially mostly plus-end-out and often connected to centrioles. As neurons mature, these microtubules detach, slide and gradually reorient to become predominantly minus-end-out within the future dendrites. Moreover, prior to axon specification, neurons often have one or two minor neurites with an almost uniformly plus-end-out microtubule network. Our findings show how reorganization of stable microtubules underlies the establishment of the characteristic microtubule network in mature vertebrate neurons.

Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that plays important roles in cell proliferation, differentiation and migration. EGFR overexpression or mutation is a hallmark of some cancers, leading to hyperactivation of downstream signalling. Co-regulation between EGF-dependent EGFR signalling and extracellular matrix (ECM) adhesion occurs in both healthy and malignant cells. Increasing ECM stiffness can contribute to lung cancer progression and is sensed by integrins to promote proliferation and invasion. Emerging evidence suggests non-canonical roles for EGFR in mechano-sensing, but the molecular mechanisms and functional consequences remain unclear. Here, we demonstrate that EGFR is activated in human lung cancer cells upon early adhesion to ECM substrates with physiologically relevant stiffness (28 kPa versus 1.5 kPa), independently of canonical ligands and integrins. Mechano-induced EGFR activation correlates with and requires active Src and F-actin, and it is coupled to stiffness-dependent plasma membrane retention of EGFR within disordered lipid microdomains. Early stiffness-dependent EGFR activation is required for enhanced migration. These findings uncover a non-canonical role for EGFR in early adhesion related mechano-sensing with potential implications for treatment of lung cancer.

The ability of cells to stick to each other and form tissues is mediated by protein complexes at the plasma membrane, such as adherens junctions (AJs). Key aspects of AJ stability are the biomechanical properties of the constituent proteins and the forces generated by the associated actin cytoskeleton. This Review concisely overviews our current understanding of how these factors play out at different length scales. When actomyosin pulls on the cadherin-catenin complex, the molecular interactions within the complex lead to an increase in AJ stability. Transcellular E-cadherin clusters are dynamically maintained by constant turnover and recruitment of actin-binding proteins organises the internal actin cytoskeleton. Among these are actin polymerisers that sustain the actin network and provide the mechanical forces important for AJ integrity. Finally, the distribution of AJs around the cell periphery and the long-range organisation of the associated actin bundles could contribute to maintaining AJ stability across tissues. We conclude with a summary of recently developed biophysical tools useful for the study of AJ mechanics and a few open questions that we expect to see answered in the not-too-distant future.

Accurate cell segmentation is an essential step in the quantitative analysis of fluorescence microscopy images. Pre-trained deep learning models for automatic cell segmentation such as those offered by Cellpose perform well across a variety of biological datasets but might still introduce segmentation errors. Although training custom models can improve accuracy, it often requires programming expertise and significant time, limiting the accessibility of automatic cell segmentation for many wet lab researchers. To address this gap, we developed 'Toggle-Untoggle', a standalone desktop application that enables intuitive, code-free quality control of automated cell segmentation. Our tool integrates the latest Cellpose 'cyto3' model, known for its robust performance across diverse cell types, while also supporting the 'nuclei' model and user-specified custom models to provide flexibility for a range of segmentation tasks. Through a user-friendly graphical interface, users can interactively toggle individual segmented cells on or off, merge or draw cell masks, and export morphological features and cell outlines for downstream analysis. Here, we demonstrate the utility of Toggle-Untoggle in enabling accurate, efficient single-cell analysis on real-world fluorescence microscopy data, with no coding skills required.