Biology subjects

Paluch, E. K.

Publications and source records attributed to Paluch, E. K..

13 recordsLinked to original sources

bioRxiv2026

Fast single molecule localization microscopy in 3D

Super-resolution methods, such as single molecule localisation microscopy (SMLM), can determine the distribution of specific proteins at the nanometre scale. However, three-dimensional (3D) SMLM is typically limited to thin sections and requires prohibitively long acquisition times to image complete cells. Here we introduce a 3D SMLM imaging modality, Intrinsic3D, that combines the speed and optical sectioning of oblique plane microscopy (OPM) with the nanometre resolution of SMLM to determine the position of single molecule emitters throughout complete volumes more than an order-of-magnitude faster over regular DNA-PAINT. By scanning the oblique light sheet across the volume and combining localization information from two or more adjacent planes, an emitter's x, y and z position is determined, eliminating the need for post-processing steps to stitch multiple image stacks together. We demonstrate resolution and speed by imaging nuclear pore complexes in U2OS cells and Drosophila egg chambers. As a proof-of-concept, we map the 3D distribution of the cell-adhesion protein DE-cadherin in the Drosophila follicular epithelium and show that spot adherens junctions are enriched near tricellular junctions and contain more E-cadherin than the apical adherens junctions, suggesting that spot junctions play a wider role in cell-cell adhesion than previously appreciated. Finally, we examine the organization of Gliotactin, a tricellular septate junction protein relative to E-cadherin using exchange-PAINT. These investigations demonstrate that Intrinsic3D delivers rapid 3D protein mapping in whole cells. Combining Intrinsic3D with existing multiplex and resolution enhancing methods (e.g. exchange-PAINT and RESI) will enable multicolour, 3D in situ spatial proteomics in tissue samples and bridge the gap between SMLM and systems-level cell biology.

cell biology↗

bioRxiv2026

Morphospace analysis reveals divergent cellular behaviours driving tissue internalisation during insect gastrulation

During gastrulation, embryonic epithelia undergo large-scale remodelling to internalise the mesoderm. Whether the cellular mechanisms underlying these tissue deformations are conserved across species remains unclear. Using quantitative cell-shape embedding, we compare cellular behaviours underlying gastrulation in two insects. We show that while Drosophila mesoderm cells follow a coordinated shape change sequence during gastrulation, mesoderm cells in the beetle Tribolium display heterogeneous shapes with no clear trajectory. This heterogeneity arises from two distinct internalisation modes: canonical apical constriction-driven tissue invagination, and early internalisation through out-of-plane cell divisions. Interpretable machine learning together with in vivo perturbations identify nuclear crowding as a key predictor of out-of-plane divisions and show that, in both species, proliferation slows tissue folding while promoting individual cell internalisation. Our study showcases morphospace analysis as a powerful tool for dissecting the cellular basis of tissue morphogenesis and reveals a conserved antagonistic relationship between cell divisions and tissue folding during gastrulation.

developmental biology↗

bioRxiv2026

Basement membrane mechanics drives patterned response to developmental signalling

Precise spatiotemporal patterning of cell fate decisions during development, such as those accompanying gastrulation, has traditionally been attributed to morphogen gradients. Emerging evidence also points to a crucial role for mechanics in regulating cell fate decisions. Here, we uncover that basement membrane mechanics in human pluripotent stem cells (hPSCs) regulates the spatiotemporal response to gastrulation-inducing BMP4. Reducing mechanosensation via soft substrates or chemical inhibitors abolishes stereotypical spatial patterning of the BMP4 response in hPSC colonies. This loss arises from disrupted epithelial polarity and increased permeability, which enhance BMP receptor accessibility. Strikingly, apical exposure to soluble laminin similarly disrupts polarity and suppresses hPSC mechanosensing, phenocopying the soft substrates effect and suggesting feedback between polarity signaling and mechanosensing. Finally, softening the basement membrane in mouse embryos triggers ectopic and premature mesoderm differentiation, disrupting gastrulation patterning. Together, this study describes a mechanochemical feedback mechanism that establishes basement membrane mechanics as a key regulator of developmental patterning.

cell biology↗

bioRxiv2024

Cell shape noise strength regulates shape dynamics during EMT-associated cell spreading

Cellular shape is intimately linked to cell function and state, and transitions between cell states are tightly coupled to shape changes. Yet, shape has been largely overlooked in state transitions studies. Here, we combine morphometric analysis with theoretical modeling and molecular perturbations to interrogate cell shape dynamics during epithelial-to-mesenchymal transition (EMT). Using stochastic inference, we extract the morphogenetic landscape underlying EMT. We show that within this landscape, EMT-associated cell spreading reflects a transition between shape attractors. Strikingly, we observe a peak in cell shape noise strength concomitant with spreading, and show that higher shape noise accelerates transitions between shape attractors. Our morphometric analysis framework will be widely applicable to quantitative cell shape investigations in physiology and disease. Together, our results identify a key role for cellular stochasticity as a regulator of shape change rate, and highlight that shape dynamics yield rich phenotypic information that enhances our understanding of cellular states.

biophysics↗

bioRxiv2024

Cryo-electron tomography reveals the nanoscale architecture of the actin cortex in cellular blebs

In animal cells, cellular deformations driving cytokinesis, migration, and epithelial constriction are driven by contractile tension in the actomyosin cortex, a thin network of actin and myosin underlying the plasma membrane. Cortical tension results from myosin-generated forces and as such, cortical myosin organization and dynamics have received significant attention. However, recent studies highlight that alongside myosin motor activity, the organization of the cortical actin network is a key regulator of tension. Yet, very little is known about the structural arrangement of cortical actin filaments. This is mostly due to the small thickness and high density of the cortex, which makes the visualization of cortical actin filaments challenging. Here, we use cryo-electron tomography (cryo-ET) to unveil the structural organization of cortical actin. As a model, we use isolated cellular blebs, which assemble an actin cortex comparable to the cortex of entire cells, but are small enough to be amenable to cryo-ET. We find that the bleb actin cortex is mostly composed of short and straight actin filaments. We then characterize cortex structural parameters, including the density of potential cross-linking and membrane attachment points. Our study unveils the nanoscale three-dimensional organization of the cortical actin network in cellular blebs. As such, it provides a quantitative framework for models of cortical tension generation, and will help understanding the nanoscale basis of cell surface contractions.

biophysics↗

bioRxiv2023

Synergistic action of actin binding proteins regulate actin network organization and cell shape

Animal cell shape is largely determined by the organization of the actin cytoskeleton. Spread shapes result from a balance between protrusive actin networks and contractile stress fibers, while rounded shapes are supported by a contractile actomyosin cortex. The assembly and regulation of distinct types of actin networks have been extensively studied, yet, what determines which networks dominate in a given cell remains unclear. In this Brief Report, we explore the molecular regulation of overall actin organization and resulting cell shape. We use our recently published comparison of the F-actin interactome in spread interphase and rounded mitotic cells to establish a list of candidate regulators of actin networks in spread cells. Utilizing micropatterning and automated image analysis we quantitatively analyze how these candidates affect actin organization. Out of our initial 16 candidates, we identify subsets of proteins promoting stress fibers or regulating their arrangement. Interestingly, no single regulator depletion caused significant cell shape change. However, perturbing two hits simultaneously, supervillin and myosin II, led to stress fiber disassembly and cell rounding. Overall, our systematic investigation shows that actin networks are robust to perturbations, and identifies regulatory modules controlling overall actin organization and resulting cell shape.

cell biology↗

bioRxiv2023

POLCAM: Instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

biophysics↗

bioRxiv2022

Proteomic analysis of the actin cortex in interphase and mitosis

In animal cells, many shape changes are driven by gradients in the contractile tension of the actomyosin cortex, a thin cytoskeletal network that supports the plasma membrane. Elucidating how cortical tension is controlled is thus essential for understanding cell and tissue morphogenesis. Increasing evidence shows that alongside myosin II activity, actin network organisation and composition are key to cortex tension regulation. However, how cortex composition changes when cortical tension changes remains poorly understood. Here, we compared cortices from cells in interphase and in mitosis, as mitosis entry is associated with a strong increase in cortical tension. We purified cortex-enriched cellular fractions and analysed their composition using mass spectrometry, identifying 922 proteins consistently represented in both interphase and mitotic cortices. We curated this dataset by focusing on actin-related proteins, narrowing down to 238 candidate regulators of the cortex during the mitotic cortical tension increase. Among these candidates, our analysis pointed to a role for septins, and in particular septin 9, in the control of mitotic cell rounding. Overall, our study brings insight into the regulation of mitotic rounding, and paves the way for systematic investigations of the regulation of cell surface mechanics.

cell biology↗

bioRxiv2020

Mouse embryonic stem cells switch migratory behaviour during early differentiation

Development relies on a series of precisely orchestrated cell fate changes. While studies of fate transitions often focus on changes in gene regulatory networks, most transitions are also associated with changes in cell shape and cell behaviour. Here, we investigate changes in migratory behaviour in mouse embryonic stem (ES) cells during their first developmental fate transition, exit from ES cell state. We show that naive pluripotent ES cells cannot efficiently migrate on 2-dimensional substrates but are able to migrate in an amoeboid fashion when placed in confinement. Exit from ES cell state, typically characterised by enhanced cell spreading, is associated with decreased migration in confinement and acquisition of mesenchymal-like migration on 2D substrates. Interestingly, confined, amoeboid-like migration of ES cells strongly depends on Myosin IIA, but not Myosin IIB. In contrast mesenchymal-like migration of cells exiting the ES cell state does not depend on Myosin motor activity but relies on the activity of the Arp2/3 complex. Together, our data suggest that during early differentiation, cells undergo a switch in the regulation of the actin cytoskeleton, leading to a transition from amoeboid-to mesenchymal-like migration. Summary statementNaive mouse embryonic stem cells display amoeboid-like migration in confinement, but switch to mesenchymal-like migration as they exit the ES cell state.

cell biology↗

bioRxiv2020

Cell surface fluctuations regulate early embryonic lineage sorting

In development, lineage segregation of multiple lineages must be coordinated in time and space. An important example is the mammalian inner cell mass (ICM), in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the foetus). The physical mechanisms that determine this spatial segregation between EPI and PrE are still poorly understood. Here, we identify an asymmetry in cell-cell affinity, a mechanical property thought to play a significant role in tissue sorting in other systems, between EPI and PrE precursors (pEPI and pPrE). However, a computational model of cell sorting indicated that these differences alone appeared insufficient to explain the spatial segregation. We also observed significantly greater surface fluctuations in pPrE compared to pEPI. Including the enhanced surface fluctuation in pPrE in our simulation led to robust cell sorting. We identify phospho-ERM regulated membrane tension as an important mediator of the increased surface fluctuations in pPrE. Using aggregates of engineered cell lines with different surface fluctuation levels cells with higher surface fluctuations were consistently excluded to the outside of the aggregate. These cells behaved similarly when incorporated in the embryo. Surface fluctuations-driven segregation is reminiscent of activity-induced phase separation, a sorting phenomenon in colloidal physics. Together, our experiments and model identify dynamic cell surface fluctuations, in addition to static mechanical properties, as a key factor for orchestrating the correct spatial positioning of the founder embryonic lineages.

developmental biology↗

bioRxiv2019

Membrane tension mediated mechanotransduction drives fate choice in embryonic stem cells

Changes in cell shape and mechanics frequently accompany cell fate transitions. Yet how mechanics affects the regulatory path-ways controlling cell fate is poorly understood. To probe the interplay between shape, mechanics and fate, we used embryonic stem (ES) cells, which spread as they undergo early differentiation. We found that this spreading is regulated by a beta-catenin mediated decrease in plasma membrane tension. Strikingly, preventing the membrane tension decrease obstructs early differentiation of ES cells. We further find that blocking the decrease in membrane tension inhibits endocytosis of FGF signalling components, which direct the exit from the ES cell state. The early differentiation defects we observed can be rescued by increasing Rab5a-facilitated endocytosis. Thus, we show that a mechanically-triggered increase in endocytosis regulates fate transitions. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling, and therefore cell fate.

cell biology↗

bioRxiv2019

Abscission couples cell division to embryonic stem cell fate

Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cells, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here we show that exit from naive pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naive pluripotency. Finally, interfering with abscission impairs exit from naive pluripotency. Altogether, our data indicate that a switch in the division machinery leading to faster abscission is crucial for pluripotency exit. Our study identifies abscission as a key step coupling cell division to fate transitions.

cell biology↗

bioRxiv2019

Tissue mechanics regulate mitotic nuclear dynamics during epithelial development

Cell divisions are essential for tissue growth. In pseudostratified epithelia, where nuclei are staggered across the tissue, each nucleus migrates apically before undergoing mitosis. Successful apical nuclear migration is critical to preserve tissue integrity during cell division. Most previous investigations have focused on the local cellular mechanisms controlling nuclear migration. Yet, inter-species and inter-organ comparisons of different pseudostratified epithelia suggest global tissue architecture may influence nuclear dynamics, but the underlying mechanisms remain elusive. Here, we use the developing Drosophila wing disc to systematically investigate, in a single epithelial type, how changes in tissue architecture during growth influence mitotic nuclear migration. We observe distinct nuclear dynamics at discrete developmental stages, as epithelial morphology changes. We then use genetic and physical perturbations to show a direct effect of cell density on mitotic nuclear positioning. We also find Rho kinase and Diaphanous, which facilitate mitotic cell rounding in confined cell conditions, are essential for efficient apical nuclear movement. Strikingly, perturbation of Diaphanous causes increasing defects in apical nuclear migration as the tissue grows, and these defects can be reversed by acute physical reduction of cell density. Our findings reveal how the mechanical environment imposed on cells within a tissue alters the molecular and cellular mechanisms adopted by single cells for mitosis. We speculate that mechanical regulation of apical mitotic positioning could be a global mechanism for tissue growth control.

developmental biology↗