Plasma membrane and cell surface mechanics in embryonic stem cells

Changes in cell shape frequently accompany cell fate transitions. Cell shape changes are regulated by cell surface mechanics. One of the main determinants of cell surface mechanics is membrane tension, which is regulated by the interaction between the plasma membrane and the cytoskeleton. Yet how mechanics, and in particular membrane tension, affects the regulatory pathways controlling cell fate is poorly understood. In my PhD, I investigated the role of cell surface mechanics in regulating cell fate transition in early development. In order, to probe the interplay between shape, mechanics and fate, I used mouse embryonic stem (ES) cells, which spread as they undergo early differentiation. In order to asses cell surface mechanical changes during exit form naïve pluripotency, I helped establish a membrane pulling assay using an optical tweezer. Using this assay, I found that cell spreading during exit from naïve pluripotency is regulated by a decrease in plasma membrane tension. Higher tension appears to be due to higher expression and activity of proteins regulating membrane-to-cortex attachment, such as Ezrin-Radixin- Moesin. Next I demonstrated using Ezrin mutants that preventing this decrease in membrane tension obstructs early differentiation of ES cells. I confirmed these results using micropatterning to physically prevent the cells from changing their shape and membrane tension. I next investigated which membrane tension-mediated mechanosensitive pathway could explain these results. I found that decrease in membrane tension results in an increase in endocytosis which is a major regulator of signalling events. Specifically, I found that if cell membrane tension is not decreased, endocytosis of FGF signalling components, which direct exit from the ES cell state, is significantly inhibited. This results in defects in exiting naïve pluripotency as the ERK pathway requires endocytosis for full activation. Strikingly, the early differentiation defects I observed can be rescued by increasing Rab5a-facilitated endocytosis. Thus, I show that a mechanically-triggered increase in endocytosis regulates fate transitions. My findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling during cell fate changes

Membrane Tension Orchestrates Rear Retraction in Matrix-Directed Cell Migration.

In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices

Interplay between mechanics and signalling in regulating cell fate.

Mechanical signalling affects multiple biological processes during development and in adult organisms, including cell fate transitions, cell migration, morphogenesis and immune responses. Here, we review recent insights into the mechanisms and functions of two main routes of mechanical signalling: outside-in mechanical signalling, such as mechanosensing of substrate properties or shear stresses; and mechanical signalling regulated by the physical properties of the cell surface itself. We discuss examples of how these two classes of mechanical signalling regulate stem cell function, as well as developmental processes in vivo. We also discuss how cell surface mechanics affects intracellular signalling and, in turn, how intracellular signalling controls cell surface mechanics, generating feedback into the regulation of mechanosensing. The cooperation between mechanosensing, intracellular signalling and cell surface mechanics has a profound impact on biological processes. We discuss here our understanding of how these three elements interact to regulate stem cell fate and development

Membrane Tension Gates ERK-Mediated Regulation of Pluripotent Cell Fate.

Cell fate transitions are frequently accompanied by changes in cell shape and mechanics. However, how cellular mechanics affects the instructive signaling pathways controlling cell fate is poorly understood. To probe the interplay between shape, mechanics, and fate, we use mouse embryonic stem cells (ESCs), which change shape as they undergo early differentiation. We find that shape change is regulated by a β-catenin-mediated decrease in RhoA activity and subsequent decrease in the plasma membrane tension. Strikingly, preventing a decrease in membrane tension results in early differentiation defects in ESCs and gastruloids. Decreased membrane tension facilitates the endocytosis of FGF signaling components, which activate ERK signaling and direct the exit from the ESC state. Increasing Rab5a-facilitated endocytosis rescues defective early differentiation. Thus, we show that a mechanically triggered increase in endocytosis regulates early differentiation. Our findings are of fundamental importance for understanding how cell mechanics regulates biochemical signaling and therefore cell fate.This work was supported b\ the European Union¶s Hori]on 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641639 (ITN Biopol, HdB and EKP), the Medical Research Council UK (MRC programme award MC_UU_12018/5, HdB and EKP), the Human Frontier Science Program (Young InvestigatorGrant RGY 66/2013 to EKP), the Leverhulme Trust (Prize in Biological Sciences to EKP), an ERC Consolidator Grant (CellFateTech, 772798, KC), a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute (KJC). KJC is a Royal Society University Research Fellow

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival

International audienceHow growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival

Early-life stress triggers long-lasting organismal resilience and longevity via tetraspanin.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here, we show that early-life thermal stress strongly up-regulates tsp-1, a gene encoding the conserved transmembrane tetraspanin in C. elegans. TSP-1 forms prominent multimers and stable web-like structures critical for membrane barrier functions in adults and during aging. Increased TSP-1 abundance persists even after transient early-life heat stress. Such regulation requires CBP-1, a histone acetyltransferase that facilitates initial tsp-1 transcription. Tetraspanin webs form regular membrane structures and mediate resilience-promoting effects of early-life thermal stress. Gain-of-function TSP-1 confers marked C. elegans longevity extension and thermal resilience in human cells. Together, our results reveal a cellular mechanism by which early-life thermal stress produces long-lasting memory-like impact on organismal resilience and longevity

Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics.

Emergent physical properties of tissues are not readily understood by reductionist studies of their constituent cells. Here, we show molecular signals controlling cellular, physical, and structural properties and collectively determine tissue mechanics of lymph nodes, an immunologically relevant adult tissue. Lymph nodes paradoxically maintain robust tissue architecture in homeostasis yet are continually poised for extensive expansion upon immune challenge. We find that in murine models of immune challenge, cytoskeletal mechanics of a cellular meshwork of fibroblasts determine tissue tension independently of extracellular matrix scaffolds. We determine that C-type lectin-like receptor 2 (CLEC-2)-podoplanin signaling regulates the cell surface mechanics of fibroblasts, providing a mechanically sensitive pathway to regulate lymph node remodeling. Perturbation of fibroblast mechanics through genetic deletion of podoplanin attenuates T cell activation. We find that increased tissue tension through the fibroblastic stromal meshwork is required to trigger the initiation of fibroblast proliferation and restore homeostatic cellular ratios and tissue structure through lymph node expansion

Cell protrusions and contractions generate long-range membrane tension propagation

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation ofmembrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows

The smell of us – crowdsourcing human body odor evaluation

International audienceHuman body odor is produced when sweat-secreted compounds are metabolized by bacteria present on the skin. The resulting volatile mixture is often negatively perceived, motivating the use of personal cosmetic deodorants. Yet body odor may also be positively perceived in some contexts, and is proposed to play a role in sexual attraction, kin identification and social bonding. Because only human smellers can report the hedonic qualities of body odor, their persceptions are a valualbe complement to modern GC-MS-based quantitative chemical measurements. Here we present a crowdsourcing framework that engages volunteer smellers to characterize human sweat samples. Our approach seeks to reward both the sweat donor and the smeller with a web-based graphical interface that is informative, interesting, and fun. 300 samples from 87 individual donors were scored by 93 smellers for intensity, pleasantness, and a variety of odor descriptors. Body odor intensity and pleasantness were determined to vary with age, gender, and self-reported deodorant use. Counterintuitively, deodorant use showed no effect on the perceived intensity of body odor, and was associated with a decrease in the perceived pleasantness. From these data, we determine the precision and dynamic range of the volunteer nose as a body odor evaluation instrument and estimate the scale of crowdsourcing effort that would be required to precisely quantify the public perception of body odors