Mechano-regulation by clathrin pit-formation and passive cholesterol-dependent tubules during de-adhesion.

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.

Cholesterol Depletion by MßCD Enhances Cell Membrane Tension and Its Variations-Reducing Integrity.

Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) remodels the plasma membrane's mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it's unclear if MßCD alters membrane tension or only enhances membrane-cytoskeleton interactions-and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MßCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations' frequency spectrum. This helps us conclude that MßCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MßCD also has a strong impact on the cell membrane's tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters-an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.

Mapping Cell Membrane Fluctuations Reveals Their Active Regulation and Transient Heterogeneities.

Shape fluctuations of the plasma membrane occur in all cells, are incessant, and are proposed to affect membrane functioning. Although studies show how membrane fluctuations are affected by cellular activity in adherent cells, their spatial regulation and the corresponding change in membrane mechanics remain unclear. In this article, we study how ATP-driven activities and actomyosin cytoskeleton impact basal membrane fluctuations in adherent cells. Using interference imaging, we map height fluctuations within single cells and compare the temporal spectra with existing theoretical models to gain insights about the underlying membrane mechanics. We find that ATP-dependent activities enhance the nanoscale z fluctuations but stretch out the membrane laterally. Although actin polymerization or myosin-II activity individually enhances fluctuations, the cortex in unperturbed cells stretches out the membrane and dampens fluctuations. Fitting with models suggest this dampening to be due to confinement by the cortex. However, reduced fluctuations on mitosis or on ATP-depletion/stabilization of cortex correlate with increased tension. Both maps of fluctuations and local temporal autocorrelation functions reveal ATP-dependent transient short-range (<2 µm) heterogeneities. Together, our results show how various ATP-driven processes differently affect membrane mechanics and hence fluctuations, while creating distinct local environments whose functional role needs future investigation.

Early tension regulation coupled to surface myomerger is necessary for the primary fusion of C2C12 myoblasts.

Here, we study the time-dependent regulation of fluctuation-tension during myogenesis and the role of the fusogen, myomerger. We measure nanometric height fluctuations of the basal membrane of C2C12 cells after triggering differentiation. Fusion of cells increases fluctuation-tension but prefers a transient lowering of tension (at ~2-24 h). Cells fail to fuse if early tension is continuously enhanced by methyl-ß-cyclodextrin (MßCD). Perturbing tension regulation also reduces fusion. During this pre-fusion window, cells that finally differentiate usually display lower tension than other non-fusing cells, validating early tension states to be linked to fate decision. Early tension reduction is accompanied by low but gradually increasing level of the surface myomerger. Locally too, regions with higher myomerger intensity display lower tension. However, this negative correlation is lost in the early phase by MßCD-based cholesterol depletion or later as differentiation progresses. We find that with tension and surface-myomerger's enrichment under these conditions, myomerger clusters become pronouncedly diffused. We, therefore, propose that low tension aided by clustered surface-myomerger at the early phase is crucial for fusion and can be disrupted by cholesterol-reducing molecules, implying the potential to affect muscle health.

Squeezing the eggs to grow: The mechanobiology of mammalian folliculogenesis.

The formation of functional eggs (oocyte) in ovarian follicles is arguably one of the most important events in early mammalian development since the oocytes provide the bulk genetic and cytoplasmic materials for successful reproduction. While past studies have identified many genes that are critical to normal ovarian development and function, recent studies have highlighted the role of mechanical force in shaping folliculogenesis. In this review, we discuss the underlying mechanobiological principles and the force-generating cellular structures and extracellular matrix that control the various stages of follicle development. We also highlight emerging techniques that allow for the quantification of mechanical interactions and follicular dynamics during development, and propose new directions for future studies in the field. We hope this review will provide a timely and useful framework for future understanding of mechano-signalling pathways in reproductive biology and diseases.

Effect of heterogeneous substrate adhesivity of follower cells on speed and tension profile of leader cells in primary keratocyte collective cell migration.

In single keratocyte motility, membrane tension is reported to be high at cell-fronts and believed to establish front coherence. To understand role of membrane mechanics in collective cell migration, we study membrane height fluctuations in cell sheets from fish scales using interference reflection microscopy (IRM). We report the monolayer to have cells lacking substrate adhesion and show that such 'non-sticky' cells can form bridges between leader cells and far-away follower cells. Do such interactions alter motility and membrane mechanics in such leaders? We find non-significant, but reduced speed for leaders with 'non-sticky' followers in comparison to other leaders. Cells show high phenotypic variability in their membrane fluctuation tension profiles. On average, this tension is found to be lower at cell fronts than the mid-section. However, leaders with non-sticky followers are more prone to display higher tension at their front and have a negative correlation between cell speed and front-mid tension difference. Thus, we conclude that intracellular tension gradients are heterogeneous in cell sheets and substrate adhesivity of followers can control the coupling of the gradient to cell speed.

Cellular and Nuclear Forces: An Overview.

Biological cells sample their surrounding microenvironments using nanoscale force sensors on the cell surfaces. These surface-based force and stress sensors generate physical and chemical responses inside the cell. The inherently well-connected cytoskeleton and its physical contacts with the force elements on the nuclear membrane lead these physicochemical responses to cascade all the way inside the cell nucleus, physically altering the nuclear state. These physical alterations of the cell nucleus, through yet-unknown complex steps elicit physical and functional response from the chromatin in the form of altered gene expression profiles. This mechanism of force/stress sensing by the cell and then its nuclear response has been shown to play a vital role in maintaining robust cellular homeostasis, controlling gene expression profiles during developmental phases as well as cell differentiation. Over the last few years, there has been appreciable progress toward identification of the molecular players responsible for force sensing. However, the actual sensing mechanism of cell surface bound force sensors and more importantly cascading of the signals, both physical (via cytosolic force sensing elements such as microtubule and actin framework) and chemical (cascade of biochemical signaling from cell surface to nuclear surface and further to the chromatin), inside the cell is poorly understood. In this chapter, we present a review of the currently known molecular players in cellular as well as nuclear force sensing repertoire and their possible mechanistic aspects. We also introduce various biophysical concepts that are used to describe the force/stress sensing and response of a cell. We hope this will help asking clearer questions and designing pointed experiments for better understanding of the force-dependent design principles of the cell surface, nuclear surface, and gene expression.