Understanding the regulation of membrane mechanical heterogeneity in live cells

Ghosh, Tanmoy (2026) Understanding the regulation of membrane mechanical heterogeneity in live cells. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Tanmoy Ghosh (20RS117)) 20RS117.pdf - Submitted Version Restricted to Repository staff only Download (11MB)

Abstract

Cells continuously sense and adapt to the mechanical properties of their environment, actively modulating their own mechanical state. This ability is fundamentally important for various cellular processes, including endocytosis and cell migration. However, recent work reveal any equilibration of local mechanical perturbations on the cell membrane to be not instantaneous – but of varying rates depending on cell type. This can potentially create inhomogeneities in its mechanical state, which in turn affects its function (1). This thesis focuses on developing methodologies of measuring membrane mechanical heterogeneities and understanding their connection to the cellular functional state and their regulation by select relevant membrane associated proteins. A central theme is to analyze spontaneous heterogeneities, extract rates of equilibration of mechanical perturbations at the membrane as well as to characterize the non-equilibrium nature of local membrane fluctuations (2). To investigate the three-dimensional heterogeneity of tension in HeLa cells, we first employed optical tweezers for tether pulling and interference reflection microscopy (IRM) for basal tension measurements on the same setup. This method revealed a significant apico-basal membrane tension gradient. This gradient is absent in cell-derived plasma membrane spheres and mirrors both the spatial distribution of cortical contractility and the pattern of transferrin endocytosis seen in Stimulated Emission Depletion (STED) microscopy. Inhibition of dynamin-dependent endocytosis perturbs, but does not abolish, the tension gradient, and complete inhibition of clathrin increases the tension gradient, indicating that endocytosis plays an opposite role to contractility for maintaining the tension gradient. Thus, the actin cortex contractility, combined with endocytosis, plays a dominant role in shaping tension profiles. For such heterogeneities to be created by actomyosin contractility, the connection of actin to the membrane (by p-Ezrin) must be crucial. However, it remains less explored how the membrane pinning resists the flow of tension or maintains the tension heterogeneity. To address this, in the second part of my thesis, a custom-built TIRF setup was added on the IRM setup. Correlative TIRF-IRM microscopy revealed that regions with high p-Ezrin-mediated membrane–cortex pinning exhibit elevated fluctuation amplitudes and reduced local membrane tension for many cells. Contrary to previous reports based on STED microscopy, we identify two distinct modes of p-Ezrin–F-actin pinning capable of generating the localized tension gradients at the base of the cell. The correlative IRM-TIRF has also helped reveal subtle regulation of membrane fluctuations by Cu-transporter CTR1’s clustering on the PM (3). Besides membrane-actin attachments affecting flow of perturbations to fluctuations or tension, this thesis also addresses the role of Cav-1 protein and caveolae (invaginations on the plasma membrane created primarily by Cav-1). In the third part of this thesis, we study the distribution of the Cav-1 structures in a cell with structured tension profile. We micropattern cells to circular shapes and show that they develop radial tension gradients. In such cells, we investigate how caveolae/ Cav1-scaffolds affect propagation of membrane fluctuations. Under the localized mechanical stress, on a prevalent tension gradient, we observe that caveolae flatten at high tension regions increasing the smaller scaffold structures at these regions. The flattening was validated using two super-resolution microscopy techniques, STED and Stochastic Optical Reconstruction Microscopy (STORM) while also been supported by a gradual reduction of colocalization with its neck protein - EHD-2. Experiments with Cav1 and Cavin-1 knockout cells demonstrate that caveolae flatten preferentially in high-tension regions, promoting homogenization of tension flow. The work proceeds to characterize equilibration rates of locally lowered tension. While Cav1 knockout cells show higher timescales, we bring deeper insights by comparing regions within the same cells. We propose that flattening of caveolae to scaffolds significantly affects tension equilibration. The functional footprint of caveolae-flattening is investigated by imaging clathrin pits together with Cav1. Clathrin-mediated endocytosis exhibits larger and more static pits at high-tension edges, where there are a greater number of scaffolds. However, whether caveolae may crosstalk mechanically to clathrin pits by releasing their excess membrane locally to the system during membrane need for endocytosis – needs future validation. Slow equilibration, heterogeneities are all emergent properties of a membrane-actin composite system or do underlying active processes directly affect membrane fluctuations, giving rise to the contrasting characteristics in comparison to actin-free membrane? The thesis finally shows measurements of entropy production rate associated with membrane fluctuations captured by IRM. Utilizing particle swarm optimization and the thermodynamic uncertainty relation, the entropy production rate is mapped out in cells (for the first time to the best of our knowledge). Comparing after ATP depletion, we demonstrate that a relatively low but significant active contribution could be measured that is reduced on ATP-depletion or actin depolymerization by Cytochalasin D (2). Collectively, these findings reveal that cytoskeletal distribution can create patterned heterogeneities in cells while, endocytic and caveolar distribution can effectively reduce heterogeneities in a background where equilibration of tension is slow.

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