Role of membrane fluctuations and tension in cell fusion during myogenesis

Chakraborty, Madhura (2023) Role of membrane fluctuations and tension in cell fusion during myogenesis. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Madhura Chakraborty (17RS067)) 17RS067.pdf - Submitted Version Restricted to Repository staff only Download (7MB)

Abstract

Myogenesis is a complex process involving the fusion of myoblasts to form multinucleated myotubes, which serves as the fundamental units of skeletal muscles. The fusion of myoblasts is a critical step in myogenesis that involves overcoming thermodynamic barriers to enable the fusion of cells. This process is regulated by several proteins, including myomaker and myomerger, which play a crucial role in cell surface remodelling and fusion. Despite the importance of proper membrane dynamics in facilitating membrane interactions, the regulation and function of membrane mechanics and fluctuations during this process remain poorly understood. To address this gap in knowledge, we investigated the membrane mechanics involved in myoblast differentiation and fusion using the non-invasive microscopy technique of Interference reflection microscopy (IRM). Our study used measurements of nanometric height fluctuations and the effective fluctuation tension of the basal membrane of C2C12 cells during differentiation to understand their regulation and functional linkage with the fusogen myomerger. Some myoblasts fuse to form myotubes during myoblast differentiation, while others remain undifferentiated. In addition to studying how early tension regulation affects fusion or differentiation, we have focused on the role fluctuations and tension play in cells' fate determination (whether they would finally fuse or not). Our research revealed that cells that have formed myotubes exhibit higher tension than cells that fail to fuse or myoblasts at the start of differentiation. Furthermore, the tension profiles of cells that have undergone fusion and those that have not can be distinguished during differentiation. Fusion of cells causes a gradual rise in tension but is preceded by a brief dip in tension at early time points of differentiation (~2-24 hours) in such cells. Perturbing tension during this pre-fusion window, either by cholesterol depletion with MCD or inhibiting cdc42, completely blocks fusion or reduces the fusion index. We also report that even before fusion, cells that finally differentiate exhibit lower tension than other cells that later do not fuse. This indicates a link between early tension states and fate decisions. The early tension reduction is accompanied by a low but gradually increasing surface myomerger. Myomerger expression negatively correlates with tension at early time points for whole cell averages. Even in single cells, regions with higher myomerger intensity exhibit lower tension. However, this negative correlation is lost as differentiation progresses. Going beyond correlative results, we show that the initial tension reduction depends on myomerger expression, as siRNA knockdown of myomerger causes a rise in early tension and prevents fusion. Imaging surface distrubtion of myomerger shows that their clustering depends on tension and differentiation phase and is thus altered by cholesterol depletion or as differentiation progresses. These findings suggest that low tension aided by clustered surface myomerger during the early stage is crucial for fusion and can be disrupted by cholesterol-reducing molecules. In summary, our work establishes the role of membrane tension during C2C12 myoblast differentiation and its potential association with surface myomerger for mediating successful differentiation. We believe this study opens many directions, some of which may be relevant for studies on muscle health.

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