Understanding spatiotemporal coupling of tension regulation and endocytosis

Mandal, Tithi (2024) Understanding spatiotemporal coupling of tension regulation and endocytosis. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Tithi Mandal (18RS012)) 18RS012.pdf - Submitted Version Restricted to Repository staff only Download (9MB)

Abstract

Cellular mechanoregulation is central to cell survival and functioning and uses the plasma membrane for mechanoregulation. Endocytosis, besides playing this pivotal role in regulating the flow of molecules in the cell, also directly impacts the mechanical state of the cell. This thesis addresses the global and local crosstalk of endocytosis with the cell surface mechanics. HeLa cells are studied using a combination of interference reflection microscopy (IRM), optical trapping and fluorescence experiments. To understand the effect of enhanced endocytosis on tension, the cargo, transferrin (Tf), of the constitutive clathrin-mediated endocytosis (CME) is used. Pulsed administration of Tf resulted in a fast increase in cell surface tension followed by a slower decrease, finally returning to original values by 30 min. However, tension rose similarly, but an oscillation followed the peak when exposure to Tf. The oscillating feature was abrogated when fast recycling was suppressed by knocking down Rab4. While continuous exposure reduced the intracellular tension variations, pulsed endocytosis or Rab4 knockdown failed to do so. We also establish that the pre-existing tension profile strongly determines the positioning of the pits and is partially regulated by AP2. We next address the build-up and resolution of mechanical changes by de-adhesion endocytosis, respectively. De-adhesion was shown to reduce tension when the cortex was intact and decreased tether force at the apical side while enhancing endocytosis. Blocking dynamin-dependent scission of endocytic pits did not halt the regulation, but knocking down AP2-dependent pit formation halted tension recovery. Notably, individually, ATP or cholesterol depletion did not suppress regulation but both depleted and arrested it. Our data strongly support the central role of Clathrin and AP2-dependent pit formation in reducing fluctuations, as confirmed by super-resolution microscopy. Furthermore, we show that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions. We finally explore the spatiotemporal crosstalk of tension with another mechanosensory system – showing a focal adhesion kinase-dependent rise in tension on chemokine addition in T cells followed by a redistribution of mechanosensory channel - Piezo and its activation resulting in Ca2+ flux. This thesis addresses key aspects of crosstalk between cell surface mechanics and endocytosis. It lays the foundation for future work addressing the role of mechanics in the possible crosstalk between different endocytic pathways.

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