Illukkumbura, Rukshala Vidumini; (2021) Membrane dynamics and advective transport of the PAR polarity proteins. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The establishment of cell architecture, whether in a migrating cell, a polarized epithelial cell, or an asymmetrically dividing stem cell, requires proper intracellular patterning. One mechanism by which cells are able to locally concentrate molecules in space is through directed transport, typically through the action of cytoskeletal-motor networks. Using polarization of the C. elegans embryo as a model system, I sought to understand how actomyosin cortical flows drive efficient segregation of polarity proteins to one side of the cell. Prior data established that anterior PAR proteins are segregated into the anterior by cortical actomyosin flows, yet the mechanisms underlying this transport are unclear. More recent work suggested that oligomerization of PAR-3 and its ability to recruit other aPAR proteins is specifically required for efficient segregation. This data raised additional questions: Do all membrane-associated molecules sense flows? Do particular features of molecules such as clustering enable their segregation by advection? How is this regulated to enable correct spatiotemporal control of protein targeting? We combined single molecule tracking methods with perturbation of PAR-3 cluster dynamics to directly assess the ability of polarity molecules to be advected by flows and how this may be affected by cluster regulation. My results suggest that a variety of polarity molecules are advected by cortical flows, allowing flow to shape molecular distributions. At the same time, not all molecules are advected, or at least not advected efficiently, indicating that specific molecular features of protein complexes facilitate advection. Surprisingly, despite being required for efficient segregation, clustering of PAR-3 is not required to sense flows. Moreover, although clustering alters diffusivity, the observed changes are minimal and would not be expected to substantially alter their ability to be segregated. Rather, clustering is most likely required to shape the pattern of membrane association, potentially through positive feedback, though this remains to be definitively explored.

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