Mechanisms regulating cell shape and architecture in health and disease.

Much research on cell organization has been using tissue culture epithelial or fibroblast cells. However, many other types of cells have a unique cellular organization tailored to their function. Blood cells have a special morphology that allows them to withstand mechanical stress associated with bloodstream flow. Blood platelets have a disk shape contributed by a ring of microtubules assembled into the marginal band. The forces from the microtubules contribute to flattening the cell. Upon activation from endothelial injury, the platelets switch to a sphere shape by altering the microtubule cytoskeleton. In blood platelets, contraction and rearrangement of the cytoskeleton plays an important mechanical role for cellular function. However there is no structural information on how the architecture of the marginal band, the molecular composition of the band and the molecular mechanism underlying the activation switch to sphere shape after injury.

The student will isolate platelets from mouse blood. They will define the cytological architecture of platelets using Correlation-Light electron microscopy (CLEM) and tomography. Using proteomics, the student will characterise the actin and microtubule cytoskeleton proteome to define the molecular components that dictate the specific cellular organization. The project will then focus on reconstituting in vitro the cytoskeletal organization of microtubules into a marginal band structure with cross-linkers and motors to mediate changes in microtubule organization in vitro. Using TIRF-microscopy and purified motors and crosslinkers, the aim will be to reconstitute a minimal microtubule-motor-crosslinker complex that has properties to withstand mechanical force in vitro. Understanding the underlying principles of cellular organization in platelets will contribute to defining the molecular mechanism of platelet activation and function. By comparing and contrasting microtubule bundle organization in different cell types such as the cytokinetic midzone and the marginal band, it will also enable the student to define the molecular basis for the functional cellular diversity.

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