| Summary: | The cortex is a ~100nm thick layer of F-actin located under the cell membrane, rich in myosin II and actin-binding proteins. It is essential for cytokinesis, cell locomotion, and tissue morphogenesis. Despite its importance, our knowledge of the cortex is poor. Two actin nucleators, Diaph1 and the Arp2/3 complex, have been shown to generate actin filaments in the cortex. The actin networks generated by these nucleators display clear differences in their organisation and the actin-binding proteins they recruit. Therefore, cells may be able to rapidly control their cortical mechanical properties by regulating the activity of each nucleator. When I searched for regulators of mDia1 and Arp2/3 in a proteomic analysis of the cortex, I found four candidate nucleation promotion factors (NPFs): IQGAP1, NCKIPSD, Fli-I and the WAVE complex. Interestingly, IQGAP1 and NCKIPSD interact with both Diaph1 and the Arp2/3 complex, suggesting they may participate in crosstalk. I examined the role of each NPF in the generation and maintenance of cortical actin. After examining the localisation of each NPF, I determined their impact on cortical assembly by examining how their depletion affected bleb size. Indeed, reduction in Arp2/3 activity leads to smaller blebs, while large blebs result from a decrease in Diaph1 activity. IQGAP1 and NCKIPSD depletion yielded large bleb phenotypes, WAVE gave a small bleb phenotype consistent with its role in regulating Arp2/3, and Fli-I depletion had no phenotype. Next, I examined changes in F-actin network organisation after NPF depletion with scanning electron microscopy. The density and interconnection of the network were altered, further suggesting the importance of IQGAP1, NCKIPSD and WAVE in controlling the organisation of the actin cortex. I showed that NPF depletion significantly affected successful completion of mitosis in HeLa cells. Finally, in collaboration with other students, I examined how NPFs controlled properties of cell mechanics.
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