Download files
Abstract
The actin cytoskeleton is involved in virtually every biological process. Assembly of
actin structures in cells is mediated by actin-associated proteins (AAPs) that
collaborate to assemble and regulate actin filaments. Actin nucleators generate linear
and branched actin filaments while the tropomyosins (Tpms), known as the master
regulators of actin filaments, stabilise and confer specific functions to filaments by
governing their interaction with other AAPs. The dynamics of actin assembly has been
studied in in vitro and ex vivo systems; however, no studies have investigated the de
novo assembly kinetics of functional actin structures in vivo in mammals. Furthermore,
no study has thoroughly investigated the relationship between cytoskeletal Tpms and
actin filaments in cells. This thesis employed cutting-edge subcellular intravital
microscopy to investigate the recruitment kinetics of the linear and branched actin
nucleators, formins mDia1, mDia2 and the Arp2/3 complex, respectively, Tpms 3.1 and
4.2, myosin IIA, and the crosslinker alpha-actinin 4 during de novo actin scaffold
assembly that drives regulated secretory granule exocytosis in rodent salivary glands,
as well as the relationship between Tpm3.1 and actin filaments. This was achieved by
developing a novel mouse salivary gland gene delivery technique using both viral and
non-viral vectors. The findings provide insights into Tpm regulation of actin filaments,
suggesting that multiple functionally distinct actin populations exist and work in
tandem. Actin scaffold assembly requires the collaborative effort between multiple
actin nucleators that have unique recruitment kinetics and activities. Remarkably, actin
scaffold-driven regulated secretory granule exocytosis in vivo was shown to occur in
two distinct phases, which revealed novel functions and interdependence between the
branched and linear actin networks.