Cross-linked Profilin:actin - A tool to study actin dynamics in non-muscle cells

Abstract: The microfilament system, consisting of actin and a number of auxiliary proteins, is fundamental for cell motility. Its dynamic organization depends on receptor-mediated signals, leading to rapid polymerizations and depolymerizations of actin. Profilin binds to non-filamentous actin, inhibits spontaneous filament formation, and functions as a regulator of actin polymerization. The profilin:actin complex, is thought to be the principal source of actin for filament formation although the role of profilin is not fully elucidated.In this thesis, a cross-linked profilin:actin complex (PxA), that retains the properties of ordinary profilin:actin, except for being non-dissociable, has been used to characterize the role of profilin and profilin:actin in non-muscle cells. A rapid screening method, employing PxA and based on the far western technique and mass-spectrometry, was designed to identify cellular components that specifically bind profilin:actin. Microinjection of PxA into cells infected with the bacteria Listeria monocytogenes impaired bacterial motility but a mutant PxA, unable to bind proline-rich sequences had no effect, demonstrating that profilin:actin is vital for the activity of the actin polymer-forming complex that the pathogen recruits to its surface upon infection. Fluorescence microscopy using two distinct sets of affinity-purified actin and profilin antibodies generated against PxA enabled localization of monomeric actin in cells. One of the actin and both profilin antibodies resulted in a dotted pattern of fluorescence partially aligning with microtubules whereas the other actin antibody detected filamentous actin. The result demonstrates extensive variability in epitope recognition, and indicates that unpolymerized actin, i.e. profilin:actin and maybe other complex-bound forms of actin, distributes in small packages that might be transported along microtubules. Microinjection of PxA into lamprey axons demonstrated the involvement of actin polymerization during synaptic signaling.