Chemotaxis is a fundamental process involved in a diverse range of biological phenomena such as nutrient finding in unicellular organisms, development and morphogenesis of multicellular organisms, wound healing and immune response. It is also related to a number of diseases including immunological disorders and cancer metastasis. It is a complex cellular process which is composed of several steps including signal detection and amplification, cell polarisation and cytoskeleton reorganisation. There are several signal transduction pathways working in parallel to control chemotaxis, which involve a multitude of regulatory factors and effectors modulating the cytoskeletal dynamics. The final outcome is actin polymerisation at the leading edge, depolymerisation at the trailing edge and myosin II filament assembly and contraction in the back of the cell, which together drive the directional cell movement in the direction of the chemotactic gradient. One of the poorly understood aspects of the chemotactic response is a biphasic character of the chemoattractant-induced actin polymerisation. The two temporally distinct phases of actin polymerisation reveal very different characteristics and regulation, but the exact mechanisms responsible for these differences have not been determined. We have taken advantage of recent advances in a quantitative proteomic technique and adapted the SILAC experimental approach to analyse the fast translocation dynamics of proteins associated with a crude cytoskeleton preparation after stimulation with the chemoattractant in Dictyostelium discoideum. These experiments detected, among many other proteins, a wide range of cytoskeletal components including structural constituents, actin binding proteins and regulatory factors associated with the cortical cytoskeleton. The SILAC results provide valuable quantitative information about the translocation dynamics for hundreds of cytoskeletal components in the same experiment. Most of those proteins follow the generic biphasic profile of enrichment reflecting actin dynamics, but subsets of proteins show specific incorporation during only one of the two phases of actin polymerisation. We identify several positive regulators of actin polymerisation showing enrichment specific to the first phase of chemotactic response and other regulatory components incorporated to the cytoskeleton exclusively during the second phase of cAMP-induced actin polymerisation. Detailed analysis of the major structural components of the actin cytoskeleton reveal that about 25% of the primary actin nucleator – Arp2/3 complex, incorporated to the cortex during the first phase, do not trigger formation of a new filament. In order to verify the proteomic analysis 55 known cytoskeletal proteins, which showed strong enrichment in the SILAC experiments, were selected for tagging with GFP followed by in vivo fluorescence microscopy imaging. Their analyses served as positive controls validating the translocation dynamics patterns measured with the SILAC experiments and also provided further detailed spatio-temporal characterisation of the cAMP-mediated protein translocation. Regulatory factors involved in the first phase of actin polymerisation can be classified into two groups exhibiting distinct incorporation dynamics detected by TIRFM imaging. The first group includes DocA, DocB and GacR proteins, which show very rapid and transient translocation to the cortex and are most likely involved in the earliest events following cAMP stimulation. The second group, which is composed of Roco7, PakB, XacB and GacA proteins, shows slower enrichment and longer occupancy at the cortex. TIRFM analysis also led to characterisation of a new class of cytoskeletal components, which bind exclusively to the pre-existing actin filaments and do not associate with the structures formed during response to cAMP stimulation. These proteins, which include MhcA, ZizA, RapGAP1 and two novel cytoskeletal components, translocate only to the inner cortex upon cAMP stimulation and are likely involved in regulation of the actin depolymerisation phase. In order to identify novel components of the cytoskeleton 54 uncharacterised proteins showing response to cAMP stimulation in the SILAC experiments, were cloned and tagged with GFP. Most of those proteins showed specific localisations to various cellular compartments including the nucleus, contractile vacuoles and various types of vesicles. 12 of the selected proteins showed colocalisation with the cortical structures and clear translocation in response to cAMP stimulation. A small number of these proteins contain actin binding domains identified by sequence homology analysis, but none of them were previously characterised. These novel cytoskeletal components need to be further investigated in order to determine their functions in the cytoskeleton and chemotaxis.
|Date of Award
|Kees Weijer (Supervisor)