Recursive interaction between encapsulated dynamic microtubule asters and deformable membranes

Abstract

Cytoskeletal networks, the filamentous systems in the cytoplasm of cells, are giving the cell its shape and supporting the mechanical structure of the cell, as well as organizing the cytoplasmic organelles in the cell. The cytoskeleton microtubules are reorganizing and maintaining the global morphology of the cell. This is the result of the signals that regulate microtubule filaments. The geometry of the membrane also affects these signaling pathways, which leads to the recursive interaction of the microtubules and signals at the membrane. To reconstitute a minimal system for the purpose of studying this recursive interaction, purified pig brain tubulin together with purified centrosomes, as microtubule organizing centers, was encapsulated in giant unilamellar vesicles (GUVs) using the cDICE method (Abkarian et al., 2011). To study how microtubule dynamics is regulated by tubulin concentration, different concentrations of tubulin dimers, with and without different concentrations of the microtubule regulator stathmin (Daub et al., 2001) in both unphosphorylated and phosphorylated states, were used for single filament TIRF microscopy (Bieling et al., 2010) to study the microtubule dynamics, as well as confocal laser scanning microscopy (CLSM) to investigate the effect of regulators and confinement on the microtubule asters. For further investigation of the effect of a phosphorylated stathmin gradient on the positioning of microtubule asters and the morphology of GUVs, dynamic microtubule asters were encapsulated together with a signaling system which mimics the Rac1-Pak1 pathway that controls the microtubule regulator stathmin (Daub et al., 2001). Stathmin sequesters tubulin dimers, thus decreasing the effective concentration of tubulin, which affects microtubule dynamics. This inhibitory effect of stathmin is regulated by its phosphorylation. As a result, the size of microtubule asters is smaller in the presence of stathmin as compared to the phosphorylated stathmin. This results in the reorganization of microtubule asters in confinement. Introducing the light-activated phosphorylated stathmin gradient in the system, which forms upon kinase translocation to the membrane, shows: the pre-formed microtubule based protrusions are stable, newly formed protrusions are caused by the signaling system and the response of the system to the activation of the signaling system depends on the initial morphology of the confinement.