New Roles for Actin-Binding Proteins and PIP₂ in Intracellular Calcium Homeostasis

Abstract

Spatiotemporal increase of the intracellular Ca2+ is the most universal way to regulate the function of a eukaryotic cell. Owing to a host of actin-binding proteins and enzymes whose activities are modulated by the local concentration of Ca2+, free Ca2+ in cytosol serves as a pivotal second messenger in a variety of cell functions. The rise and fall of intracellular Ca2+ wave has been best illustrated in eggs at fertilization. However, the molecular mechanism by which intracellular Ca2+ is increased in the fertilized egg is largely unknown despite the discoveries of the distinct Ca2+-mobilizing second messengers in the past 30 years. In this thesis, I have used the starfish oocytes to study how Ca2+ signaling can be modulated by the actin cytoskeleton, which is known to be dynamically remodelled during meiotic maturation and fertilization of the egg. The principal issues of my experimental work are: (i) to establish the role of actin-binding proteins and PIP2 in the regulation of the Ca2+ signaling; (ii) to study the effect of the Ca2+-store depletion on Ca2+ signaling and on the structure and function of the actin cytoskeleton, and (iii) to study the role of the actin-cytoskeleton in establishing the block to polyspermy. Microinjected into starfish eggs, actin-binding protein gelsolin, function-blocking antibody to depactin, and the PHVsequestering fusion protein that indirectly alters the actin cytoskeleton, all changed a certain aspect of Ca2+ signaling. Depletion of the Ca2+ store with ionomycin in turn drastically changed the cortical structure and the actin cytoskeleton of the eggs, eventually leading to a deleterious effect on egg activation and early development. Finally, the alteration of the actin cytoskeleton led to failure to establish a fast and slow block to polyspermy. Taken together, this study indicated that the actin cytoskeleton is an important factor that optimizes the Ca2+ response at egg activation and guides monospermic fertilization