Dynamics of Morphological Cell States

Abstract

Cellular proliferation greatly depends on the intake of extracellular material. One very distinct method of such endocytosis is a process known as macropinocytosis. During this process, large circular waves of newly polymerized actin form into cup-like, three dimensional structures, protruding the cellular membrane upwards from the lamellipodium. Such structures are known as Circular Dorsal Ruffles (CDRs). These transient and dynamic rings of intense actin polymerization typically last for several minutes and span over diameters in the micrometer range. Upon finalization of the structure, recruited motor proteins facilitate a contraction of the upper rim, enclosing material inside of the structure in a large vesicle of cellular membrane. Stimulation of cells to express these CDRs is facilitated by exposing cells to so-called growth factors. These growth factors trigger a signaling cascade within the cell, leading to recruitment of several key proteins involved in actin polymerization to assemble into the aforementioned circular shape. In this thesis, the focus will be laid on how the expression of CDRs depends on the concentration of added growth factors and how individual cells react to such stimuli. As will be shown, a primary effect of increased stimulant concentration is a trend towards expression of longer lasting, less motile CDRs. The effect of stimulation on individual cells actively expressing CDRs is, counterintuitively, a reduction in CDR activity, while previously inactive cells are stimulated to begin expressing CDRs as would be expected. Here, the amount of live single cell data acquired during the study plays an important role in identification of such patterns due to the large variance of cellular processes inherent to biological systems, as well as the inability to determine protein concentrations within cells during experiments. A crucial consideration for performed experiments was generation of large datasets per experiment. This was facilitated by utilizing a microfluidic perfusion system in conjunction with microcontact printing, forcing cells into predetermined shapes an locations on the provided substrate. These methods in combination with usage of sophisticated image processing algorithms allowed for extraction of a dataset of significant size. Usage of statistical methods such as clustering, allowed for deeper insights into behavior of cells and yielded similarities of cellular behavior to excitable reaction-diffusion systems.