Explorations Into The Life Of Dictyostelium Discoideum.
In this dissertation, we explore several aspects of the Dictyostelium discoideum life cycle: growth, aggregation, and differentiation: We present experiments that question the conventional wisdom that vegetative D. discoideum are solitary individuals. We examine evidence - the cell density dependent transition from lag to exponential growth - which suggests that growth is stimulated by cell to cell interactions. Using a conditioned medium assay, we rule out the possibility that this interaction is through growth factors. Instead, we find that the interactions are short ranged. Next, we examine chemotactic, aggregation-stage cells. Using a microfluidic platform for cell stimulation, we investigate the timing of the biochemical signals involved in direction sensing, and we attempt to alter this timing. We flatten cells to various degrees and apply an external pulse of chemoattractant. The response is then monitored by imaging a fluorescent protein (PHCRAC -GFP or LimE-GFP) which translocates to the leading edge of the cell. We expect a dependence between the response time and the area of the cell, since the size should affect the time it takes for biochemical messengers to diffuse across the cell. The results were not definitive, because of the large variability in time it takes for the cell to respond to a signal. The large variability might be due to variations between the time the chemical signal is applied and the time it reaches the cell. To evaluate this possibility, we apply the theory of G.I. Taylor  and R. Aris  to answer the question of how quickly we are able to apply chemical signals to the target. We show that our platform for cell stimulation is the fastest available, and we rule out the pos- sibility that the variability in the response times is due to experimental setup. Rather, the source of variability must be biological, either intrinsic to each cell, or due to variability between cells (individuality). Recently, it was shown that D. discoideum cells are also capable of swimming chemotactically . Using low Reynolds number flow simulations, we find evidence that supports the claim that the mechanisms employed by D. discoideum to swim are the same mechanisms employed when they crawl. Finally, we conclude our investigations by examining how D. discoideum sort out into regions of prestalk cells and prespore cells. Using a microfluidic flattening device with cells that express a fluorescent protein (CbpD::GFP) only if they are prestalk, we find indications that cell sorting is intrinsically a three dimensional process, as cell sorting was never observed in monolayer aggregates.
Dictyostelium; Chemotaxis; Low Reynolds number swimming
Sethna, James Patarasp; Franck, Carl Peter
Ph.D. of Physics
Doctor of Philosophy
dissertation or thesis