This thesis describes four projects aimed towards understanding different mechanisms underlying chemotaxis.
The first chapter tackles the question of whether chemotaxing HL-60s move by spatially biasing the generation of new protrusions, or whether they move by random generation of protrusions followed by spatially biasing the selection of these protrusions. We found that both modes can occur during establishment of the first protrusions, and the dominant mode is determined by three factors: external agonist gradients, pre-existing morphological polarity, and unknown intrinsic cues. We further identified a role for the actin cytoskeleton in establishing polarity of the WAVE complex, an actin-nucleation promoting factor.
In the second chapter, we try to identify molecules which have an instructive role in establishing polarity, by generating spatial asymmetries of candidate molecules across a cell. Other members of the lab have tackled this question using a phytochrome-based light system to create asymmetric distributions of exogenous signaling proteins on the plasma membrane. The work here represents a complementary approach using a combination of microfluidics and drug inhibitors to carve out asymmetries in distributions of endogenous signaling proteins.
In the third chapter, we tried to determine the spatial range of signaling from activated receptors for different steps in the chemotactic cascade. Chemotaxing cells must integrate information across their surface to determine the proper direction of movement. Knowing how receptor occupancy is translated into activation of downstream signals at different points in the cascade is crucial for understanding this process. We used a variety of experimental techniques to generate agonist distributions that were confined to a subcellular region and visualized spread of the signals beyond the agonist-bound region.
The fourth chapter deals with ongoing efforts to characterize the feedback roles of actin in affecting different levels in the chemotactic cascade, and to distinguish between functions that depend on dynamic processes involving actin rearrangements, versus those where having a static actin cytoskeleton is sufficient. So far, we have identified a role for dynamic actin processes in the resensitization of agonist-bound receptors. We further hope to determine a role for the static actin cytoskeleton in the desensitization of agonist-bound receptors.