Quantitative and statistical analysis of the molecular and physical processes that steer immune cell migration

Neutrophils are the most abundant circulating leukocytes in humans, comprising the first line of innate immune defense. As neutrophils migrate towards sites of infection and inflammation, they encounter a highly heterogeneous environment. During interactions with obstacles, neutrophils may be forced to split their front into multiple competing leading edges, raising the question of how the cell selects which front to maintain and which front(s) to abandon. This is not a simple dilemma, as apart from the mechanical considerations the cells must integrate receptor inputs, while maintaining polarity. To understand how motile cells process information and make directional decisions I took a highly interdisciplinary approach, combining microfluidics, sub-cellular optogenetic strategies, quantitative time-lapse microscopy, data science and machine learning techniques. I challenged single HL-60 neutrophil-like cells with microfluidic devices containing obstacles. From time-lapse microscopy data, I extracted hundreds of image features that report on cell shape and distributions of cytoskeletal components. Through a supervised statistical learning approach, I identified a small subset of features that carry predictive power. Surprisingly, one can predict the cell's turning direction with accuracy greater than 70% only during the last third of the decision-making process (about 15 s before the initiation of retraction). In this context, cell decision-making does not require amplification of pre-existing cytoskeletal asymmetries. Using subcellular optogenetic receptor activation, I show that receptor inputs can bias the direction of cells, with 75% success rate. I found that administering stimulation only during the early phase of competition did not bias the cell's stochastic choice, suggesting that inducing an early transient asymmetry is not sufficient to drive the system out of its steady state. Cdc42 activity measurements suggest that the two fronts are independently executing their protrusion programs and are amenable to biasing only late in the competition, upon cell stretching. In addition, I found that, once a cell has made a directional decision, the losing front may enter a refractory period that requires complete retraction before stimulation can encourage a new protrusion; this refractory period is particularly pronounced for faster-moving cells. To further probe this refractory nature of a retracting edge, I moved to a simpler geometry. Using subcellular optogenetic receptor activation, I attempted to overwrite the front-rear polarity in cells migrating inside 1-D straight channels. I demonstrated a context-dependent "listening" to receptor inputs. I found that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 activity and reorient migrating neutrophils. I showed that by tuning the phosphorylation of myosin regulatory light chain one can modulate the activity and localization of myosin II and thus the amenability of the cell rear to "listen" to receptor inputs and respond to directional reprogramming. Collectively my work suggests a "selective listening" model in which both actively protruding cell fronts and actively retracting cell rears have strong commitment to their current migratory program. In addition, my work revealed a new dimension of the interplay between the cytoskeleton and signal transduction, providing evidence for an "upstream" role for the cytoskeleton in limiting signal transmission

Keywords: Bioengineering.
Advisor: Fordyce Polly
Theriot Julie
Prakash Manu
Tibshirani Robert