Organizing morphogenesis: Mechanisms of actomyosin patterning by RhoGTPase signaling

Morphogenesis is an astonishing orchestration of molecules, cells and tissues, of signaling and mechanics, across space and time. How are its components choreographed across scales? This thesis investigates this question in the context of Drosophila ventral furrow formation, a well-established, simple model of tissue folding. First, we demonstrated how, on the tissue level, combinatorial activation of two transcriptional tissue patterns sets up actomyosin distribution. This tissue-level actomyosin pattern is tuned by the balance of two RhoA GTPase regulators, RhoGEF2 and C-GAP and in turn regulates the curvature of the resulting fold. We then investigated how myosin organization on the cell-level is concerted by RhoGEF2 and C-GAP interplay. We found that the balance of RhoGEF2 and C-GAP regulates the size of an active myosin patch at the constricting apical cell surface, but that both regulators act partially synergistically in promoting temporal myosin dynamics. Overexpression of both regulators together causes a distinct myosin spatiotemporal pattern, suggesting that RhoGEF2 and C-GAP are more than simple antagonists and their regulatory interactions provide essential components of myosin patterning and organization. In summary, this thesis provides insight into how a simple and common regulatory module organizes contractility in the ventral furrow across multiple scales, in space and time.Ph.D

Divergent and combinatorial mechanical strategies that promote epithelial folding during morphogenesis

Folding is an important way by which epithelia are sculpted into three-dimensional shapes during development. Recent studies have integrated quantitative image analysis and physical modeling to uncover contributing mechanisms. It is becoming clear that folding processes often employ multiple mechanisms of force generation. Here, we review divergent modes of epithelial folding. We argue that, in many cases, several mechanisms interact at different time-scales and length-scales to generate a fold. In other cases, very similar folds are generated by different folding mechanisms. How one (or a specific combination of) mechanism(s) might be advantageous in one case versus another is not understood, and future work using quantitative analyses and modeling will be required to determine reasons for distinct modes of folding

Combinatorial patterns of graded RhoA activation and uniform F-actin depletion promote tissue curvature

ABSTRACT During development, gene expression regulates cell mechanics and shape to sculpt tissues. Epithelial folding proceeds through distinct cell shape changes that occur simultaneously in different regions of a tissue. Here, using quantitative imaging in Drosophila melanogaster, we investigate how patterned cell shape changes promote tissue bending during early embryogenesis. We find that the transcription factors Twist and Snail combinatorially regulate a multicellular pattern of lateral F-actin density that differs from the previously described Myosin-2 gradient. This F-actin pattern correlates with whether cells apically constrict, stretch or maintain their shape. We show that the Myosin-2 gradient and F-actin depletion do not depend on force transmission, suggesting that transcriptional activity is required to create these patterns. The Myosin-2 gradient width results from a gradient in RhoA activation that is refined through the balance between RhoGEF2 and the RhoGAP C-GAP. Our experimental results and simulations of a 3D elastic shell model show that tuning gradient width regulates tissue curvature.</jats:p

CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

The Scar/WAVE complex drives actin nucleation during cell migration. Interestingly, the same complex is important in forming membrane ruffles during macropinocytosis, a process mediating nutrient uptake and membrane receptor trafficking. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but its other paralogue, CYRI-A, has not been characterized. Here, we implicate CYRI-A as a key regulator of macropinosome formation and integrin internalization. We find that CYRI-A is transiently recruited to nascent macropinosomes, dependent on PI3K and RAC1 activity. CYRI-A recruitment precedes RAB5A recruitment but follows sharply after RAC1 and actin signaling, consistent with it being a local inhibitor of actin polymerization. Depletion of both CYRI-A and -B results in enhanced surface expression of the α5β1 integrin via reduced internalization. CYRI depletion enhanced migration, invasion, and anchorage-independent growth in 3D. Thus, CYRI-A is a dynamic regulator of macropinocytosis, functioning together with CYRI-B to regulate integrin trafficking