A Molecular Model for Cell Intercalation during Convergence and Extension: Interacting Cytoskeletal Elements and Myosin Regulation.

Abstract:
My doctoral research investigates the organization, behavior, force production, and regulation of the contractile actomyosin cytoskeleton and the dynamic adhesions underlying cell intercalation in the African clawed frog, Xenopus laevis. Cell intercalation is the process by which the cells of a tissue actively wedge themselves between one another (intercalate) along a specific dimension to produce a narrower, longer tissue, a process often called “convergence (narrowing) and extension (lengthening)” or CE.
CE in Xenopus occurs through progressive expression of Mediolateral Intercalation Behavior (MIB), a cycle of several iterated cell behaviors that drive the intercalation of the presumptive mesodermal cells on the dorsal lip of the blastopore. I examined the mechanism of MIB from the perspective of both the contractile cytoskeleton and dynamic cell adhesions. I have approached the question of the interaction and function of contractile actomyosin and cell adhesions by testing mechanisms of myosin regulation, tropomyosin activity, actin compartmentalization, and cadherin turnover based adhesions.
There is a role for a contractile actin cytoskeleton in both epithelial cell (Blankenship et al, 2006; Lavayer and Lecuit, 2012), and mesodermal cell rearrangement (Skoglund et al., 2008; Rolo et al., 2009; Kim et al, 2010). Myosin II function in the “node and cable” cytoskeletal network (NCN) in Xenopus mesodermal cells is critical for CE of both mesodermal and neural tissue (Skoglund et al, 2008). In Chapter 2 of this dissertation, I show that regulation of myosin contractility through phosphorylation of Serine-19 on the Myosin Regulatory Light Chain (MRLC) is necessary for organization of the NCN, for cell bipolarity and for cell intercalation. Additionally, cell intercalation is dependent on cadherin cell adhesions and I present a working model of how tension generated by myosin contractility is transmitted through cadherin plaques on the mediolateral surfaces of mesodermal cells to generate tissue-scale tension in this axis. Removing regulation of intracellular C-Cadherin through expression of a constitutively active C-Cadherin resulted in rearrangements of the actin cytoskeleton and failure of CE.
I present evidence that a nonmuscle isoform of the actin-binding protein Tropomyosin (XTm5) is present and necessary for the NCN. Tm5 has been shown to enhance myosin association and activity with the cytoskeleton in mammalian cells (Bryce et al, 2003). Consistent with this hypothesis, Morpholino-knockdown of XTm5 results in loss of the NCN and in CE defects. I posit that XTm5 plays a critical role in the endogenous myosin-actin interaction during MIB in Xenopus.
Using Total Internal Reflection Fluorescence Microscopy (TIRFM) on the mesodermal cells exhibiting MIB, I show that there is an additional actin cytoskeleton between the plasma membrane and the NCN, that I term the TIRF-Imaged Cortical Actin Network (TCAN). The TCAN movement is significantly different than the NCN oscillations and the movement is regulated by actin polymerization and less so by myosin activity. I present possible functions for this cytoskeleton, including spatial regulation of membrane proteins.
In the first chapter, I will summarize the current state of knowledge about CE and cell intercalation, with focus on the pertinent topics relating to my work.