Molecular model for force production and transmission during vertebrate gastrulation

Abstract

Vertebrate embryos undergo dramatic shape changes at gastrulation that require locally produced and anisotropically applied forces, yet how these forces are produced and transmitted across tissues remains unclear. We show that depletion of myosin regulatory light chain (RLC) levels in the embryo blocks force generation at gastrulation through two distinct mechanisms: destabilizing the myosin II (MII) hexameric complex and inhibiting MII contractility. Molecular dissection of these two mechanisms demonstrates that normal convergence force generation requires MII contractility and we identify a set of molecular phenotypes correlated with both this failure of convergence force generation in explants and of blastopore closure in whole embryos. These include reduced rates of actin movement, alterations in C-cadherin dynamics and a reduction in the number of polarized lamellipodia on intercalating cells. By examining the spatial relationship between C-cadherin and actomyosin we also find evidence for formation of transcellular linear arrays incorporating these proteins that could transmit mediolaterally oriented tensional forces. These data combine to suggest a multistep model to explain how cell intercalation can occur against a force gradient to generate axial extension forces. First, polarized lamellipodia extend mediolaterally and make new C-cadherin-based contacts with neighboring mesodermal cell bodies. Second, lamellipodial flow of actin coalesces into a tension-bearing, MII-contractility-dependent node-and-cable actin network in the cell body cortex. And third, this actomyosin network contracts to generate mediolateral convergence forces in the context of these transcellular arrays.

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

“…Surprisingly, at 10 µM doses of Morpholino there were no F-Actin rich structures observed in these cells, even when two different fluorescent actin probes were used ( Fig 3C'). At a lower (2μM) dose of MO, the F-Actin cytoskeleton mimicked an early stage of NCN development (Fig 3C'', diagrammed in Fig 3D-modified from Pfister et al, 2016).…”

“…The Serine 19 residue on MRLC is the major activating residue for myosin contractility (Somlyo et al, 2003), but an additional phosphorylation on the Threonine 18 residue stabilizes contractile bundles, like those seen in stress fibers (Mizutani et al, 2006). From my experiments with a single phosphonull mutation on S19 of MRLC, I show that Myosin II can still assemble and function in cytokinesis in the absence of a phosphorylatable MRLC, but force production during the power stroke is significantly decreased (Pfister 2016 (Bresnick, 1999;Yamashiro et al, 2003). The dephosphorylation of MRLC has consistently been attributed to the activity of Myosin Phosphatase (MP) (Ito et al, 1998).…”

“…At the onset of Mediolateral Intercalation Behavior (MIB), cell protrusive activity is confined to the mediolateral axis and the large lamelliform protrusions exert traction on neighboring cells, pulling the cells together and, in the process, driving cell shape change (Shih and Keller, 1992a;Keller et al, 2000). A dynamic, mediolaterally polarized cortical actin cytoskeleton, which I call the Node and Cable Network (NCN), is necessary for cell intercalation in Xenopus Pfister et al, 2016). The characteristic structure and behavior of the NCN, cell intercalation, and the resulting CE in both mesodermal and neural (Rolo et al, 2009) …”

“…I have previously shown that the node and cable network (NCN) in MIBexpressing cells functions in both contractility and formation of new adhesions on neighboring cells (Pfister et al, 2016). The combination of cross-linked actin filaments in the mediolateral lamellipodia and the cables of actin filaments in the tensile oscillatory actomyosin node and cable network (NCN) suggests local regulation of actin behavior.…”

“…In this study I examine the function of the Xenopus non-muscle orthologue of Tm5 (XTm5) in the formation of the force-generating NCN during cell intercalation. (Hardy et al, 1991;Pfister et al, 2016).…”