Pfister K et al. (2016), Molecular model for force production and transm...

XB-ART-51877

Development 2016 Feb 15;1434:715-27. doi: 10.1242/dev.128090.

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Molecular model for force production and transmission during vertebrate gastrulation.

Pfister K, Shook DR, Chang C, Keller R, Skoglund P.

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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.

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Species referenced: Xenopus laevis
Genes referenced: actb cdh3 mib1 myh10 myh6 myl12b myl2 mylip slc25a20
???displayArticle.antibodies??? Cdh3 Ab1 Myh10 Ab1 Myl12b/Myl9 Ab1 Phospho- Myl2 Ab1
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	Fig. 1. Actin and C-cadherin dynamics during mediolateral intercalation behavior. Representative images of actin organization (A-D), C-cadherin distribution (E-H) and double-labeling (red, actin; green, C-cadherin) (I-L) in cells in dorsal marginal zone explants during the four phases of MIB (M). Scale bar: 20â€…Âµm. (N) Linear arrays of actin spanning multiple cells are seen in a late stage dorsal marginal explant. Node condensation is represented by an increase in fluorescent intensity in an ROI, Oâ€² is 30â€…s after O. Quantification of rate of mediolateral displacement (P) and time C-cadherin is displaced (Q) in cases where one cell contracts and the neighbor relaxes, when both cells contract or when both cells relax (arrows below bars from left to right, respectively). (R) Proportion of displacement towards a node contraction in the specific cases where one cell contracts and the neighbor relaxes compared with contraction events, regardless of the behavior of the neighbor cell. For P, Q and R, n is a minimum of 12 events from 6 cell pairs. Both a contraction (arrow)/relaxation (asterisk) pair (Fig. 1S,Sâ€²) and a contraction/contraction pair (arrows) are shown (Fig. 1T,Tâ€²).
	Fig. 2. Regulatory light chain molecular and cellular phenotypes. (A) Western blotting reveals RLC protein decreases in a MO dose-dependent manner compared with COMO (n=13 gels). (B) Scattered COMO (B) or RLC morphant (Bâ€²) cells in intact stage 17 embryos visualized in 15â€…Î¼m Z-projections of stacks by means of co-injected fluorescent Rhodamine-dextran reveals a cell shape dependence on RLC levels. (C) Myosin IIB heavy chain levels exhibit an RLC MO dose-dependent decrease associated with an increase in proteolytic degradation. (D) Injecting wtRLC-mCherry or pnRLC-mCherry mRNA into developing embryos leads to protein expression as detected by anti-mCherry antibody. (E,F) Two methodologies show that wtRLC and pnRLC expression rescues MHC-IIB stability; quantification of RLC and myosin IIB immunofluorescence levels in RLC morphant dorsal marginal zone explants (E; n=7 explants/condition) and western blot analysis of myosin IIB heavy chain levels in RLC MO embryos (F). (G) RLC morphant cells have a larger surface area than control cells in explants and this phenotype can be rescued by either wtRLC or pnRLC expression (n is at least 52 cells/condition). (H) However, RLC morphant cells have a reduced length-to-width ratio. Expression of wtRLC but not pnRLC substantially rescued this cell shape phenotype (n, at least 14 cells/condition). (I) Morphant cells expressing pnRLC labeled with Rhodamine-dextran (red) display lower length-to-width ratios than corresponding control cells (green). Error bars represent s.e.m. Cont, control. Scale bar: 100 Î¼m in B and 20 Î¼m in I.
	Fig. 3. Reduced convergence forces affect whole embryo morphology. (A) Schematic of the â€˜tractor-pullâ€™ device to measure convergence forces. Explants made from RLC morphant embryos generate less pulling force than controls (B) and co-expressing wtRLC but not pnRLC rescues these pulling forces (C) as measured in sandwich explants at 4 and 6â€…h after the onset of gastrulation (n=6 axes/condition). (D) The length of tailbud stage embryos is reduced in RLC morphants; this effect is rescued by wtRLC but not pnRLC co-expression (n, at least 9 embryos/condition). (E-I) Time-lapse movies show normal blastopore closure (E), delayed blastopore closure in an RLC morphant (F) and rescue by expressing wtRLC (G). This delay is not rescued by expression of pnRLC (H), although an embryo co-injected with pnRLC and COMO gastrulates normally (I). Times indicate hours post stage 10, and dorsal is up in E-I. P<0.05, **P<0.001.
	Fig. 4. Actin structures depend on RLC phosphorylation. (A) Imaging of the cortical actin structures in control cells reveals a normal cortical actin structure. (B) An RLC morphant cell exhibits a reorganization of cortical actin. This effect is partially rescued by wtRLC expression (C), but not pnRLC expression (D). (E) These differences are quantified by comparing the number of node structures per cell (n, at least 22 cells assayed/condition). Dots represent the starting point of the node and the lines represent the node displacement for a control cell (F) and RLC morphant cell (G) are shown. (H) Rate of node movement (at least 9 nodes assayed per condition). Quantification of kymograph analysis of cortical actin in the regions shown (I), shows a reduction in the rate of actin movement during node formation that depends on RLC phosphorylation (J). (K) By contrast, actin movement in lamellipodia is not sensitive to RLC depletion. For J,K, at least 48 kymographs were averaged for each condition. (L) The number of new lamellipodia per cell also depends on RLC phosphorylation (n is at least 31 lamellipodia for each condition). All MOs are at 10â€…ÂµM. Scale bars: 20â€…Î¼m. Error bars represent s.e.m. P<0.05, P<0.005, **P<0.001.
	Fig. 5. Colocalization and interdepencies of transcellular array components. (A,B) Imaging of F-actin with moe-GFP (A) and RLC with wtRLC-mCherry (Aâ€²) shows colocalization (Aâ€²) in both node-and-cable structures with the same relative concentrations in both (B; n=8 regions of interest). (C) Both pRLC and MHC localize to cell cortices in stage 13 explants using both MHC-IIB (red) and mono-phosphorylated RLC (green) (S19-P) antibodies. Levels of both are reduced in RLC morphants (D), and rescued by expression of wtRLC (E), whereas pnRLC expression rescues heavy chain IIB but not pRLC levels (F). At both stage 10 (G,H) and stage 12 (I,J), C-cadherin localization by immunostaining is perturbed in RLC morphant cells (H). Expression of red (K)- and green (L)-labeled C-cadherin in neighboring cells reveals that they colocalize at the membrane (yellow in M), as expected if they have a role in adhesion. Scale bars: 20 Âµm in A*,G-I and 10 Âµm in L.
	Fig. 6. C-cadherin localization on lamellipodia depends on RLC. Actin is red and C-cadherin or caC-cadherin is green. (A,B) Cadherin forms puncta (arrow) on lamellipodia (A) that resolve into long plaques (arrow), linking actin cables from neighboring cells (B, asterisk). (C) By contrast, lamellipodia on RLC morphant cells maintain a cloud of diffuse C-cadherin signal (arrow) that does not resolve into puncta. (D) Time-lapse microscopy of C-cadherin dynamics in adjacent control cells labeled singly for actin (left) or C-cadherin (right) display adhesions (arrows) that when broken, snap backwards rapidly (asterisk). (E) RLC MO-treated cells (co-injected with a blue dextran) do not make adhesions with neighboring control cells. (F) pnRLC-expressing morphant cells display little protrusive activity or adhesion remodeling (arrow). (G-I) Cells expressing caC-cadherin exhibit longer linear adhesion plaques than C-cadherin-expressing cells (arrows in G,H), which are associated with thicker actin cables (arrow in Gâ€², quantified in I; n=20 widths per condition). Merged images are in Gâ€²,Hâ€². Scale bar in A is 20â€…Î¼m for all images. Error bars represent s.e.m. ***P<0.001.
	Fig. 7. Summary of the roles of myosin II contractility, actin cytoskeleton and C-cadherin dynamics in generating the convergence force driving cell intercalation during CE. (A) Deep dorsal mesodermal cells extend large filo-lamelliform protrusions in the medial and lateral directions (white arrows), a protrusive activity characterized by a diffuse actin network (red cross-hatching). These attach to neighboring cells by de novo formation of small, nascent C-cadherin puncta (bright green). As the actin network undergoes retrograde flow toward the cell body, it coalesces into a series of intersecting arcs, called â€˜proto-nodesâ€™ (black arrows), which mature into a characteristic â€˜node-and-cableâ€™ actin cytoskeleton spanning the cell body (red cables and intersections). (B) The node-and-cable system undergoes a mediolaterally oriented, actomyosin-mediated contraction (blue arrows, A,B), thereby generating tension that shortens the cell, exerts traction on the neighboring cells, drives mediolateral cell intercalation, and generates a tissue-level, tensile convergence force. This mechanism consists of two interlinked and iterated molecular cycles, first an adhesion cycle (A) consisting of nascent, C-cadherin plaques arising on polarized lamellipodia, which mature into larger, linear actin containing plaques that link the cytoskeletons of individual cells together in a tissue-level system in a contraction-dependent manner, and second, a cytoskeletal cycle (B) consisting of a mediolaterally oriented tension-generating actomyosin contraction. Both cycles are dependent on a contractile myosin II complex (blue arrows in A,B), as the maturation of cadherin puncta into mature adhesion plaques, the maturation of the protrusive cytoskeleton into protonodes and those into the polarized node-and-cable system; all fail in RLC morphants (C).

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