Weng S et al. (2022), Convergent extension requires adhesion-dependen...

XB-ART-58881

Cell Rep 2022 Apr 26;394:110666. doi: 10.1016/j.celrep.2022.110666.

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Convergent extension requires adhesion-dependent biomechanical integration of cell crawling and junction contraction.

Weng S , Huebner RJ , Wallingford JB .

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Convergent extension (CE) is an evolutionarily conserved collective cell movement that elongates several organ systems during development. Studies have revealed two distinct cellular mechanisms, one based on cell crawling and the other on junction contraction. Whether these two behaviors collaborate is unclear. Here, using live-cell imaging, we show that crawling and contraction act both independently and jointly but that CE is more effective when they are integrated via mechano-reciprocity. We thus developed a computational model considering both crawling and contraction. This model recapitulates the biomechanical efficacy of integrating the two modes and further clarifies how the two modes and their integration are influenced by cell adhesion. Finally, we use these insights to understand the function of an understudied catenin, Arvcf, during CE. These data are significant for providing interesting biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.

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Species referenced: Xenopus laevis
Genes referenced: arvcf
GO keywords: convergent extension
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	Graphical Abstract.
	Figure 1. Mosaic labeling showing dynamics of distinct actin populations for crawling and contraction during convergent extension (A) Illustration of convergent extension showing tissue elongation in the AP axis by cell intercalation in the orthogonal direction. (B) Sketch showing crawling mode of convergent extension in four cells with actin in ML protrusions in green. (C) Sketch showing contraction mode of convergent extension in four cells with actin at AP interfaces in purple. (D) Schematic illustrating mosaic labeling technique in a Xenopus embryo. (E and F) Representative images showing uniform labeling of membrane-BFP (E) and mosaic labeling of different colors of an actin biosensor (Lifeact-GFP [F] and Lifeact-RFP [G]). (H) Example showing actin in AP cells labeled in one color and ML cells in another. Asterisks mark representative tCRs for later analysis. (I–K) Still images from a representative time-lapse movie (Video S1, Example 1) showing membrane (I, blue), actin in the ML protrusions for crawling (J, green, arrowheads), or actin at the AP interface for contraction (K, purple). Dashed lines mark cell-cell interfaces; boxes mark tricellular regions (“tCR”) and the middle region (“Mid”); see text for details. (L and M) Kymograph along the AP cell interface showing spatiotemporal dynamics of actin from the ML protrusions representing the “crawling” signal (L) and actin from the AP cells representing the “contraction” signal (M).
	Figure 2. Direct quantification of crawling- and contraction-based cell intercalation during convergent extension (A) Method for intercalation step analysis. Left, intercalation steps were identified as individual peaks on the trace of intercalation velocity. Right, each step is categorized based on its correlation with peaks in the crawling and/or contraction signals (green and purple, respectively). Gray boxes mark the 40-s time window for correlational analysis. (B–E) Examples of crawling-only (B), contraction-only (C), and concurrent intercalation steps (D and E). Each shows traces of intercalation velocity and actin dynamics (left), a schematic (top-right), and still frames (bottom-right) from time-lapse data. Asterisks mark the correlated peaks of velocity, crawling, and/or contraction. (F) Stack plots showing in percentile the number of steps, accumulative time of each step, and total displacement of cell intercalation for each category as indicated. Intercalation steps having no correlation with crawling or tCR contraction were labeled as “Others.”
	Figure 3. Concurrent crawling and contraction improve the efficacy of cell intercalation (A) Concurrent steps produce significantly greater intercalation than do crawling or contraction steps. (B and C) Concurrent steps increase both the step duration and average intercalation velocity. (D) Sketch showing multiple concurrent crawling and contraction pulses (“3 + Concur. peaks”). (E and F) Multiple concurrent crawling and contraction pulses (“3 + peaks”) further improve the intercalation displacement and duration. p values are calculated using Wilcoxon rank sum test (A.K.A. Mann-Whitney U test). ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; NS, not significant.
	Figure 4. Integration of crawling and contraction enhances actin assembly (A) Schematic showing contraction pulses (purple) in contraction-only and concurrent steps. (B and C) Violin plots showing increased peak actin intensity (B) and pulse duration (C) of contraction pulses when they occurred concurrently with crawling pulses compared with contraction pulses occurring alone. (D) Schematic showing crawling protrusions (green) in crawling-only and concurrent steps. (E and F) Increased peak actin intensity (E) and pulse duration (F) of crawling protrusions when they occurred concurrently with contraction pulses compared with crawling protrusion occurring alone. (G) Schematic showing amplified actin assembly in crawling protrusions and contraction cortex when they are integrated during concurrent steps. p values are calculated using Wilcoxon rank sum test (A.K.A. Mann-Whitney U test). ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; NS, not significant.
	Figure 5. A vertex model provides insights into the biomechanics of crawling-contraction integration (A) Each individual cell was modeled as a 90-vertex polygon. (B) A four-cell model, with cell-cell interfaces modeled as independent entities. (C–E) Schematic focused on the boxed region in (B), showing the design for subcellular modeling behaviors. (C) Cell-cell interfaces were connected via cell adhesion clusters of , holding adhesive forces of . (D) Crawling forces were applied to ML vertices around tricellular regions. (E) Contraction forces were added to cell cortex, which is a function of the amount of actomyosin density including the pulsatile component . (F) Representative simulation result of a four-cell model; magnification in insets reveals minimal extracellular gaps between cells. (G) Representative simulation result of a 27-cell model, showing not only cell intercalation but also tissue-wide convergent extension. (H) Schematic showing timepoints for crawling forces and contraction pulses, so crawling-only or contraction-only intercalations steps can be distinguished from concurrent steps. (I and J) The model recapitulates enhanced intercalation displacement and higher velocity for concurrent steps compared with crawling or contraction alone. (K and L) Violin plots showing synergistic effect of integration on effective protrusion intensity (K) and AP contraction force (L), recapitulating experimental observations (see Figure 4). p values are calculated using Wilcoxon rank sum test (A.K.A. Mann Whitney U test). ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; NS, not significant. See also Figures S5 and S6.
	Figure 6. Role of cell adhesion in crawling and contraction (A) Representative simulation result of a four-cell model with low adhesion showing complete cell intercalation. Magnification in insets shows enlarged voids in multi-cellular regions. (B) Simulation result of a 27-cell model with low adhesion reduced tissue-wide convergent extension. (C–E) Effect of increasing adhesion on intercalation (vertex displacement) during crawling-only, contraction-only, and concurrent steps in the model. “WT” marks the value used for simulating the wild-type condition in Figure 5. “Low” marks the value used for simulating the low-adhesion condition in (A) and (B). (F) Effect of increasing adhesion on the effective protrusion intensity in crawling-only and concurrent steps. Enhanced protrusion intensity during concurrent steps is only observed with medium-high adhesion. (G) Effect of adhesion on contraction force in contraction-only and concurrent steps. Contraction force during concurrent steps is robustly increased in concurrent steps. p values are calculated using Wilcoxon rank sum test (A.K.A. Mann Whitney U test). ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; NS, not significant. See also Videos S4 and S5.
	Figure 7. The Arvcf catenin is specifically required for integration of crawling and contraction (A) Still images of membrane-GFP-labeled cells showing that both wild-type and Arvcf-depleted cells were mediolaterally elongated. (B) Arvcf knockdown decreased average intercalation velocity. (C) Violin plots of intercalation (vertex displacement) in crawling-only, contraction-only, and concurrent steps. (D) Simulation results recapitulating the concurrent step-specific defect in intercalation. “KD” marks the results using a set of parameters for simulating Arvcf depletion. (E) Representative simulation result of a 27-cell model with parameters simulating Arvcf depletion. Tissue-wide convergent extension is reduced. p values are calculated using Wilcoxon rank sum test (A.K.A. Mann Whitney U test). ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005; NS, not significant. See also Figure S7.
	Figure S1. Representative images from a second example. Related to Figure 1 and Movie S1. (A-C) Still images from another representative time-lapse movie (Movie S1, Example 2) showing in (A) membrane (blue), in (B) actin in the ML protrusions for crawling (green, arrowheads) and in (C) actin at the AP interface for contraction (purple). Dashed lines mark cell-cell interfaces. Boxes mark tCRs and the middle region (“Mid”). (D, E) Kymograph along the AP cell interface shows spatiotemporal dynamics of actin from the ML protrusions representing the “crawling” signal (D) and actin from the AP cells representing the “contraction” signal (E). White lines outline the tCRs.

	Figure S3. Cross-correlation analysis of cell intercalation velocity and bulk actin dynamics. Related to STAR Methods. (A) Low cross-correlation between intercalation velocity from a paired left and right tricellular vertices sharing the same AP interface. n = 9. (B-D) Low cross-correlation between cell intercalation velocity and bulk actin dynamics for crawling (B), tCR contraction (C), and Mid contraction (D). (E) Strong cross-correlation between actin dynamics for crawling and tCR contraction. (F) Low cross-correlation between actin dynamics for tCR contraction and Mid contraction, demonstrating spatial heterogeneity along the AP interface. All show a wide range of correlation with variable lag time. Each line represents cell intercalation and actin dynamics at one tCR. n > 53.
	Figure S4. Classification of intercalation steps. Related to Figure 2 and Movie S2, S3. (A) Representative intercalation step analysis for the tCRs in Figure 1I-M. For each tCR, the intercalation velocity, crawling signal, and contraction signal were plotted verses time. Peaks and valleys were detected using customized scripts and individual peaks were separated with dashed lines at the adjacent valleys. An intercalation step was defined as an individual peak on the velocity trace and was classified based on its cross-correlation with pulses on the crawling and/or contraction signals. The correlated peaks were color-coded as indicated. Intercalation steps that had no cross-correlation with crawling nor contraction pulses were filled with diagonal strips, while contraction and crawling pulses that had no cross-correlation with any intercalation step were filled with dots. Brown arrows label correlated contraction and crawling pulses. Brown double-line arrows mark correlated crawling and contraction pulses that are associated with different velocity peaks. Daggers mark crawling and contraction pulses that were correlated successively. Lag-time window for all cross-correlation analysis was 40 sec, and the threshold for cross- correlation coefficient was 0.5. (B) Examples showing an intercalation step correlated with a contraction pulse from the middle region of an AP interface, and not correlated with actin dynamics in the tCR. See Movie S3. (C) Figure 2F replotted in which Mid contraction (“ContractMid”) was also considered. The majority of cell intercalation is attributed to actin dynamics at tCRs and therefore the Mid contraction was neglected in this study.
	Figure S5. Design of crawling and contraction in a four-cell model. Related to Figure 5 and STAR Methods. (A) Schematic showing the application of crawling force and actomyosin pulses . Crawling forces on the left and right cells were applied on the tricellular vertices at randomized timepoints and toward each other exclusively. Actomyosin pulses were applied at both tricellular regions and the middle of the AP interface. (B) Representative simulation input of crawling force and actomyosin pulses. Green traces show the profile of crawling forces on the left and right tricellular vertices. Purple kymograph shows the spatiotemporal dynamics of the applied actomyosin pulses along the shortening AP interface. (C) Simulation results from the input in (B). Purple kymograph shows the calculated contraction forces along the AP interface. Green traces mark the tip of the protrusions from the left and right cells. (D) Bar plot showing the insignificance of actomyosin pulses from the middle region of an AP interface. (E) Schematic showing the definition of the proxy for protrusion actin assembly. Depending on the cell-cell interaction, protrusion profile varies with the same applied crawling force. The integral of protrusion length over time, defined as the effective protrusion intensity, was used as a proxy for the protrusion actin assembly dynamics.
	Figure S6. Biomechanical synergy of crawling contraction integration over a wide range of crawling forces and contraction pulses in the in-silico model. Related to Figure 5. (A, B) Effect of increasing crawling force (A) or increasing actomyosin pulses (B) on intercalation (vertex displacement) during crawling only, contraction only, and concurrent steps in the model. (C, D) Synergistic effect of concurrent crawling and contraction on the protrusion dynamics over a wide range of crawling forces and actomyosin pulses. (E, F) Synergistic effect of concurrent crawling and contraction on the contraction force over a wide range of crawling forces and actomyosin pulses. “WT” marks the values used for simulating the wildtype condition.
	Figure S7. Supplementary results of Arvcf knockdown. Related to Figure 7. (A) Violin plots showing that elongated protrusion duration during concurrent steps normally observed was disrupted in Arvcf depleted cells. (B) Violin plots showing that amplified protrusion assembly during concurrent steps normally observed was diminished in Arvcf depleted cells. (C) Violin plots showing that elongated contraction pulse duration during concurrent steps was not altered in Arvcf depleted cells. (D) Violin plots showing that amplified contraction pulses during concurrent steps was not altered in Arvcf depleted cells. (E) Modeling results showing diminished amplification of the effective protrusion intensity during concurrent steps with the “KD” parameters. (F) Modeling results showing no difference on the amplification of contraction force during concurrent steps with the “KD” parameters.

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