Landino J et al. (2023), Neighbor cells restrain furrowing during epithe...

XB-ART-59819

bioRxiv 2023 Jun 08; doi: 10.1101/2023.06.08.544077.

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Neighbor cells restrain furrowing during epithelial cytokinesis.

Landino J , Misterovich E , Chumki S , Miller AL .

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UNLABELLED: Cytokinesis challenges epithelial tissue homeostasis by generating forces that pull on neighboring cells via cell-cell junctions. Previous work has shown that junction reinforcement at the furrow in Xenopus laevis epithelia regulates the speed of furrowing 1 . This suggests the cytokinetic array that drives cell division is subject to resistive forces from epithelial neighbor cells. We show here that contractility factors accumulate in neighboring cells near the furrow during cytokinesis. Additionally, increasing neighbor cell stiffness, via ɑ-actinin overexpression, or contractility, through optogenetic Rho activation in one neighbor cell, slows or asymmetrically pauses furrowing, respectively. Notably, optogenetic stimulation of neighbor cell contractility on both sides of the furrow induces cytokinetic failure and binucleation. We conclude that forces from the cytokinetic array in the dividing cell are carefully balanced with restraining forces generated by neighbor cells, and neighbor cell mechanics regulate the speed and success of cytokinesis. HIGHLIGHTS: Neighboring cells assemble actomyosin arrays adjacent to the cytokinetic furrowOverexpression of an F-actin cross-linker in neighbor cells slows furrowingOptogenetic activation of contractility in one neighbor pauses furrow ingressionHyper-contractility in both neighbors restrains furrowing & cells fail cytokinesis.

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Genes referenced: actn1 rho

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	Figure 2:. Overexpression of ɑ-actinin in neighbor cells slows cytokinetic furrow ingression.A) Micrographs from time-lapse confocal imaging of cytokinesis with neighbors overexpressing low or high levels of GFP-ɑ-actinin and stained with a membrane dye (CellMask Deep Red). B) Kymographs of membrane and ɑ-actinin signal during furrowing generated from the dashed lines shown in (A). C) Quantification of ɑ-actinin signal intensity normalized to membrane in neighbor cells used in the analysis of rate of furrow ingression. Mean ± SEM. For low overexpression: n = 14 cells, 5 embryos. For high overexpression: n = 18 cells, 7 embryos. ** p ≤ 0.005. D) Quantification of the rate of furrow ingression for junctions with neighbors expressing endogenous, low overexpression, or high overexpression of ɑ-actinin. Mean ± SEM. For junctions with endogenous neighbors: n = 50, 32, 19 (junctions, dividing cells, embryos), for low overexpression: n = 16, 11, 5, for high overexpression: n = 23, 17, 8. * p ≤ 0.05 and p ≤ 0.005. E) Ratio of furrow ingression rate for cells with two neighbors with endogenous levels of ɑ-actinin, one endogenous and one low overexpression neighbor, and one endogenous and one high overexpression neighbor. Mean ± SEM. For two endogenous neighbors: n = 19 cells, 10 embryos, for one low overexpression/one endogenous neighbor: n = 6 cells, 3 embryos, for one high overexpression/one endogenous neighbor: n = 7 cells, 6 embryos. p ≤ 0.005.
	Figure 3:. Optogenetic activation of contractility in one neighbor cells stalls furrowing.A) Micrographs from time-lapse confocal imaging of cytokinesis with optogenetic stimulation of Rho-mediated actomyosin contractility in neighbors on one side of the furrow. Orange triangle and orange line indicate the region and duration of stimulation, respectively. Time shown in minutes:seconds. B) Kymograph of furrow ingression generated from the dashed line shown in (A). Orange arrowhead indicates side of furrow with a stimulated neighbor, blue arrowhead indicates side of furrow with unstimulated neighbor. Orange line indicates the duration of stimulation. C) Quantification of the rate of furrow ingression before, during, and after neighbor cells were optogenetically stimulated. Junctions neighboring the stimulated cells are shown in warm colors, while corresponding junctions with unstimulated neighbors are shown in cool colors. Mean ± SEM. n = 7 junctions, 7 cells, 5 embryos. * p ≤ 0.05 and ** p ≤ 0.005. D) Total rate of furrow ingression for junctions from cells with stimulated (orange) or unstimulated (blue) neighbors, as shown in (C), or for junctions in control cells selected from a region in the field of view that was well separated from the region of optogenetic stimulation. Mean ± SEM. For stimulated and unstimulated junctions: n = 7 junctions, 7 cells, 5 embryos, for control cells: n = 20 junctions, 10 cells, 6 embryos. * p ≤ 0.05 and ** p ≤ 0.005.
	Figure 4:. Optogenetic activation of contractility in both neighbors causes cytokinetic failure.A) Micrographs from time-lapse confocal imaging of cytokinesis with optogenetic stimulation of Rho-mediated actomyosin contractility in neighbors on both sides of the furrow. Orange triangles and orange line indicate the regions and duration of stimulation, respectively. Time shown in minutes:seconds. B) Kymograph of furrow ingression generated from the dashed line shown in (A). Orange arrowheads indicate both sides of the furrow with a stimulated neighbor. Orange line indicates the duration of stimulation. Dashed lines indicate example time points used to measure cell width at Anaphase (1), Maximum Ingression (2), and After Stimulation (3). C) Quantification of cell width at the equator relative to the initial anaphase width. Arrowheads indicate the maximum ingression width and width after stimulation for the cell shown in (B). D) Quantification of the success of cytokinesis for dividing cells with stimulated neighbors, dividing cells in the same field of view with unstimulated neighbors, and cells expressing the optogenetic system but dividing in tissue that did not experience light stimulation. Mean ± SEM. For stimulated neighbors: n = 12 cells, 12 embryos, for unstimulated neighbors: n = 28 cells, 10 embryos, and for unstimulated tissue: n = 26 cells, 8 embryos, * p ≤ 0.05.

References [+] :

Arnold, Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function. 2017, Pubmed