Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling.

During organogenesis, precise control of spindle orientation balances proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events occurring early in mitosis. Here, we identify a novel orientation mechanism which corrects erroneous anaphase orientations during telophase. The directionality of reorientation correlates with the maintenance or loss of basal contact by the apical daughter. While the scaffolding protein LGN is known to determine initial spindle positioning, we show that LGN also functions during telophase to reorient oblique divisions toward perpendicular. The fidelity of telophase correction also relies on the tension-sensitive adherens junction proteins vinculin, α-E-catenin, and afadin. Failure of this corrective mechanism impacts tissue architecture, as persistent oblique divisions induce precocious, sustained differentiation. The division orientation plasticity provided by telophase correction may enable progenitors to adapt to local tissue needs.

Oral epithelia protect against constant challenges by bacteria, viruses, toxins and injury while also contributing to the formation of ectodermal appendages such as teeth, salivary glands and lingual papillae. Despite increasing evidence that differentiation pathway genes are frequently mutated in oral cancers, comparatively little is known about the mechanisms that regulate normal oral epithelial development. Here, we characterize oral epithelial stratification and describe multiple distinct functions for the mitotic spindle orientation gene LGN (Gpsm2) in promoting differentiation and tissue patterning in the mouse oral cavity. Similar to its function in epidermis, apically localized LGN directs perpendicular divisions that promote stratification of the palatal, buccogingival and ventral tongue epithelia. Surprisingly, however, in dorsal tongue LGN is predominantly localized basally, circumferentially or bilaterally and promotes planar divisions. Loss of LGN disrupts the organization and morphogenesis of filiform papillae but appears to be dispensable for embryonic hair follicle development. Thus, LGN has crucial tissuespecific functions in patterning surface ectoderm and its appendages by controlling division orientation.

The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.

During organogenesis, precise control of spindle orientation ensures a proper balance of proliferation and differentiation. In the developing murine epidermis, planar and perpendicular divisions yield symmetric and asymmetric fate outcomes, respectively. Classically, division axis specification involves centrosome migration and spindle rotation, events that occur Author ContributionsKJL and SEW designed, and KJL, CPD, DCS, and AJB performed the experiments. KJL and SEW analyzed data. LFK provided Afdnfl/fl mice for afadin knockout experiments. KMB provided extensive intellectual input at project onset and throughout the course of the work. KJL and SEW made the figures and wrote the manuscript with input from all authors. Declaration of InterestsWe have no interests or conflicts to declare.

Cytokinesis and other cell shape changes are driven by the actomyosin contractile cytoskeleton.The molecular rearrangements that bring about contractility in non-muscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal crosslinking by myosins and non-motor crosslinkers, are thought to promote contractility. Here, we examined how the abundance of motor and non-motor crosslinkers controls the speed of cytokinetic furrowing. We built a minimal model to simulate the contractile dynamics of the C. elegans zygote cytokinetic ring. This model predicted that intermediate levels of non-motor crosslinkers would allow maximal contraction speed, which we found to be the case for the scaffold protein anillin, in vivo. Our model also demonstrated a non-linear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of non-muscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, both motor and non-motor crosslinkers promote cytokinetic ring closure when present at low levels, but act as a brake when present at higher levels. Together, our findings extend the growing appreciation for the roles of crosslinkers, but reveal that they not only drive but also brake cytoskeletal remodeling.

Oriented cell divisions balance self-renewal and differentiation in stratified epithelia such as the skin epidermis. During peak epidermal stratification, the distribution of division angles among basal keratinocyte progenitors is bimodal, with planar and perpendicular divisions driving symmetric and asymmetric daughter cell fates, respectively. An apically-restricted, evolutionarily-conserved spindle orientation complex that includes the scaffolding protein LGN/Pins/Gpsm2 plays a central role in promoting perpendicular divisions and stratification, but why only a subset of cell polarize LGN is not known. Here, we demonstrate that the LGN paralog, AGS3/Gpsm1, is a novel negative regulator of LGN, and inhibits perpendicular divisions. Static and ex vivo live imaging reveal that AGS3 overexpression displaces LGN from the apical cortex and increases planar orientations, while AGS3 loss prolongs cortical LGN localization and leads to a perpendicular orientation bias. Genetic epistasis experiments in double mutants confirm that AGS3 operates through LGN. Finally, clonal lineage tracing shows that LGN and AGS3 promote asymmetric and symmetric fates, respectively, while also influencing differentiation through delamination. Collectively, these studies shed new light into how spindle orientation influences epidermal stratification.

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