Centrosomes comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Here, we have performed a microscopy-based genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins required for centriole duplication and mitotic PCM recruitment. We analysed 92% of the Drosophila genome (13,059 genes) and identified 32 genes involved in centrosome function. An extensive series of secondary screens classified these genes into four categories: (1) nine are required for centriole duplication, (2) 11 are required for centrosome maturation, (3) nine are required for both functions, and (4) three genes regulate centrosome separation. These 32 hits include several new centrosomal components, some of which have human homologs. In addition, we find that the individual depletion of only two proteins, Polo and Centrosomin (Cnn) can completely block centrosome maturation. Cnn is phosphorylated during mitosis in a Polo-dependent manner, suggesting that the Polo-dependent phosphorylation of Cnn initiates centrosome maturation in flies.

Because physical form and function are intimately linked, mechanisms that maintain cell shape and size within strict limits are likely to be important for a wide variety of biological processes. However, while intrinsic controls have been found to contribute to the relatively well-defined shape of bacteria and yeast cells, the extent to which individual cells from a multicellular animal control their plastic form remains unclear. Here, using micropatterned lines to limit cell extension to one dimension, we show that cells spread to a characteristic steady-state length that is independent of cell size, pattern width, and cortical actin. Instead, homeostatic length control on lines depends on a population of dynamic microtubules that lead during cell extension, and that are aligned along the long cell axis as the result of interactions of microtubule plus ends with the lateral cell cortex. Similarly, during the development of the zebrafish neural tube, elongated neuroepithelial cells maintain a relatively well-defined length that is independent of cell size but dependent upon oriented microtubules. A simple, quantitative model of cellular extension driven by microtubules recapitulates cell elongation on lines, the steady-state distribution of microtubules, and cell length homeostasis, and predicts the effects of microtubule inhibitors on cell length. Together this experimental and theoretical analysis suggests that microtubule dynamics impose unexpected limits on cell geometry that enable cells to regulate their length. Since cells are the building blocks and architects of tissue morphogenesis, such intrinsically defined limits may be important for development and homeostasis in multicellular organisms.

Coordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.

As they enter mitosis, animal cells undergo profound actin-dependent changes in shape to become round. Here we identify the Cdk1 substrate, Ect2, as a central regulator of mitotic rounding, thus uncovering a link between the cell-cycle machinery that drives mitotic entry and its accompanying actin remodeling. Ect2 is a RhoGEF that plays a well-established role in formation of the actomyosin contractile ring at mitotic exit, through the local activation of RhoA. We find that Ect2 first becomes active in prophase, when it is exported from the nucleus into the cytoplasm, activating RhoA to induce the formation of a mechanically stiff and rounded metaphase cortex. Then, at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation. Ect2 localization therefore defines the stage-specific changes in actin cortex organization critical for accurate cell division.

Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling.

Animal cells undergo a dramatic series of shape changes as they divide, which depend on re-modeling of cell-substrate adhesions. Here, we show that while focal adhesion complexes are disassembled during mitotic rounding, integrins remain in place. These integrin-rich contacts connect mitotic cells to the underlying substrate throughout mitosis, guide polarized cell migration following mitotic exit, and are functionally important, since adherent cells undergo division failure when removed from the substrate. Further, the ability of cells to re-spread along pre-existing adhesive contacts is essential for division in cells compromised in their ability to construct a RhoGEF-dependent (Ect2) actomyosin ring. As a result, following Ect2 depletion, cells fail to divide on small adhesive islands but successfully divide on larger patterns, as the connection between daughter cells narrows and severs as they migrate away from one another. In this way, regulated re-modeling of cell-substrate adhesions during mitotic rounding aids division in animal cells.

Cells going through mitosis undergo precisely timed changes in cell shape and organisation, which serve to ensure the fair partitioning of cellular components into the two daughter cells. These structural changes are driven by changes in actin filament and microtubule dynamics and organisation. While most evidence suggests that the two cytoskeletal systems are remodelled in parallel during mitosis, recent work in interphase cells has implicated the centrosome in both microtubule and actin nucleation, suggesting the potential for regulatory crosstalk between the two systems. Here, by using both in vitro and in vivo assays to study centrosomal actin nucleation as cells pass through mitosis, we show that mitotic exit is accompanied by a burst in cytoplasmic actin filament formation that depends on WASH and the Arp2/3 complex. This leads to the accumulation of actin around centrosomes as cells enter anaphase and to a corresponding reduction in the density of centrosomal microtubules. Taken together, these data suggest that the mitotic regulation of centrosomal WASH and the Arp2/3 complex controls local actin nucleation, which may function to tune the levels of centrosomal microtubules during passage through mitosis.

Under conditions of homeostasis, dynamic changes in the length of individual adherens junctions (AJs) provide epithelia with the fluidity required to maintain tissue integrity in the face of intrinsic and extrinsic forces. While the contribution of AJ remodeling to developmental morphogenesis has been intensively studied, less is known about AJ dynamics in other circumstances. Here, we study AJ dynamics in an epithelium that undergoes a gradual increase in packing order, without concomitant large-scale changes in tissue size or shape. We find that neighbor exchange events are driven by stochastic fluctuations in junction length, regulated in part by junctional actomyosin. In this context, the developmental increase of isotropic junctional actomyosin reduces the rate of neighbor exchange, contributing to tissue order. We propose a model in which the local variance in tension between junctions determines whether actomyosin-based forces will inhibit or drive the topological transitions that either refine or deform a tissue.

Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. We show that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions.

Despite decades of research, how mammalian cell size is controlled remains unclear because of the difficulty of directly measuring growth at the single-cell level. Here we report direct measurements of single-cell volumes over entire cell cycles on various mammalian cell lines and primary human cells. We find that, in a majority of cell types, the volume added across the cell cycle shows little or no correlation to cell birth size, a homeostatic behavior called "adder". This behavior involves modulation of G1 or S-G2 duration and modulation of growth rate. The precise combination of these mechanisms depends on the cell type and the growth condition. We have developed a mathematical framework to compare size homeostasis in datasets ranging from bacteria to mammalian cells. This reveals that a near-adder behavior is the most common type of size control and highlights the importance of growth rate modulation to size control in mammalian cells.

Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows. Here, we present a robust, open-source approach for treating, labelling and imaging live or fixed cells in automated sequences. NanoJ-Fluidics is based on low-cost Lego hardware controlled by ImageJ-based software, making high-content, multimodal imaging easy to implement on any microscope with high reproducibility. We demonstrate its capacity on event-driven, super-resolved live-to-fixed and multiplexed STORM/DNA-PAINT experiments.

Epithelia are continuously self-renewed, but how epithelial integrity is maintained during the morphological changes that cells undergo in mitosis is not well understood. Here, we show that as epithelial cells round up when they enter mitosis, they exert tensile forces on neighboring cells. We find that mitotic cell-cell junctions withstand these tensile forces through the mechanosensitive recruitment of the actin-binding protein vinculin to cadherin-based adhesions. Surprisingly, vinculin that is recruited to mitotic junctions originates selectively from the neighbors of mitotic cells, resulting in an asymmetric composition of cadherin junctions. Inhibition of junctional vinculin recruitment in neighbors of mitotic cells results in junctional breakage and weakened epithelial barrier. Conversely, the absence of vinculin from the cadherin complex in mitotic cells is necessary to successfully undergo mitotic rounding. Our data thus identify an asymmetric mechanoresponse at cadherin adhesions during mitosis, which is essential to maintain epithelial integrity while at the same time enable the shape changes of mitotic cells.

Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA-adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodeling.

Proper orientation of the mitotic spindle plays a crucial role in embryos, during tissue development, and in adults, where it functions to dissipate mechanical stress to maintain tissue integrity and homeostasis. While mitotic spindles have been shown to reorient in response to external mechanical stresses, the subcellular cues that mediate spindle reorientation remain unclear. Here, we used a combination of optogenetics and computational modeling to investigate how mitotic spindles respond to inhomogeneous tension within the actomyosin cortex. Strikingly, we found that the optogenetic activation of RhoA only influences spindle orientation when it is induced at both poles of the cell. Under these conditions, the sudden local increase in cortical tension induced by RhoA activation reduces pulling forces exerted by cortical regulators on astral microtubules. This leads to a perturbation of the balance of torques exerted on the spindle, which causes it to rotate. Thus, spindle rotation in response to mechanical stress is an emergent phenomenon arising from the interaction between the spindle positioning machinery and the cell cortex.

Collective motions of epithelial cells are essential for morphogenesis. Tissues elongate, contract, flow, and oscillate, thus sculpting embryos. These tissue level dynamics are known, but the physical mechanisms at the cellular level are unclear. Here, we demonstrate that a single epithelial monolayer of MDCK cells can exhibit two types of local tissue kinematics, pulsations and long range coherent flows, characterized by using quantitative live imaging. We report that these motions can be controlled with internal and external cues such as specific inhibitors and substrate friction modulation. We demonstrate the associated mechanisms with a unified vertex model. When cell velocity alignment and random diffusion of cell polarization are comparable, a pulsatile flow emerges whereas tissue undergoes long-range flows when velocity alignment dominates which is consistent with cytoskeletal dynamics measurements. We propose that environmental friction, acto-myosin distributions, and cell polarization kinetics are important in regulating dynamics of tissue morphogenesis.

Animal cells undergo repeated shape changes, for example by rounding up and respreading as they divide. Cell rounding can be also observed in interphase cells, for example when cancer cells switch from a mesenchymal to an ameboid mode of cell migration. Nevertheless, it remains unclear how interphase cells round up. In this article, we demonstrate that a partial loss of substrate adhesion triggers actomyosin-dependent cortical remodeling and ERM activation, which facilitates further adhesion loss causing cells to round. Although the path of rounding in this case superficially resembles mitotic rounding in involving ERM phosphorylation, retraction fiber formation, and cortical remodeling downstream of ROCK, it does not require Ect2. This work provides insights into the way partial loss of adhesion actives cortical remodeling to drive cell detachment from the substrate. This is important to consider when studying the mechanics of cells in suspension, for example using methods like real-time deformability cytometry (RT-DC).

The orderly packing and precise arrangement of epithelial cells is essential to the functioning of many tissues, and refinement of this packing during development is a central theme in animal morphogenesis. The mechanisms that determine epithelial cell shape and position, however, remain incompletely understood. Here, we investigate these mechanisms in a striking example of planar order in a vertebrate epithelium: The periodic, almost crystalline distribution of cone photoreceptors in the adult teleost fish retina. Based on observations of the emergence of photoreceptor packing near the retinal margin, we propose a mathematical model in which ordered columns of cells form as a result of coupling between planar cell polarity (PCP) and anisotropic tissue-scale mechanical stresses. This model recapitulates many observed features of cone photoreceptor organization during retinal growth and regeneration. Consistent with the model's predictions, we report a planar-polarized distribution of Crumbs2a protein in cone photoreceptors in both unperturbed and regenerated tissue. We further show that the pattern perturbations predicted by the model to occur if the imposed stresses become isotropic closely resemble defects in the cone pattern in zebrafish lrp2 mutants, in which intraocular pressure is increased, resulting in altered mechanical stress and ocular enlargement. Evidence of interactions linking PCP, cell shape, and mechanical stresses has recently emerged in a number of systems, several of which show signs of columnar cell packing akin to that described here. Our results may hence have broader relevance for the organization of cells in epithelia. Whereas earlier models have allowed only for unidirectional influences between PCP and cell mechanics, the simple, phenomenological framework that we introduce here can encompass a broad range of bidirectional feedback interactions among planar polarity, shape, and stresses; our model thus represents a conceptual framework that can address many questions of importance to morphogenesis.

Cell migration is essential during development and in human disease progression including cancer. Most cell migration studies concentrate on known or predicted components of migration pathways.

As tissues develop, they are subjected to a variety of mechanical forces. Some of these forces are instrumental in the development of tissues, while others can result in tissue damage. Despite our extensive understanding of force-guided morphogenesis, we have only a limited understanding of how tissues prevent further morphogenesis once the shape is determined after development. Here, through the development of a tissue-stretching device, we uncover a mechanosensitive pathway that regulates tissue responses to mechanical stress through the polarization of actomyosin across the tissue. We show that stretch induces the formation of linear multicellular actomyosin cables, which depend on Diaphanous for their nucleation. These stiffen the epithelium, limiting further changes in shape, and prevent fractures from propagating across the tissue. Overall, this mechanism of force-induced changes in tissue mechanical properties provides a general model of force buffering that serves to preserve the shape of tissues under conditions of mechanical stress.