Sarah Woolner

Myosin-10 and actin filaments are essential for mitotic spindle function

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.

Unconventional myosins acting unconventionally

Unconventional myosins are proteins that bind actin filaments in an ATP-regulated manner. Because of their association with membranes, they have traditionally been viewed as motors that function primarily to transport membranous organelles along actin filaments. Recently, however, a wealth of roles for myosins that are not obviously related to organelle transport have been uncovered, including organization of F-actin, mitotic spindle regulation and gene transcription. Furthermore, it has also become apparent that the motor domains of different myosins vary strikingly in their biophysical attributes. We suggest that the assumption that most unconventional myosins function primarily as organelle transporters might be misguided.

Sisyphus, the drosophila myosin XV homolog, traffics within filopodia transporting key sensory and adhesion cargos

Unconventional myosin proteins of the MyTH-FERM superclass are involved in intrafilopodial trafficking, are thought to be mediators of membrane-cytoskeleton interactions, and are linked to several forms of deafness in mammals. Here we show that the Drosophila myosin XV homolog, Sisyphus, is expressed at high levels in leading edge cells and their cellular protrusions during the morphogenetic process of dorsal closure. Sisyphus is required for the correct alignment of cells on opposing sides of the fusing epithelial sheets, as well as for adhesion of the cells during the final zippering/fusion phase. We have identified several putative Sisyphus cargos, including DE-cadherin (also known as Shotgun) and the microtubule-linked proteins Katanin-60, EB1, Milton and aPKC. These cargos bind to the Sisyphus FERM domain, and their binding is in some cases mutually exclusive. Our data suggest a mechanism for Sisyphus in which it maintains a balance between actin and microtubule cytoskeleton components, thereby contributing to cytoskeletal cross-talk necessary for regulating filopodial dynamics during dorsal closure.

Spindle Position in Symmetric Cell Divisions during Epiboly Is Controlled by Opposing and Dynamic Apicobasal Forces

Summary Orientation of cell division is a vital aspect of tissue morphogenesis and growth. Asymmetric divisions generate cell fate diversity and epithelial stratification, whereas symmetric divisions contribute to tissue growth, spreading, and elongation. Here, we describe a mechanism for positioning the spindle in symmetric cell divisions of an embryonic epithelium. We show that during the early stages of epiboly, spindles in the epithelium display dynamic behavior within the plane of the epithelium but are kept firmly within this plane to give a symmetric division. This dynamic stability relies on balancing counteracting forces: an apically directed force exerted by F-actin/myosin-2 via active cortical flow and a basally directed force mediated by microtubules and myosin-10. When both forces are disrupted, spindle orientation deviates from the epithelial plane, and epithelial surface is reduced. We propose that this dynamic mechanism maintains symmetric divisions while allowing the quick adjustment of division plane to facilitate even tissue spreading.

Gene induction following wounding of wild-type versus macrophage-deficient Drosophila embryos

By using a microarray screen to compare gene responses after sterile laser wounding of wild‐type and ‘macrophageless’ serpent mutant Drosophila embryos, we show the wound‐induced programmes that are independent of a pathogenic response and distinguish which of the genes are macrophage dependent. The evolutionarily conserved nature of this response is highlighted by our finding that one such new inflammation‐associated gene, growth arrest and DNA damage‐inducible gene 45 (GADD45), is upregulated in both Drosophila and murine repair models. Comparison of unwounded wild‐type and serpent mutant embryos also shows a portfolio of ‘macrophage‐specific’ genes, which suggest analogous functions with vertebrate inflammatory cells. Besides identifying the various classes of wound‐ and macrophage‐related genes, our data indicate that sterile injury per se, in the absence of pathogens, triggers induction of a ‘pathogen response’, which might prime the organism for what is likely to be an increased risk of infection.

Force and the spindle: Mechanical cues in mitotic spindle orientation

The mechanical environment of a cell has a profound effect on its behaviour, from dictating cell shape to driving the transcription of specific genes. Recent studies have demonstrated that mechanical forces play a key role in orienting the mitotic spindle, and therefore cell division, in both single cells and tissues. Whilst the molecular machinery that mediates the link between external force and the mitotic spindle remains largely unknown, it is becoming increasingly clear that this is a widely used mechanism which could prove vital for coordinating cell division orientation across tissues in a variety of contexts.

Imaging the cytoskeleton in live Xenopus laevis embryos.

Historically, much of our understanding of actin filaments, microtubules and intermediate filaments has come from the study of fixed cells and tissues. But the cytoskeleton is inherently dynamic, and so developing the means to image it in living cells has proved crucial. Advances in confocal microscopy and fluorescent protein technologies have allowed us to dynamically image the cytoskeleton at high resolution and so learn much more about its cellular functions. However, most of this work has been performed in cultured cells, and a critical next step is to understand how the cytoskeleton functions in the context of an intact organism. We, and others, have developed methods to image the cytoskeleton in living vertebrate embryos. Here, we describe an approach to image the cytoskeleton in embryos of the frog, Xenopus laevis, using mRNA to express fluorescently tagged cytoskeletal probes and confocal microscopy to visualize their dynamic behavior.

Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion.

Cytoplasmic dynein light intermediate chains are required for the maintenance of centriole cohesion and the formation of a bipolar spindle in both human cells and Xenopus embryos.

Relating cell shape and mechanical stress in a spatially disordered epithelium using a vertex-based model

Abstract Using a popular vertex-based model to describe a spatially disordered planar epithelial monolayer, we examine the relationship between cell shape and mechanical stress at the cell and tissue level. Deriving expressions for stress tensors starting from an energetic formulation of the model, we show that the principal axes of stress for an individual cell align with the principal axes of shape, and we determine the bulk effective tissue pressure when the monolayer is isotropic at the tissue level. Using simulations for a monolayer that is not under peripheral stress, we fit parameters of the model to experimental data for Xenopus embryonic tissue. The model predicts that mechanical interactions can generate mesoscopic patterns within the monolayer that exhibit long-range correlations in cell shape. The model also suggests that the orientation of mechanical and geometric cues for processes such as cell division are likely to be strongly correlated in real epithelia. Some limitations of the model in capturing geometric features of Xenopus epithelial cells are highlighted.

Decoupling the roles of cell shape and mechanical stress in orienting and cueing epithelial mitosis

Distinct mechanisms involving cell shape and mechanical force are known to influence the rate and orientation of division in cultured cells. However, uncoupling the impact of shape and force in tissues remains challenging. Combining stretching of Xenopus laevis tissue with a novel method of inferring relative mechanical stress, we find separate roles for cell shape in orientating division and mechanical stress in cueing division. We demonstrate that division orientation is best predicted by an axis of cell shape defined by the position of tricellular junctions, which aligns exactly with the principal axis of local cell stress rather than the tissue-level stress. The alignment of division to cell shape requires functional cadherin, but is not sensitive to relative cell stress magnitude. In contrast, cell proliferation rate is more directly regulated by mechanical stress, being correlated with relative isotropic stress, and can be decoupled from cell shape when myosin II is depleted.Distinct mechanisms involving cell shape and mechanical force are known to influence the rate and orientation of division in cultured cells. However, uncoupling the impact of shape and force in tissues remains challenging. Combining stretching of Xenopus laevis tissue with a novel method of inferring relative mechanical stress, we find separate roles for cell shape in orientating division and mechanical stress in cueing division. We demonstrate that division orientation is best predicted by an axis of cell shape defined by the position of tricellular junctions (TCJs), which aligns exactly with the principal axis of local cell stress rather than the tissue-level stress. The alignment of division to cell shape requires functional cadherin and the localisation of the spindle orientation protein, LGN, to TCJs, but is not sensitive to relative cell stress magnitude. In contrast, cell proliferation rate is more directly regulated by mechanical stress, being correlated with relative isotropic stress, and can be decoupled from cell shape when myosin II is depleted.