Projective light-sheet microscopy with flexible parameter selection.

Projection imaging accelerates volumetric interrogation in fluorescence microscopy, but for multi-cellular samples, the resulting images may lack contrast, as many structures and haze are summed up. Here, we demonstrate rapid projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle. This allows us to selectively image different sub-volumes of a sample, rapidly switch between them and exclude background fluorescence.

Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling.

The Hippo pathway is an evolutionarily conserved regulator of tissue growth that integrates inputs from both polarity and actomyosin networks. An upstream activator of the Hippo pathway, Kibra, localizes at the junctional and medial regions of the apical cortex in epithelial cells, and medial accumulation promotes Kibra activity. Here, we demonstrate that cortical Kibra distribution is controlled by a tug-of-war between apical polarity and actomyosin dynamics.

Aurora A and cortical flows promote polarization and cytokinesis by inducing asymmetric ECT-2 accumulation.

In the early embryo, cell polarization and cytokinesis are interrelated yet distinct processes. Here, we sought to understand a poorly understood aspect of cleavage furrow positioning. Early embryos deficient in the cytokinetic regulator centralspindlin form furrows, due to an inhibitory activity that depends on aster positioning relative to the polar cortices.

Small GTPases modulate intrinsic and extrinsic forces that control epithelial folding in embryos.

Epithelial folding is a common means to execute morphogenetic movements. The gastrulating Drosophila embryo offers many examples of epithelial folding events, including the ventral, cephalic, and dorsal furrows. Each of these folding events is associated with changes in intracellular contractility and/or cytoskeleton structures that autonomously promote epithelial folding.

Cortical recruitment of centralspindlin and RhoA effectors during meiosis I of primary spermatocytes.

Haploid male gametes are produced through meiosis during gametogenesis. Whereas the cell biology of mitosis and meiosis is well studied in the nematode , comparatively little is known regarding the physical division of primary spermatocytes during meiosis I. Here, we investigated this process using high-resolution time-lapse confocal microscopy and examined the spatiotemporal regulation of contractile ring assembly in primary spermatocytes.

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of embryos.

Ventral furrow formation, the first step in gastrulation, is a well-studied example of tissue morphogenesis. Rho1 is highly active in a subset of ventral cells and is required for this morphogenetic event. However, it is unclear whether spatially patterned Rho1 activity alone is sufficient to recapitulate all aspects of this morphogenetic event, including anisotropic apical constriction and coordinated cell movements.

Competition between kinesin-1 and myosin-V defines posterior determination.

Local accumulation of ) mRNA in the oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for mRNA posterior localization. In this study, we use Staufen, an RNA-binding protein that colocalizes with mRNA, as a proxy for mRNA.

PLK1 plays dual roles in centralspindlin regulation during cytokinesis.

Cytokinesis begins upon anaphase onset. An early step involves local activation of the small GTPase RhoA, which triggers assembly of an actomyosin-based contractile ring at the equatorial cortex. Here, we delineated the contributions of PLK1 and Aurora B to RhoA activation and cytokinesis initiation in human cells.

Spatiotemporal Regulation of RhoA during Cytokinesis.

The active form of the small GTPase RhoA is necessary and sufficient for formation of a cytokinetic furrow in animal cells. Despite the conceptual simplicity of the process, the molecular mechanisms that control it are intricate and involve redundancy at multiple levels. Here, we discuss our current knowledge of the mechanisms underlying spatiotemporal regulation of RhoA during cytokinesis by upstream activators.

A GAP that Divides.

Cytokinesis in metazoan cells is mediated by an actomyosin-based contractile ring that assembles in response to activation of the small GTPase RhoA. The guanine nucleotide exchange factor that activates RhoA during cytokinesis, ECT-2, is highly regulated. In most metazoan cells, with the notable exception of the early embryo, RhoA activation and furrow ingression require the centralspindlin complex.

Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization.

Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF.

Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres.

Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation.

Cytokinesis in Metazoa and Fungi.

SUMMARYCell division-cytokinesis-involves large-scale rearrangements of the entire cell. Primarily driven by cytoskeletal proteins, cytokinesis also depends on topological rearrangements of the plasma membrane, which are coordinated with nuclear division in both space and time. Despite the fundamental nature of the process, different types of eukaryotic cells show variations in both the structural mechanisms of cytokinesis and the regulatory controls.

Local RhoA activation induces cytokinetic furrows independent of spindle position and cell cycle stage.

The GTPase RhoA promotes contractile ring assembly and furrow ingression during cytokinesis. Although many factors that regulate RhoA during cytokinesis have been characterized, the spatiotemporal regulatory logic remains undefined. We have developed an optogenetic probe to gain tight spatial and temporal control of RhoA activity in mammalian cells and demonstrate that cytokinetic furrowing is primarily regulated at the level of RhoA activation.

Cytokinesis: Placing the Furrow in Context.

A Caenorhabditis elegans mutant has been identified in which an ectopic myosin cap shifts the cleavage furrow relative to the spindle center. Surprisingly, the molecules that suppress this cap in wild-type embryos generate a cap in other asymmetrically dividing cells.

Optical Control of Peroxisomal Trafficking.

The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light.

The RhoGAP activity of CYK-4/MgcRacGAP functions non-canonically by promoting RhoA activation during cytokinesis.

Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood.

Aurora B kinase promotes cytokinesis by inducing centralspindlin oligomers that associate with the plasma membrane.

In metazoans, cytokinesis is triggered by activation of the GTPase RhoA at the equatorial plasma membrane. ECT-2, the guanine nucleotide exchange factor (GEF) required for RhoA activation, is activated by the centralspindlin complex that concentrates on spindle midzone microtubules. However, these microtubules and the plasma membrane are not generally in apposition, and thus the mechanism by which RhoA is activated at the cell equator remains unknown.

Binding of the CYK-4 subunit of the centralspindlin complex induces a large scale conformational change in the kinesin subunit.

Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly.

Cytokinesis: centralspindlin moonlights as a membrane anchor.

The hardest working complex in animal cell division has a new gig. This extraordinary machine, the centralspindlin complex, works overtime, contributing to nearly every step in cytokinesis. It has now been shown to stabilize an association between the plasma membrane and the midbody microtubules prior to abscission.

Centralspindlin: at the heart of cytokinesis.

The final step in the cell cycle is the formation of two genetically identical daughter cells by cytokinesis. At the heart of cytokinesis in animal cells is the centralspindlin complex which is composed of two proteins, a kinesin-like protein, Mitotic kinesin-like protein 1, and a Rho GTPase activating protein (RhoGAP), CYK-4. Through its targeted localization to a narrow region of antiparallel microtubule overlap immediately following chromosome segregation, centralspindlin initiates central spindle assembly.

RhoA activation during polarization and cytokinesis of the early Caenorhabditis elegans embryo is differentially dependent on NOP-1 and CYK-4.

The GTPase RhoA is a central regulator of cellular contractility in a wide variety of biological processes. During these events, RhoA is activated by guanine nucleotide exchange factors (GEFs). These molecules are highly regulated to ensure that RhoA activation occurs at the proper time and place.

TULIPs: tunable, light-controlled interacting protein tags for cell biology.

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser.

The RhoGAP domain of CYK-4 has an essential role in RhoA activation.

Cytokinesis in animal cells is mediated by a cortical actomyosin-based contractile ring. The GTPase RhoA is a critical regulator of this process as it activates both nonmuscle myosin and a nucleator of actin filaments [1]. The site at which active RhoA and its effectors accumulate is controlled by the microtubule-based spindle during anaphase [2].