Syncytial Drosophila Embryos Research Articles

Centrosome-organized plasma membrane infoldings linked to growth of a cortical actin domain.

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.

Regulation of centrosome size by the cell-cycle oscillator in Drosophila embryos

Mitotic centrosomes assemble when centrioles recruit large amounts of pericentriolar material (PCM) around themselves. In early C. elegans embryos, mitotic centrosome size appears to be set by the limiting amount of a key component. In Drosophila syncytial embryos, thousands of mitotic centrosomes are assembled as the embryo proceeds through 13 rounds of rapid nuclear division, driven by a core cell cycle oscillator. These divisions slow during nuclear cycles 11–13, and we find that centrosomes respond by reciprocally decreasing their growth rate, but increasing their growth period—so that they grow to a relatively consistent size at each cycle. At the start of each cycle, moderate CCO activity initially promotes centrosome growth, in part by stimulating Polo/PLK1 recruitment to centrosomes. Later in each cycle, high CCO activity inhibits centrosome growth by suppressing the centrosomal recruitment and/or maintenance of centrosome proteins. Thus, in fly embryos, mitotic centrosome size appears to be regulated predominantly by the core cell cycle oscillator, rather than by the depletion of a limiting component.

Kinesin-1 patterns Par-1 and Rho signaling at the cortex of syncytial embryos of Drosophila.

The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. Here, we found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, our data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern.

Sponge/DOCK-dependent regulation of F-actin networks directing cortical cap behaviors and syncytial furrow ingression

In the early syncytial Drosophila embryo, rapid changes in filamentous actin networks and membrane trafficking pathways drive the formation and remodeling of cortical and furrow morphologies. Interestingly, genomic integrity and the completion of mitoses during cell cycles 10–13 depends on the formation of transient membrane furrows that serve to separate and anchor individual spindles during division. While substantial work has led to a better understanding of the core network components that are responsible for the formation of these furrows, less is known about the regulation that controls cytoskeletal and trafficking function. The DOCK protein Sponge was one of the first proteins identified as being required for syncytial furrow formation, and disruption of Sponge deeply compromises F-actin populations in the early embryo, but how this occurs is less clear. Here, we perform quantitative analysis of the effects of Sponge disruption on cortical cap growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. We find that membrane trafficking is relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but that Sponge acts as a master regulator of a diverse cohort of Arp2/3 regulatory proteins. As DOCK family proteins have been implicated in regulating GTP exchange on small GTPases, we also suggest that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, we demonstrate the phasic requirements for branched F-actin and linear F-actin networks in potentiating furrow ingression. In total, these results provide quantitative insights into how a large DOCK scaffolding protein coordinates the activity of a variety of different actin regulatory proteins to direct the remodeling of the apical cortex into cytokinetic-like furrows.

Pseudocleavage furrows restrict plasma membrane-associated PH domain in syncytial Drosophila embryos

Syncytial cells contain multiple nuclei and have local distribution and function of cellular components despite being synthesized in a common cytoplasm. The syncytial Drosophila blastoderm embryo shows reduced spread of organelle and plasma membrane-associated proteins between adjacent nucleo-cytoplasmic domains. Anchoring to the cytoarchitecture within a nucleo-cytoplasmic domain is likely to decrease the spread of molecules; however, its role in restricting this spread has not been assessed. In order to analyze the cellular mechanisms that regulate the rate of spread of plasma membrane-associated molecules in the syncytial Drosophila embryos, we express a pleckstrin homology (PH) domain in a localized manner at the anterior of the embryo by tagging it with the bicoid mRNA localization signal. Anteriorly expressed PH domain forms an exponential gradient in the anteroposterior axis with a longer length scale compared with Bicoid. Using a combination of experiments and theoretical modeling, we find that the characteristic distribution and length scale emerge due to plasma membrane sequestration and restriction within an energid. Loss of plasma membrane remodeling to form pseudocleavage furrows shows an enhanced spread of PH domain but not Bicoid. Modeling analysis suggests that the enhanced spread of the PH domain occurs due to the increased spread of the cytoplasmic population of the PH domain in pseudocleavage furrow mutants. Our analysis of cytoarchitecture interaction in regulating plasma membrane protein distribution and constraining its spread has implications on the mechanisms of spread of various molecules, such as morphogens in syncytial cells.

Dia- and Rok-dependent enrichment of capping proteins in a cortical region.

ABSTRACTRho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II (MyoII, encoded by zip in flies) control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos, Rho1 signaling is high between actin caps, i.e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. In the present study, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok-MyoII control Capping protein enrichment and support a model that Dia and Rok-MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling. This article has an associated First Person interview with the first authors of the paper.

Emergence of a smooth interface from growth of a dendritic network against a mechanosensitive contractile material.

Structures and machines require smoothening of raw materials. Self-organized smoothening guides cell and tissue morphogenesis and is relevant to advanced manufacturing. Across the syncytial Drosophila embryo surface, smooth interfaces form between expanding Arp2/3-based actin caps and surrounding actomyosin networks, demarcating the circumferences of nascent dome-like compartments used for pseudocleavage. We found that forming a smooth and circular boundary of the surrounding actomyosin domain requires Arp2/3 in vivo. To dissect the physical basis of this requirement, we reconstituted the interacting networks using node-based models. In simulations of actomyosin networks with local clearances in place of Arp2/3 domains, rough boundaries persisted when myosin contractility was low. With addition of expanding Arp2/3 network domains, myosin domain boundaries failed to smoothen, but accumulated myosin nodes and tension. After incorporating actomyosin mechanosensitivity, Arp2/3 network growth locally induced a surrounding contractile actomyosin ring that smoothened the interface between the cytoskeletal domains, an effect also evident in vivo. In this way, a smooth structure can emerge from the lateral interaction of irregular active materials.

Cytoskeletal mechanics and dynamics in the Drosophila syncytial embryo.

Cell and tissue functions rely on the genetic programmes and cascades of biochemical signals. It has become evident during the past decade that the physical properties of soft material that govern the mechanics of cells and tissues play an important role in cellular function and morphology. The biophysical properties of cells and tissues are determined by the cytoskeleton, consisting of dynamic networks of F-actin and microtubules, molecular motors, crosslinkers and other associated proteins, among other factors such as cell-cell interactions. The Drosophila syncytial embryo represents a simple pseudo-tissue, with its nuclei orderly embedded in a structured cytoskeletal matrix at the embryonic cortex with no physical separation by cellular membranes. Here, we review the stereotypic dynamics and regulation of the cytoskeleton in Drosophila syncytial embryos and how cytoskeletal dynamics underlies biophysical properties and the emergence of collective features. We highlight the specific features and processes of syncytial embryos and discuss the applicability of biophysical approaches.

The Emergent Yo-yo Movement of Nuclei Driven by Cytoskeletal Remodeling in Pseudo-synchronous Mitotic Cycles

Many aspects in tissue morphogenesis are attributed to a collective behavior of the participating cells. Yet, the mechanism for emergence of dynamic tissue behavior is not well understood. Here, we report that the "yo-yo"-like nuclear movement in the Drosophila syncytial embryo displays emergent features indicative of collective behavior. Following mitosis, the array of nuclei moves away from the wave front by several nuclear diameters only to return to its starting position about 5min later. Based on experimental manipulations and numerical simulations, we find that the ensemble of elongating and isotropically oriented spindles, rather than individual spindles, is the main driving force for anisotropic nuclear movement. ELMO-dependent F-actin restricts the time for the forward movement and ELMO- and dia-dependent F-actin is essential for the return movement. Our study provides insights into how the interactions among the cytoskeleton as individual elements lead to collective movement of the nuclear array on a macroscopic scale.

First person – Bipasha Dey

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Bipasha Dey is first author on ‘DE-cadherin and Myosin II balance regulates furrow length for onset of polygon shape in syncytial Drosophila embryos’, published in JCS. Bipasha has recently completed her PhD and is continuing to work in in the lab of Dr Richa Rikhy at Indian Institute of Science Education and Research, Maharashtra, India, where she is broadly interested in studying how mechanical forces in cells and tissues guide morphogenesis.

DE-cadherin and Myosin II balance regulates furrow length for onset of polygon shape in syncytial Drosophila embryos.

Cell shape morphogenesis, from spherical to polygonal, occurs in epithelial cell formation in metazoan embryogenesis. In syncytial Drosophila embryos, the plasma membrane incompletely surrounds each nucleus and is organized as a polygonal epithelial-like array. Each cortical syncytial division cycle shows a circular to polygonal plasma membrane transition along with furrow extension between adjacent nuclei from interphase to metaphase. In this study, we assess the relative contribution of DE-cadherin (also known as Shotgun) and Myosin II (comprising Zipper and Spaghetti squash in flies) at the furrow to polygonal shape transition. We show that polygonality initiates during each cortical syncytial division cycle when the furrow extends from 4.75 to 5.75 μm. Polygon plasma membrane organization correlates with increased junctional tension, increased DE-cadherin and decreased Myosin II mobility. DE-cadherin regulates furrow length and polygonality. Decreased Myosin II activity allows for polygonality to occur at a lower length than controls. Increased Myosin II activity leads to loss of lateral furrow formation and complete disruption of the polygonal shape transition. Our studies show that DE-cadherin-Myosin II balance regulates an optimal lateral membrane length during each syncytial cycle for polygonal shape transition.This article has an associated First Person interview with the first author of the paper.

F-BAR domain protein Syndapin regulates actomyosin dynamics during apical cap remodeling in syncytial Drosophila embryos.

Branched actin networks driven by Arp2/3 interact with actomyosin filaments in processes such as cell migration. Similar interactions occur in the syncytial Drosophila blastoderm embryo where expansion of apical caps by Arp2/3-driven actin polymerization occurs in interphase, and cap buckling at contact edges by Myosin II to form furrows takes place in metaphase. Here, we study the role of Syndapin (Synd), an F-BAR domain-containing protein, in apical cap remodeling prior to furrow extension. We found that depletion of synd resulted in larger apical caps. Super-resolution and TIRF microscopy showed that control embryos had long apical actin protrusions in caps during interphase and short protrusions during metaphase, whereas synd depletion led to formation of sustained long protrusions, even during metaphase. Loss of Arp2/3 function in synd mutants partly reverted defects in apical cap expansion and protrusion remodeling. Myosin II levels were decreased in synd mutants, an observation consistent with the expanded cap phenotype previously reported for Myosin II mutant embryos. We propose that Synd function limits branching activity during cap expansion and affects Myosin II distribution in order to bring about a transition in actin remodeling activity from apical cap expansion to lateral furrow extension.

Cyto-architecture constrains the spread of photoactivated tubulin in the syncytial Drosophila embryo.

Drosophila embryogenesis begins with nuclear division in a common cytoplasm forming a syncytial cell. Morphogen gradient molecules spread across nucleo-cytoplasmic domains to pattern the body axis of the syncytial embryo. The diffusion of molecules across the syncytial nucleo-cytoplasmic domains is potentially constrained by association with the components of cellular architecture. However, the extent of restriction has not been examined. Here we use photoactivation (PA) to generate a source of cytoplasmic or cytoskeletal molecules in order to monitor the kinetics of their spread in the syncytial Drosophila embryo. Photoactivated PA-GFP and PA-GFP-Tubulin generated within a fixed anterior area diffused along the antero-posterior axis. These molecules were enriched in the cortical cytoplasm above the yolk-filled center, suggesting that the cortical cytoplasm is phase separated from the yolk-filled center. The length scales of diffusion were extracted using exponential fits under steady state assumptions. PA-GFP spread a greater distance as compared to PA-GFP-Tubulin. Both molecules were more restricted when generated in the center of the embryo. The length scale of spread for PA-GFP-Tubulin increased in mutant embryos containing short plasma membrane furrows and a disrupted tubulin cytoskeleton. PA-GFP spread was unaffected by cyto-architecture perturbation. Taken together, these data show that PA-GFP-Tubulin spread is restricted by its incorporation in the microtubule network and intact plasma membrane furrows. This photoactivation based analysis of protein spread allows for interpretation of the dependence of gradient formation on syncytial cyto-architecture.

Quantitative analysis of subcellular distributions with an open-source, object-based tool.

ABSTRACTThe subcellular localization of objects, such as organelles, proteins, or other molecules, instructs cellular form and function. Understanding the underlying spatial relationships between objects through colocalization analysis of microscopy images is a fundamental approach used to inform biological mechanisms. We generated an automated and customizable computational tool, the SubcellularDistribution pipeline, to facilitate object-based image analysis from three-dimensional (3D) fluorescence microcopy images. To test the utility of the SubcellularDistribution pipeline, we examined the subcellular distribution of mRNA relative to centrosomes within syncytial Drosophila embryos. Centrosomes are microtubule-organizing centers, and RNA enrichments at centrosomes are of emerging importance. Our open-source and freely available software detected RNA distributions comparably to commercially available image analysis software. The SubcellularDistribution pipeline is designed to guide the user through the complete process of preparing image analysis data for publication, from image segmentation and data processing to visualization.This article has an associated First Person interview with the first author of the paper.

Microtubules are necessary for proper Reticulon localization during mitosis.

During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.

Par-1 controls the composition and growth of cortical actin caps during Drosophila embryo cleavage.

Cell structure depends on the cortex, a thin network of actin polymers and additional proteins underlying the plasma membrane. The cell polarity kinase Par-1 is required for cells to form following syncytial Drosophila embryo development. This requirement stems from Par-1 promoting cortical actin caps that grow into dome-like metaphase compartments for dividing syncytial nuclei. We find the actin caps to be a composite material of Diaphanous (Dia)-based actin bundles interspersed with independently formed, Arp2/3-based actin puncta. Par-1 and Dia colocalize along extended regions of the bundles, and both are required for the bundles and for each other's bundle-like localization, consistent with an actin-dependent self-reinforcement mechanism. Par-1 helps establish or maintain these bundles in a cortical domain with relatively low levels of the canonical formin activator Rho1-GTP. Arp2/3 is required for displacing the bundles away from each other and toward the cap circumference, suggesting interactions between these cytoskeletal components could contribute to the growth of the cap into a metaphase compartment.

Lighting Up Gene Activation in Living Drosophila Embryos.

With its rapid development, ease of collection, and the presence of a unique layer of nuclei located close to the surface, the Drosophila syncytial embryo is ideally suited to study the establishment of gene expression patterns during development. Recent improvements in RNA labeling technologies and confocal microscopy allowfor visualizing gene activation and quantifying transcriptional dynamics in living Drosophila embryos. Here we review the available tools for mRNA fluorescent labeling and detection in live embryos and precisely describe the overall procedure, from design to mounting and confocal imaging.

Solitary waves in morphogenesis: Determination fronts as strain-cued strain transformations among automatous cells

We present a simple theory of a strain pulse propagating as a solitary wave through a continuous two-dimensional population of cells. A critical strain is assumed to trigger a strain transformation, while, simultaneously, cells move as automata to tend to restore a preferred cell density. We consider systems in which the strain transformation is a shape change, a burst of proliferation, or the commencement of growth (which changes the shape of the population sheet), and demonstrate isomorphism among these cases. Numerical and analytical solutions describe a strain pulse whose height does not depend on how the strain disturbance was first launched, or the rate at which the strain transformation is achieved, or the rate constant in the rule for the restorative cell motion. The strain pulse is therefore very stable, surviving the imposition of strong perturbations: it would serve well as a timing signal in development. The automatous wave formulation is simple, with few model parameters. A strong case exists for the presence of a strain pulse during amelogenesis. Quantitative analysis reveals a simple relationship between the velocity of the leading edge of the pulse in amelogenesis and the known speed of migration of ameloblast cells. This result and energy arguments support the depiction of wave motion as an automatous cell response to strain, rather than as a response to an elastic energy gradient. The theory may also contribute to understanding the determination front in somitogenesis, moving fronts of convergent-extension transformation, and mitotic wavefronts in the syncytial drosophila embryo.

A rapid, membrane-dependent pathway directs furrow formation through RalA in the early Drosophila embryo

Plasma membrane furrow formation is crucial in cell division and cytokinesis. Furrow formation in early syncytial Drosophila embryos is exceptionally rapid, with furrows forming in as little as 3.75 min. Here, we use 4D imaging to identify furrow formation, stabilization, and regression periods, and identify a rapid, membrane-dependent pathway that is essential for plasma membrane furrow formation in vivo. Myosin II function is thought to provide the ingression force for cytokinetic furrows, but the role of membrane trafficking pathways in guiding furrow formation is less clear. We demonstrate that a membrane trafficking pathway centered on Ras-like protein A (RalA) is required for fast furrow ingression in the early fly embryo. RalA function is absolutely required for furrow formation and initiation. In the absence of RalA and furrow function, chromosomal segregation is aberrant and polyploid nuclei are observed. RalA localizes to syncytial furrows, and mediates the movement of exocytic vesicles to the plasma membrane. Sec5, which is an exocyst complex subunit and localizes to ingressing furrows in wild-type embryos, becomes punctate and loses its cortical association in the absence of RalA function. Rab8 also fails to traffic to the plasma membrane and accumulates aberrantly in the cytoplasm in RalA disrupted embryos. RalA localization precedes F-actin recruitment to the furrow tip, suggesting that membrane trafficking might function upstream of cytoskeletal remodeling. These studies identify a pathway, which stretches from Rab8 to RalA and the exocyst complex, that mediates rapid furrow formation in early Drosophila embryos.

The FHA domain determines Drosophila Chk2/Mnk localization to key mitotic structures and is essential for early embryonic DNA damage responses.

DNA damage responses, including mitotic centrosome inactivation, cell-cycle delay in mitosis, and nuclear dropping from embryo cortex, maintain genome integrity in syncytial Drosophila embryos. A conserved signaling kinase, Chk2, known as Mnk/Loki, is essential for the responses. Here we demonstrate that functional EGFP-Mnk expressed from a transgene localizes to the nucleus, centrosomes, interkinetochore/centromere region, midbody, and pseudocleavage furrows without DNA damage and in addition forms numerous foci/aggregates on mitotic chromosomes upon DNA damage. We expressed EGFP-tagged Mnk deletion or point mutation variants and investigated domain functions of Mnk in vivo. A triple mutation in the phosphopeptide-binding site of the forkhead-associated (FHA) domain disrupted normal Mnk localization except to the nucleus. The mutation also disrupted Mnk foci formation on chromosomes upon DNA damage. FHA mutations and deletion of the SQ/TQ-cluster domain (SCD) abolished Mnk transphosphorylations and autophosphorylations, indicative of kinase activation after DNA damage. A potent NLS was found at the C-terminus, which is required for normal Mnk function. We propose that the FHA domain in Mnk plays essential dual functions in mediating embryonic DNA damage responses by means of its phosphopeptide-binding ability: activating Mnk in the nucleus upon DNA damage and recruiting Mnk to multiple subcellular structures independently of DNA damage.