Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.

The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.

A Rab39-Klp98A-Rab35 endocytic recycling pathway is essential for rapid Golgi-dependent furrow ingression.

Ingression of the plasma membrane is an essential part of the cell topology-distorting repertoire and a key element in animal cell cytokinesis. Many embryos have rapid cleavage stages in which they are furrowing powerhouses, quickly forming and disassembling cleavage furrows on timescales of just minutes. Previous work has shown that cytoskeletal proteins and membrane trafficking coordinate to drive furrow ingression, but where these membrane stores are derived from and how they are directed to furrowing processes has been less clear. Here, we identify an extensive Rab35/Rab4>Rab39/Klp98A>trans-Golgi network (TGN) endocytic recycling pathway necessary for fast furrow ingression in the Drosophila embryo. Rab39 is present in vesiculotubular compartments at the TGN where it receives endocytically derived cargo through a Rab35/Rab4-dependent pathway. A Kinesin-3 family member, Klp98A, drives the movements and tubulation activities of Rab39, and disruption of this Rab39-Klp98A-Rab35 pathway causes deep furrow ingression defects and genomic instability. These data suggest that an endocytic recycling pathway rapidly remobilizes membrane cargo from the cell surface and directs it to the trans-Golgi network to permit the initiation of new cycles of cleavage furrow formation.

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.

Interface extension is a continuum property suggesting a linkage between AP contractile and DV lengthening processes.

In the early Drosophila embryo, the elongation of the anterior-posterior (AP) body axis is driven by cell intercalation in the germband epithelium. Neighboring cells intercalate through the contraction of AP interfaces (between AP neighbors) into higher-order vertices, which then resolve through the extension of new dorsal-ventral (DV) interfaces (between DV neighbors). Although interface contraction has been extensively studied, less is known about how new interfaces are established. Here we show that DV interface elongation behaviors initiate at the same time as AP contractions, and that DV interfaces which are newly created from resolution of higher-order vertices do not appear to possess a unique 'identity;' instead, all horizontal interfaces undergo lengthening, elongating through ratchetlike sliding behaviors analogous to those found in AP interfaces. Cortical F-actin networks are essential for high area oscillation amplitudes required for effective ratcheting. Our results suggest that, contrary to canonical models, the elongation of new DV interfaces is not produced by a mechanistically separate process. Instead, medial myosin populations drive oscillating radial forces in the cells to generate transient force asymmetries at all tricellular vertices, which-combined with planar polarized stabilization-produce directional ratcheted sliding to generate both AP interface contraction and DV interface elongation.

A PtdIns(3,4,5)P3 dispersal switch engages cell ratcheting at specific cell surfaces.

Force generation in epithelial tissues is often pulsatile, with actomyosin networks generating contractile forces before cyclically disassembling. This pulsed nature of cytoskeletal forces implies that there must be ratcheting mechanisms that drive processive transformations in cell shape. Previous work has shown that force generation is coordinated with endocytic remodeling; however, how ratcheting becomes engaged at specific cell surfaces remains unclear. Here, we report that PtdIns(3,4,5)P3 is a critical lipid-based cue for ratcheting engagement. The Sbf RabGEF binds to PIP3, and disruption of PIP3 reveals a dramatic switching behavior in which medial ratcheting is activated and epithelial cells begin globally constricting apical surfaces. PIP3 enrichments are developmentally regulated, with mesodermal cells having high apical PIP3 while germband cells have higher interfacial PIP3. Finally, we show that JAK/STAT signaling constitutes a second pathway that combinatorially regulates Sbf/Rab35 recruitment. Our results elucidate a complex lipid-dependent regulatory machinery that directs ratcheting engagement in epithelial tissues.

Combinatorial deployment of F-actin regulators to build complex 3D actin structures in vivo.

Despite extensive studies on the actin regulators that direct microfilament dynamics, how these regulators are combinatorially utilized in organismal tissues to generate 3D structures is an unresolved question. Here, we present an in-depth characterization of cortical actin cap dynamics and their regulation in vivo. We identify rapid phases of initiation, expansion, duplication, and disassembly and examine the functions of seven different actin and/or nucleator regulators (ANRPs) in guiding these behaviors. We find ANRPs provide distinct activities in building actin cap morphologies - specifically, while DPod1 is a major regulator of actin intensities, Cortactin is required for continued cortical growth, while Coronin functions in both growth and intensity and is required for Cortactin localization to the cap periphery. Unexpectedly, cortical actin populations recover more rapidly after regulator disruption, suggestive of a deep competition for limited G-actin pools, and we measure in vivo Arp2/3 recruitment efficiencies through an ectopic relocalization strategy. Our results illustrate how the coordination of multiple actin regulators can orchestrate organized and dynamic actin structures in a developmental system.

The pulse of morphogenesis: actomyosin dynamics and regulation in epithelia.

Actomyosin networks are some of the most crucial force-generating components present in developing tissues. The contractile forces generated by these networks are harnessed during morphogenesis to drive various cell and tissue reshaping events. Recent studies of these processes have advanced rapidly, providing us with insights into how these networks are initiated, positioned and regulated, and how they act via individual contractile pulses and/or the formation of supracellular cables. Here, we review these studies and discuss the mechanisms that underlie the construction and turnover of such networks and structures. Furthermore, we provide an overview of how ratcheted processivity emerges from pulsed events, and how tissue-level mechanics are the coordinated output of many individual cellular behaviors.

Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density.

Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, we have disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, we show that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7.

A Nudge or a Shove: Altering Actomyosin Pulse Profiles In Vivo.

Pulsed actomyosin contractions drive morphogenetic processes, but how cyclic frequencies and amplitudes of contractions are tuned to achieve processive shrinking of cell surfaces remains unclear. In this issue of Developmental Cell, Cavanaugh et al. (2020) use optogenetics and biophysical modeling to demonstrate how cells respond to different oscillatory force profiles.

Viscoelastic voyages - Biophysical perspectives on cell intercalation during Drosophila gastrulation.

Developmental processes are driven by a combination of cytoplasmic, cortical, and surface-associated forces. However, teasing apart the contributions of these forces and how a viscoelastic cell responds has long been a key question in developmental biology. Recent advances in applying biophysical approaches to these questions is leading to a fundamentally new understanding of morphogenesis. In this review, we discuss how computational analysis of experimental findings and in silico modeling of Drosophila gastrulation processes has led to a deeper comprehension of the physical principles at work in the early embryo. We also summarize many of the emerging methodologies that permit biophysical analysis as well as those that provide direct and indirect measurements of force directions and magnitudes. Finally, we examine the multiple frameworks that have been used to model tissue and cellular behaviors.

Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction.

During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface.

Vertex sliding drives intercalation by radial coupling of adhesion and actomyosin networks during Drosophila germband extension.

Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell-cell interfaces, while the function of tricellular vertices has remained unaddressed. Here, we identify a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, we find that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. Our results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation.

Multistate outbreak of Salmonella Paratyphi B variant L(+) tartrate(+) and Salmonella Weltevreden infections linked to imported frozen raw tuna: USA, March-July 2015.

Foodborne non-typhoidal salmonellosis causes approximately 1 million illnesses annually in the USA. In April 2015, we investigated a multistate outbreak of 65 Salmonella Paratyphi B variant L(+) tartrate(+) infections associated with frozen raw tuna imported from Indonesia, which was consumed raw in sushi. Forty-six (92%) of 50 case-patients interviewed ate sushi during the week before illness onset, and 44 (98%) of 45 who specified ate sushi containing raw tuna. Two outbreak strains were isolated from the samples of frozen raw tuna. Traceback identified a single importer as a common source of tuna consumed by case-patients; this importer issued three voluntary recalls of tuna sourced from one Indonesian processor. Four Salmonella Weltevreden infections were also linked to this outbreak. Whole-genome sequencing was useful in establishing a link between Salmonella isolated from ill people and tuna. This outbreak highlights the continuing foodborne illness risk associated with raw seafood consumption, the importance of processing seafood in a manner that minimises contamination with pathogenic microorganisms and the continuing need to ensure imported foods are safe to eat. People at higher risk for foodborne illness should not consume undercooked animal products, such as raw seafood.

Membrane trafficking in morphogenesis and planar polarity.

Our understanding of how membrane trafficking pathways function to direct morphogenetic movements and the planar polarization of developing tissues is a new and emerging field. While a central focus of developmental biology has been on how protein asymmetries and cytoskeletal force generation direct cell shaping, the role of membrane trafficking in these processes has been less clear. Here, we review recent advances in Drosophila and vertebrate systems in our understanding of how trafficking events are coordinated with planar cytoskeletal function to drive lasting changes in cell and tissue topologies. We additionally explore the function of trafficking pathways in guiding the complex interactions that initiate and maintain core PCP (planar cell polarity) asymmetries and drive the generation of systematically oriented cellular projections during development.

Differentially-dimensioned furrow formation by zygotic gene expression and the MBT.

Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. Here, we present an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. We show that the deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and we track the X-chromosomal contribution to furrow lengthening to the nullo gene product. We further show that checkpoint proteins are required for furrow lengthening; however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, we examined the turnover of maternal gene products and find that this is a minor contributor to the developmental regulation of furrow morphologies. Our results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis.

Planar polarized Rab35 functions as an oscillatory ratchet during cell intercalation in the Drosophila epithelium.

The coordination between membrane trafficking and actomyosin networks is essential to the regulation of cell and tissue shape. Here, we examine Rab protein distributions during Drosophila epithelial tissue remodeling and show that Rab35 is dynamically planar polarized. Rab35 compartments are enriched at contractile interfaces of intercalating cells and provide the first evidence of interfacial monopolarity. When Rab35 function is disrupted, apical area oscillations still occur and contractile steps are observed. However, contractions are followed by reversals and interfaces fail to shorten, demonstrating that Rab35 functions as a ratchet ensuring unidirectional movement. Although actomyosin forces have been thought to drive interface contraction, initiation of Rab35 compartments does not require Myosin II function. However, Rab35 compartments do not terminate and continue to grow into large elongated structures following actomyosin disruption. Finally, Rab35 represents a common contractile cell-shaping mechanism, as mesoderm invagination fails in Rab35 compromised embryos and Rab35 localizes to constricting surfaces.Various stages of tissue morphogenesis involve the contraction of epithelial surfaces. Here, the authors identify the Rab GTPase Rab35 as an essential component of this contractile process, which functions as a membrane ratchet to ensure unidirectional movement of intercalating cells.

GW-Bodies and P-Bodies Constitute Two Separate Pools of Sequestered Non-Translating RNAs.

Non-translating RNAs that have undergone active translational repression are culled from the cytoplasm into P-bodies for decapping-dependent decay or for sequestration. Organisms that use microRNA-mediated RNA silencing have an additional pathway to remove RNAs from active translation. Consequently, proteins that govern microRNA-mediated silencing, such as GW182/Gw and AGO1, are often associated with the P-bodies of higher eukaryotic organisms. Due to the presence of Gw, these structures have been referred to as GW-bodies. However, several reports have indicated that GW-bodies have different dynamics to P-bodies. Here, we use live imaging to examine GW-body and P-body dynamics in the early Drosophila melanogaster embryo. While P-bodies are present throughout early embryonic development, cytoplasmic GW-bodies only form in significant numbers at the midblastula transition. Unlike P-bodies, which are predominantly cytoplasmic, GW-bodies are present in both nuclei and the cytoplasm. RNA decapping factors such as DCP1, Me31B, and Hpat are not associated with GW-bodies, indicating that P-bodies and GW-bodies are distinct structures. Furthermore, known Gw interactors such as AGO1 and the CCR4-NOT deadenylation complex, which have been shown to be important for Gw function, are also not present in GW-bodies. Use of translational inhibitors puromycin and cycloheximide, which respectively increase or decrease cellular pools of non-translating RNAs, alter GW-body size, underscoring that GW-bodies are composed of non-translating RNAs. Taken together, these data indicate that active translational silencing most likely does not occur in GW-bodies. Instead GW-bodies most likely function as repositories for translationally silenced RNAs. Finally, inhibition of zygotic gene transcription is unable to block the formation of either P-bodies or GW-bodies in the early embryo, suggesting that these structures are composed of maternal RNAs.

Rab8 directs furrow ingression and membrane addition during epithelial formation in Drosophila melanogaster.

One of the most fundamental changes in cell morphology is the ingression of a plasma membrane furrow. The Drosophila embryo undergoes several cycles of rapid furrow ingression during early development that culminate in the formation of an epithelial sheet. Previous studies have demonstrated the requirement for intracellular trafficking pathways in furrow ingression; however, the pathways that link compartmental behaviors with cortical furrow ingression events are unclear. Here, we show that Rab8 has striking dynamic behaviors in vivo. As furrows ingress, cytoplasmic Rab8 puncta are depleted and Rab8 accumulates at the plasma membrane in a location that coincides with known regions of directed membrane addition. We additionally use CRISPR/Cas9 technology to N-terminally tag Rab8, which is then used to address endogenous localization and function. Endogenous Rab8 displays partial coincidence with Rab11 and the Golgi, and this colocalization is enriched during the fast phase of cellularization. When Rab8 function is disrupted, furrow formation in the early embryo is completely abolished. We also demonstrate that Rab8 behaviors require the function of the exocyst complex subunit Sec5 as well as the recycling endosome protein Rab11. Active, GTP-locked Rab8 is primarily associated with dynamic membrane compartments and the plasma membrane, whereas GDP-locked Rab8 forms large cytoplasmic aggregates. These studies suggest a model in which active Rab8 populations direct furrow ingression by guiding the targeted delivery of cytoplasmic membrane stores to the cell surface through interactions with the exocyst tethering complex.

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

Efficacy of 25-OH Vitamin D3 prophylactic administration for reducing lameness in broilers grown on wire flooring.

Bacterial chondronecrosis with osteomyelitis (BCO) is the most common cause of lameness in commercial broilers. Growing broilers on wire flooring provides an excellent experimental model for reproducibly triggering significant levels of lameness attributable to BCO. In the present study we evaluated the efficacy of adding HyD (25-OH vitamin D3) to the drinking water as a preventative/prophylactic treatment for lameness. Broiler chicks were reared on 5 x 10 ft flat wire floor panels within 6 environmental chambers. Three chambers were supplied with tap water (Control group) and the remaining chambers were supplied with HyD (HyD group: 0.06 mL HyD solution/L water; dosing based on the HyD Solution label to provide 33.9 µg 25-OHD3/L) from d 1 through 56. Feed was provided ad libitum and was formulated to meet or exceed minimum standards for all ingredients, including 5,500 IU vitamin D3/kg. Lameness initially was detected on d 28, and the cumulative incidence of lameness on d 56 was higher in the Control group than in the HyD group (34.7 vs. 22.7%, respectively; P = 0.03; Z-test of proportions; chambers pooled). The most prevalent diagnoses for lame birds were osteochondrosis and osteomyelitis (BCO) of the proximal femora (52%) and tibiae (79%), accompanied by minor incidences of tibial dyschondroplasia (0.33%), spondylolisthesis, or kinky back (0.67%), and twisted legs (1%). Broilers that survived to d 56 without developing lameness did not differ in BW when compared by group within a gender. The wire flooring model imposes a rigorous, sustained challenge that undoubtedly is much more severe than typically would be experienced by broilers under normal commercial conditions. Therefore the encouraging response to HyD supplementation in the present study supports the potential for 25-OH vitamin D3 to attenuate outbreaks of lameness caused by BCO in commercial broiler flocks.