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.

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.

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.

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.

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.

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.

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.

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.

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.

Prophylactic administration of a combined prebiotic and probiotic, or therapeutic administration of enrofloxacin, to reduce the incidence of bacterial chondronecrosis with osteomyelitis in broilers.

Bacteria entering the bloodstream via translocation from the gastrointestinal tract spread hematogenously and can trigger bacterial chondronecrosis with osteomyelitis (BCO) by infecting osteochondrotic microfractures in the epiphyseal-physeal cartilage of the proximal femora and tibiae. In experiment 1, broilers were fed control feed or the same feed containing BacPack 2X, which includes the prebiotic IMW50 (a mannan oligosaccharide beta-glucan yeast cell wall product) plus the probiotic Calsporin (Bacillus subtilis C-3102). Broilers reared on wire flooring consistently developed higher incidences of BCO than hatchmates reared on wood shavings litter (≥24 vs. ≤4%, respectively; P=0.001). Adding BacPack 2X to the feed on d 1 through 56 delayed the age of onset and reduced the cumulative incidence of BCO on wire flooring when compared with broilers fed the control feed (24.0 vs. 40.7%, respectively; P=0.003). In experiment 2, broilers reared on wire flooring received tap water on d 1 through 62 (control group) or therapeutic levels of the potent fluoroquinolone antimicrobial enrofloxacin in the water on d 35 through 54 (enrofloxacin group). During enrofloxacin administration, half as many birds developed BCO in the enrofloxacin group when compared with the control group (8.1 vs. 19.5%, respectively, on d 35 through 54; P=0.001), whereas both groups had similar BCO incidences subsequent to withdrawing enrofloxacin on d 55 through 62 (14.8 vs. 18.2% for the enrofloxacin vs. control groups; P=0.386). Cumulative lameness incidences for d 1 through 62 were higher for the control group than for the enrofloxacin group (39.0 vs. 25.8%, respectively; P=0.003). These results demonstrate that wire flooring imposes a rigorous challenge that leads to high incidences of BCO that can be difficult to suppress, even with therapeutic doses of enrofloxacin. Prophylactically adding BacPack 2X to the feed reduced the incidence of BCO lameness by a proportion similar to that achieved with enrofloxacin, indicating that probiotics potentially can provide effective alternatives to antibiotics for reducing BCO lameness attributable to bacterial translocation and hematogenous distribution.

Motion-induced interruptions and postural equilibrium in linear lateral accelerations.

This study assesses lateral tipping motion-induced interruptions (MIIs) in a simulated motion environment. The objective is to revisit MII occurrence and sway motion relationship by focusing on the frequency and acceleration of the lateral motion stimulus. Results verify that MIIs increase with increasing peak sway acceleration, but the effect of sway frequency is not as clear as that of acceleration. Complex multidirectional motions create more tipping MIIs than unidirectional motion. Research should incorporate acceleration, frequency and motion complexity as factors influencing MII occurrence. To describe a temporary loss of balance without tipping, the term 'probable' MII is introduced. This term fills the gap between the theoretical definition and a human-centred perception of an MII where loss of balance is not a binary phenomenon. The 'probable' MIIs were 16-67% more common than the 'definite' MIIs. The developed mathematical model of MII occurrence versus sway acceleration (amplitude, frequency) approximated the observed MIIs with less than 9% difference.

Nuclei as mechanical bumpers during epithelial remodeling.

The morphogenesis of developing tissues relies on extensive cellular rearrangements in shape, position, and identity. A key process in reshaping tissues is cell intercalation-driven elongation, where epithelial cells align and intercalate along a common axis. Typically, analyses focus on how peripheral cortical forces influence cell shape changes. Less attention is given to how inhomogeneities in internal structures, particularly the nucleus, impact cell shaping. Here, we examine how pulsed contractile and extension dynamics interact with the nucleus in elongating Drosophila embryos. Our data show that tightly packed nuclei in apical layers hinder tissue remodeling/oscillatory behaviors. We identify two mechanisms for resolving internuclear tensions: nuclear deformation and dispersion. Embryos with non-deformable nuclei use nuclear dispersion to maintain near-normal extensile rates, while those with non-dispersible nuclei due to microtubule inhibition exhibit disruptions in contractile behaviors. Disrupting both mechanisms leads to severe tissue extension defects and cell extrusion. These findings highlight the critical role of nuclear shape and positioning in topological remodeling of epithelia.

Lattice light sheet microscopy reveals 4D force propagation dynamics and leading-edge behaviors in an embryonic epithelium in Drosophila.

How pulsed contractile dynamics drive the remodeling of cell and tissue topologies in epithelial sheets has been a key question in development and disease. Due to constraints in imaging and analysis technologies, studies that have described the in vivo mechanisms underlying changes in cell and neighbor relationships have largely been confined to analyses of planar apical regions. Thus, how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface in three dimensions, including especially the apical-basal axis, is unclear. Here, we perform lattice light sheet microscopy (LLSM)-based analysis to determine how far and fast forces propagate across different apical-basal layers, as well as where topological changes initiate from in a columnar epithelium. These datasets are highly time- and depth-resolved and reveal that topology-changing forces are spatially entangled, with contractile force generation occurring across the observed apical-basal axis in a pulsed fashion, while the conservation of cell volumes constrains instantaneous cell deformations. Leading layer behaviors occur opportunistically in response to favorable phasic conditions, with lagging layers "zippering" to catch up as new contractile pulses propel further changes in cell topologies. These results argue against specific zones of topological initiation and demonstrate the importance of systematic 4D-based analysis in understanding how forces and deformations in cell dimensions propagate in a three-dimensional environment.

A wire-flooring model for inducing lameness in broilers: evaluation of probiotics as a prophylactic treatment.

Bacterial chondronecrosis with osteomyelitis (BCO) is the most common cause of lameness in commercial broilers. Bacteria entering the blood via translocation from the respiratory system or gastrointestinal tract spread hematogenously to the proximal epiphyseal-physeal cartilage of rapidly growing femora and tibiae, causing BCO. We tested the hypothesis that rearing broilers on wire flooring should increase the incidence of BCO by persistently imposing additional torque and shear stress on susceptible leg joints. We also tested the hypothesis that probiotics might attenuate bacterial translocation and thereby reduce the incidence of BCO. In 5 independent experiments using 4 commercial lines, broilers grown on wire flooring developed lameness attributable predominately to BCO. The fastest-growing birds were not necessarily the most susceptible to lameness on wire flooring, nor did the genders differ in susceptibility in the 2 experiments that included both male and female broilers. The pathogenesis of BCO is not instantaneous, and accordingly, many broilers that did not exhibit lameness, nevertheless, did possess early pathognomonic lesions. These subclinical lesions were equally likely to develop in the right or left leg. The lesion status of the proximal femoral head did not determine the lesion status of the ipsilateral or contralateral proximal tibial head and vice versa. Broilers reared on wire flooring consistently had higher incidences of lameness than hatch-mates reared on wood-shavings litter. Adding probiotics to the diet beginning at 1 d of age consistently reduced the incidence of lameness for broilers reared on wire flooring. These experiments indicate that probiotics administered prophylactically may constitute an alternative to antibiotics for reducing lameness attributable to BCO. Rearing broilers on wire flooring provides an important new research model for investigating the etiology, pathogenesis, and treatment strategies for BCO.

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.

Multicellular rosette formation links planar cell polarity to tissue morphogenesis.

Elongation of the body axis is accompanied by the assembly of a polarized cytoarchitecture that provides the basis for directional cell behavior. We find that planar polarity in the Drosophila embryo is established through a sequential enrichment of actin-myosin cables and adherens junction proteins in complementary surface domains. F-actin accumulation at AP interfaces represents the first break in planar symmetry and occurs independently of proper junctional protein distribution at DV interfaces. Polarized cells engage in a novel program of locally coordinated behavior to generate multicellular rosette structures that form and resolve in a directional fashion. Actin-myosin structures align across multiple cells during rosette formation, and adherens junction proteins assemble in a stepwise fashion during rosette resolution. Patterning genes essential for axis elongation selectively affect the frequency and directionality of rosette formation. We propose that the generation of higher-order rosette structures links local cell interactions to global tissue reorganization during morphogenesis.

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.