Structured RhoGEF recruitment drives myosin II organization on large exocytic vesicles.

The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern.

ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion.

Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes.

Generation and timing of graded responses to morphogen gradients.

Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos.

Exocytosis by vesicle crumpling maintains apical membrane homeostasis during exocrine secretion.

Exocrine secretion commonly employs micron-scale vesicles that fuse to a limited apical surface, presenting an extreme challenge for maintaining membrane homeostasis. Using Drosophila melanogaster larval salivary glands, we show that the membranes of fused vesicles undergo actomyosin-mediated folding and retention, which prevents them from incorporating into the apical surface. In addition, the diffusion of proteins and lipids between the fused vesicle and the apical surface is limited.

Microtubules provide guidance cues for myofibril and sarcomere assembly and growth.

Background: Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. We utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila.

Results: A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth.

Global shape of Toll activation is determined by enhancer properties.

Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed "distal pinning," achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. Here, we demonstrate a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning.

Dynamics of Spaetzle morphogen shuttling in the embryo shapes gastrulation patterning.

Establishment of morphogen gradients in the early embryo is challenged by a diffusible sextracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, we followed the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions.

The formin Fhos is a primary mediator of sarcomeric thin-filament array assembly.

Actin-based thin filament arrays constitute a fundamental core component of muscle sarcomeres. We have used formation of the indirect flight musculature for studying the assembly and maturation of thin-filament arrays in a skeletal muscle model system. Employing GFP-tagged actin monomer incorporation, we identify several distinct phases in the dynamic construction of thin-filament arrays.

Adhesion and Fusion of Muscle Cells Are Promoted by Filopodia.

Indirect flight muscles (IFMs) in Drosophila are generated during pupariation by fusion of hundreds of myoblasts with larval muscle templates (myotubes). Live observation of these muscles during the fusion process revealed multiple long actin-based protrusions that emanate from the myotube surface and require Enabled and IRSp53 for their generation and maintenance. Fusion is blocked when formation of these filopodia is compromised.

Myoblast fusion: Experimental systems and cellular mechanisms.

Fusion of myoblasts gives rise to the large, multi-nucleated muscle fibers that power and support organism motion and form. The mechanisms underlying this prominent form of cell-cell fusion have been investigated by a variety of experimental approaches, in several model systems. The purpose of this review is to describe and discuss recent progress in the field, as well as point out issues currently unresolved and worthy of further investigation.

A WntD-Dependent Integral Feedback Loop Attenuates Variability in Drosophila Toll Signaling.

Patterning by morphogen gradients relies on the capacity to generate reproducible distribution profiles. Morphogen spread depends on kinetic parameters, including diffusion and degradation rates, which vary between embryos, raising the question of how variability is controlled. We examined this in the context of Toll-dependent dorsoventral (DV) patterning of the Drosophila embryo.

Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles.

Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements.

The Edges of Pancreatic Islet β Cells Constitute Adhesive and Signaling Microdomains.

Pancreatic islet β cells are organized in rosette-like structures around blood vessels and exhibit an artery-to-vein orientation, but they do not display the typical epithelial polarity. It is unclear whether these cells present a functional asymmetry related to their spatial organization. Here, we identify murine β cell edges, the sites at which adjacent cell faces meet at a sharp angle, as surface microdomains of cell-cell adhesion and signaling.

Assessing the secretory capacity of pancreatic acinar cells.

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini.

N-wasp is required for structural integrity of the blood-testis barrier.

During spermatogenesis, the blood-testis barrier (BTB) segregates the adluminal (apical) and basal compartments in the seminiferous epithelium, thereby creating a privileged adluminal environment that allows post-meiotic spermatid development to proceed without interference of the host immune system. A key feature of the BTB is its continuous remodeling within the Sertoli cells, the major somatic component of the seminiferous epithelium. This remodeling is necessary to allow the transport of germ cells towards the seminiferous tubule interior, while maintaining intact barrier properties.

Targeting secretion to the apical surface by mDia1-built actin tracks.

The apical surface of secretory tubular epithelia is a dynamic cellular domain where massive membrane turnover takes place during exocytosis and its subsequent compensatory endocytosis. This extensive membrane flow poses a difficulty in targeting secretory vesicles efficiently to a narrow apical domain. We have studied how actin filaments mediate the secretory process in the murine exocrine pancreas, which produces and secretes digestive enzymes that are deposited into the intestine.

Apical targeting of the formin Diaphanous in Drosophila tubular epithelia.

Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface.

Sequential activation of ETS proteins provides a sustained transcriptional response to EGFR signaling.

How signal transduction, which is dynamic and fluctuating by nature, is converted into a stable trancriptional response, is an unanswered question in developmental biology. Two ETS-domain transcription factors encoded by the pointed (pnt) locus, PntP1 and PntP2, are universal downstream mediators of EGFR-based signaling in Drosophila. Full disruption of pnt function in developing eye imaginal discs reveals a photoreceptor recruitment phenotype, in which only the R8 photoreceptor cell type is specified within ommatidia.

Directing exocrine secretory vesicles to the apical membrane by actin cables generated by the formin mDia1.

The final stage in exocrine secretion involves translocation of vesicles from their storage areas to the apical membrane. We show that actin-coated secretory vesicles of the exocrine pancreas travel this distance over bundles of specialized actin cables emanating from the apical plasma membrane. These bundles are stable structures that require constant G-actin incorporation and are distinct from the actin web that surrounds the exocrine lumen.

Creating gradients by morphogen shuttling.

Morphogen gradients are used to pattern a field of cells according to variations in the concentration of a signaling molecule. Typically, the morphogen emanates from a confined group of cells. During early embryogenesis, however, the ability to define a restricted source for morphogen production is limited.

Bidirectional Notch activation represses fusion competence in swarming adult Drosophila myoblasts.

A major aspect of indirect flight muscle formation during adult Drosophila myogenesis involves transition of a semi-differentiated and proliferating pool of myoblasts to a mature myoblast population, capable of fusing with nascent myotubes and generating mature muscle fibers. Here we examine the molecular genetic programs underlying these two phases of myoblast differentiation. We show that the cell adhesion proteins Dumbfounded (Duf) and Sticks and stones (Sns), together with their paralogs Roughest (Rst) and Hibris (Hbs), respectively, are required for adhesion of migrating myoblasts to myotubes and initiation of myoblast-myotube fusion.

Orienting the direction of EGFR activation.

Morphogens are typically distributed symmetrically from their source of production. In this issue of Developmental Cell, Peng et al. (2012) demonstrate that a bias in the directionality of protrusions emanating from cells secreting the EGFR ligand Spitz leads to asymmetric activation of the pathway.