PH Domain-Arf G Protein Interactions Localize the Arf-GEF Steppke for Cleavage Furrow Regulation in Drosophila

The recruitment of GDP/GTP exchange factors (GEFs) to specific subcellular sites dictates where they activate small G proteins for the regulation of various cellular processes. Cytohesins are a conserved family of plasma membrane GEFs for Arf small G proteins that regulate endocytosis. Analyses of mammalian cytohesins have identified a number of recruitment mechanisms for these multi-domain proteins, but the conservation and developmental roles for these mechanisms are unclear. Here, we report how the pleckstrin homology (PH) domain of the Drosophila cytohesin Steppke affects its localization and activity at cleavage furrows of the early embryo. We found that the PH domain is necessary for Steppke furrow localization, and for it to regulate furrow structure. However, the PH domain was not sufficient for the localization. Next, we examined the role of conserved PH domain amino acid residues that are required for mammalian cytohesins to bind PIP3 or GTP-bound Arf G proteins. We confirmed that the Steppke PH domain preferentially binds PIP3 in vitro through a conserved mechanism. However, disruption of residues for PIP3 binding had no apparent effect on GFP-Steppke localization and effects. Rather, residues for binding to GTP-bound Arf G proteins made major contributions to this Steppke localization and activity. By analyzing GFP-tagged Arf and Arf-like small G proteins, we found that Arf1-GFP, Arf6-GFP and Arl4-GFP, but not Arf4-GFP, localized to furrows. However, analyses of embryos depleted of Arf1, Arf6 or Arl4 revealed either earlier defects than occur in embryos depleted of Steppke, or no detectable furrow defects, possibly because of redundancies, and thus it was difficult to assess how individual Arf small G proteins affect Steppke. Nonetheless, our data show that the Steppke PH domain and its conserved residues for binding to GTP-bound Arf G proteins have substantial effects on Steppke localization and activity in early Drosophila embryos.     

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.     

Anillin-mediated targeting of peanut to pseudocleavage furrows is regulated by the GTPase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. Here we demonstrate that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevented pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that did form. We found that Ran was required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin alpha and beta to anillin prevented the binding of Peanut to anillin. Furthermore, RanGTP reversed the inhibitory action of importin alpha and beta. On expression of a mutant form of anillin that lacked an importin alpha and beta binding site, inhibition of Ran no longer restricted the depth and duration of furrow ingression in those pseudocleavage furrows that formed. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran.     

UNC-45 is required for NMY-2 contractile function in early embryonic polarity establishment and germline cellularization in C. elegans

The Caenorhabditis elegans UNC-45 protein is required for proper body wall muscle assembly and acts as a molecular co-chaperone for type II myosins. In contrast to other body wall muscle components, UNC-45 is also abundant in the germline and embryo. We show that maternally provided UNC-45 acts with non-muscle myosin II (NMY-2) during embryonic polarity establishment, cytokinesis and germline cellularization. In embryos depleted for UNC-45, myosin contractility is eliminated resulting in embryonic defects in polar body extrusion, cytokinesis and establishment of polarity. Despite a lack of contractility in an unc-45(RNAi) embryo, NMY-2::GFP localizes to the cortex and accumulates at the presumptive cytokinetic furrow indicating that UNC-45 is not required for cortical localization. UNC-45 and NMY-2 are also required for fertility since the lack of either component results in complete sterility due to failed initiation of the cellularization furrows that separate syncytial nuclei into germ cells. In the absence of UNC-45, the actomyosin cytoskeleton does not contract despite non-functional myosin still directly binding actin. UNC-45 has been previously suggested to be required for the folding of the myosin head, and our results refine this hypothesis suggesting that UNC-45 is not required to fold or maintain the actin binding domain but is still required for myosin function.     

The cytoskeletal involvement in cellularization of theDrosophila melanogaster embryo

Summary The distribution and arrangement of cytoskeletal components in the early embryo ofDrosophila melanogaster were examined by thin-section electron microscopy to elucidate their involvement in the formation of the cellular blastoderm, a process called cellularization. During the final nuclear division in the cortex of the syncytial blastoderm bundles of astral microtubules were closely associated with the surface plasma membrane along the midline where a new gutter was initiated. Thus the new gutter together with the pre-formed ones compartmentalized the embryo surface to reflect underlying individual daughter nuclei. Subsequently such gutters became deeper by further invagination of the plasma membrane between adjacent nuclei to form so-called cleavage furrows. Nuclei simultaneously elongated in the direction perpendicular to the embryo surface and numerous microtubules from the centrosomes ran longitudinally between the nucleus and the cleavage furrow. Microtubules often appeared to be in close association with the nuclear envelope and the cleavage furrow membrane. The plasma membrane at the advancing tip of the furrow was always undercoated with an electron-dense layer, which could be shown to be mainly composed of 5–6 nm microfilaments. These microfilaments were decorated with H-meromyosin to be identified as actin filaments. As cleavage proceeded, each nucleus with its perikaryon became demarcated by the furrow membrane, which then extended laterally to constrict the cytoplasmic connection between each newly forming cell and the central yolk region. The cytoplasmic strand thus formed possessed a prominent circular bundle of microfilaments which were also decorated with H-meromyosin and bidirectionally arranged, similar in structure to the contractile ring in cytokinesis. These observations strongly suggest that both microtubules and actin filaments play a crucial role in cellularization ofDrosophila embryos.     

Dynamics of annulate lamellae in Drosophila syncytial embryos

The mitotic events in eukaryotic cells are controlled by a family of evolutionary conserved cyclin-dependent kinases (cdk) that phosphorylate cell proteins, which results in the structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that changes in cdk1 activity controlling the assembly and disassembly of nuclear pore complexes also affect the structure of cytoplasmic pores in annulate lamellae. Here, we report a comparative electron microscopic analysis of the dynamics of these organelles during mitosis throughout the development of a Drosophila syncytial embryo. We presume that the distribution of annulate lamellae containing mature cytoplasmic pores across the cytoplasm reflects local reductions in the mitotic kinase cdk1 activity during the development of Drosophila syncytial embryos.     

A Drosophila gene with predicted rhoGEF,pleckstrin homology and SH3 domains is highly expressed in morphogenic tissues

We have identified a Drosophila gene that has substantial sequence homology to a distinct class of proto-oncogenes that includes DBL, VAV, Tiam-1, ost and ect-2. It has predicted Rho or Rac guanine exchange factor (Rho/RacGEF) and pleckstin homology (PH) domains with the PH immediately downstream of the Rho/RacGEF. Rho/RacGEFs catalyze the dissociation of GDP from the Rho/Rac subfamily of Ras-like GTPases, thus activating the target Rho/Rac (Takai et al. (1995)Trends Biochem. Sci. 20, 227–231]. Members of the Rho/Rac subfamily regulate organization of the actin cytoskeleton, which controls the morphology, adhesion and motility of cells (Nobes et al. (1995)J. Cell Sci. 108, 225–233; Ridley and Hall (1992)Cell 70, 389–399; Ridley et al. (1992)Cell 70, 401–410]. Message from this gene is found throughout oogenesis and embryogenesis. Of particular interest, message is most abundant in furrows and folds of the embryo where cell shapes are changing and the cytoskeleton is likely to be undergoing reorganization.     

PACSIN2 regulates cell adhesion during gastrulation in Xenopus laevis

We previously identified the adaptor protein PACSIN2 as a negative regulator of ADAM13 proteolytic function. In Xenopus embryos, PACSIN2 is ubiquitously expressed, suggesting that PACSIN2 may control other proteins during development. To investigate this possibility, we studied PACSIN2 function during Xenopus gastrulation and in XTC cells. Our results show that PACSIN2 is localized to the plasma membrane via its coiled-coil domain. We also show that increased levels of PACSIN2 in embryos inhibit gastrulation, fibronectin (FN) fibrillogenesis and the ability of ectodermal cells to spread on a FN substrate. These effects require PACSIN2 coiled-coil domain and are not due to a reduction of FN or integrin expression and/or trafficking. The expression of a Mitochondria Anchored PACSIN2 (PACSIN2-MA) sequesters wild type PACSIN2 to mitochondria, and blocks gastrulation without interfering with cell spreading or FN fibrillogenesis but perturbs both epiboly and convergence/extension. In XTC cells, the over-expression of PACSIN2 but not PACSIN2-MA prevents the localization of integrin β1 to focal adhesions (FA) and filamin to stress fiber. PACSIN2-MA prevents filamin localization to membrane ruffles but not to stress fiber. We propose that PACSIN2 may regulate gastrulation by controlling the population of activated α5β1 integrin and cytoskeleton strength during cell movement.     

Arp2/3-dependent pseudocleavage [correction of psuedocleavage] furrow assembly in syncytial Drosophila embryos

BACKGROUND: In syncytial blastoderm Drosophila embryos, actin caps assemble during telophase. As the cell cycle progresses through interphase, these small caps expand and fuse to form pseudocleavage furrows that are structurally related to the cleavage furrows that assemble during somatic cell division. The molecular mechanism driving cell cycle coordinated actin reorganization from the caps to the furrows is not understood. RESULTS: We show that Drosophila embryos contain a typical Arp2/3 complex and that components of this complex localize to the margins of the expanding caps, to mature pseudocleavage furrows, and to somatic cell cleavage furrows during the postcellularization embryonic divisions. A mutation that disrupts the arpc1 subunit of Arp2/3 leads to spindle fusions that are characteristic of pseudocleavage furrow disruption. By contrast, this mutation does not significantly affect nuclear positioning during interphase, which is dependent on actin cap function. In vivo analysis of actin reorganization demonstrates that the arpc1 mutation does not prevent assembly of small actin caps but blocks cap expansion and furrow assembly as the cell cycle progresses through interphase. The scrambled gene is also required for cap expansion and furrow assembly, and Scrambled is required for Arp2/3 localization to the cap margins. CONCLUSIONS: The Drosophila Arp2/3 complex and Scrambled protein are required for actin cap expansion and pseudocleavage furrow formation during the syncytial blastoderm divisions. We propose that Scrambled-dependent localization of Arp2/3 to the margins of the expanding caps triggers local actin polymerization that drives cap expansion and pseudocleavage furrow assembly.     

The 95F unconventional myosin is required for proper organization of the Drosophila syncytial blastoderm

The 95F myosin, a class VI unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three- dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions lead to aberrant nuclear divisions and to the nuclear defects observed. We propose that 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form.     

Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos

The early Drosophila embryo undergoes two distinct membrane invagination events believed to be mechanistically related to cytokinesis: metaphase furrow formation and cellularization. Both involve actin cytoskeleton rearrangements, and both have myosin II at or near the forming furrow. Actin and myosin are thought to provide the force driving membrane invagination; however, membrane addition is also important. We have examined the role of myosin during these events in living embryos, with a fully functional myosin regulatory light-chain-GFP chimera. We find that furrow invagination during metaphase and cellularization occurs even when myosin activity has been experimentally perturbed. In contrast, the basal closure of the cellularization furrows and the first cytokinesis after cellularization are highly dependent on myosin. Strikingly, when ingression of the cellularization furrow is experimentally inhibited by colchicine treatment, basal closure still occurs at the appropriate time, suggesting that it is regulated independently of earlier cellularization events. We have also identified a previously unrecognized reservoir of particulate myosin that is recruited basally into the invaginating furrow in a microfilament-independent and microtubule-dependent manner. We suggest that cellularization can be divided into two distinct processes: furrow ingression, driven by microtubule mediated vesicle delivery, and basal closure, which is mediated by actin/myosin based constriction.     

Localization and partial characterization of acyltransferases present during rapid membrane formation in the Drosophila melanogaster embryo

Rapid membrane assembly occurs in the early, syncytial Drosophila embryo when 3500 cells are separated within a period of 90 min by the formation of plasma membranes. Acyltransferases catalyzing two of the early steps in phospholipid synthesis were studied during the course of this membrane formation. The enzymes appeared to be similar to mammalian acyltransferases in pH and substrate optima. The activity was inhibited by sulfhydryl-binding reagents and stimulated by low concentrations of magnesium, calcium, and managnese. The enzymes incorporated α-l-glycerophosphate and palmityl coenzyme A into both phospholipids and neutral lipids. The acyltransferases were also localized cytochemically under conditions shown to preserve a substantial proportion of the enzyme activity. Early in embryo development, the activity was localized in the rough endoplasmic reticulum and nuclear envelope. Reaction products were particularly frequent around mitotic nuclei. During the first phase of plasma membrane formation, the activity was localized at the furrow regions of the plasma membrane and in the apical nuclear envelope. During the second (fast) phase, the reaction products appeared mainly in the basal nuclear envelope and rough endoplasmic reticulum internal to the nuclear layer. The data were consistent with the hypothesis that phospholipids for membrane assembly can be formed in situ in several subcellular compartments.     

Centrosomes and the Scrambled protein coordinate microtubule-independent actin reorganization

In Drosophila syncytial blastoderm embryos, centrosomes specify the position of actin-based interphase caps and mitotic furrows. Mutations in the scrambled locus prevent assembly of mitotic furrows, but do not block actin cap formation. The scrambled gene encodes a protein that localizes to the mitotic furrows and centrosomes. Sced localization, actin reorganization from caps into mitotic furrows, and centrosome-coordinated assembly of actin caps are not blocked by microtubule disruption. Our results indicate that centrosomes may coordinate assembly of cortical actin caps through a microtubule-independent mechanism, and that Scrambled mediates a second microtubule-independent process that drives mitotic furrow assembly.     

Localization of Pavarotti-KLP in living Drosophila embryos suggests roles in reorganizing the cortical cytoskeleton during the mitotic cycle

Pav-KLP is the Drosophila member of the MKLP1 family essential for cytokinesis. In the syncytial blastoderm embryo, GFP-Pav-KLP cyclically associates with astral, spindle, and midzone microtubules and also to actomyosin pseudocleavage furrows. As the embryo cellularizes, GFP-Pav-KLP also localizes to the leading edge of the furrows that form cells. In mononucleate cells, nuclear localization of GFP-Pav-KLP is mediated through NLS elements in its C-terminal domain. Mutants in these elements that delocalize Pav-KLP to the cytoplasm in interphase do not affect cell division. In mitotic cells, one population of wild-type GFP-Pav-KLP associates with the spindle and concentrates in the midzone at anaphase B. A second is at the cell cortex on mitotic entry and later concentrates in the region of the cleavage furrow. An ATP binding mutant does not localize to the cortex and spindle midzone but accumulates on spindle pole microtubules to which actin is recruited. This leads either to failure of the cleavage furrow to form or later defects in which daughter cells remain connected by a microtubule bridge. Together, this suggests Pav-KLP transports elements of the actomyosin cytoskeleton to plus ends of astral microtubules in the equatorial region of the cell to permit cleavage ring formation.     

Monoclonal antibody raised against murine IL-1 α peptide cross-reacts with a 60-kDa antigen in early Drosophila melanogaster embryo

Whole-mounts of Drosophila embryos were stained with the monoclonal antibody Vmp 18, raised against the peptide 199–208 of murine interleukin 1/. Immunofluorescence observations showed that the antibody cross-reacted with an antigenic determinant that changed in localization during Drosophila development. In syncytial Drosophila embryos, the antibody recognized an epitope localized on the nuclear envelope throughout mitotic division. As cellularization occurred, the fluorescence was mainly concentrated in the apical region of the blastoderm cells. Western blot analysis of whole Drosophila embryo extracts showed that the antibody recognized a 60-kDa protein in syncytial embryos and during germ band elongation. This suggests that the 60-kDa antigen undergoes dynamic redistribution during embryogenesis.This work was supported in parts by grants from the Italian MURST (40% and 60% funds) and from the Consorzio Siena Ricerche     

Functional analysis of the Drosophila diaphanous FH protein in early embryonic development

The Drosophila Formin Homology (FH) protein Diaphanous has an essential role during cytokinesis. To gain insight into the function of Diaphanous during cytokinesis and explore its role in other processes, we generated embryos deficient for Diaphanous and analyzed three cell-cycle-regulated actin-mediated events during embryogenesis: formation of the metaphase furrow, cellularization and formation of the pole cells. In dia embryos, all three processes are defective. Actin filaments do not organize properly to the metaphase and cellularization furrows and the actin ring is absent from the base of the presumptive pole cells. Furthermore, plasma membrane invaginations that initiate formation of the metaphase furrow and pole cells are missing. Immunolocalization studies of wild-type embryos reveal that Diaphanous localizes to the site where the metaphase furrow is anticipated to form, to the growing tip of cellularization furrows, and to contractile rings. In addition, the dia mutant phenotype reveals a role for Diaphanous in recruitment of myosin II, anillin and Peanut to the cortical region between actin caps. Our findings thus indicate that Diaphanous has a role in actin cytoskeleton organization and is essential for many, if not all, actin-mediated events involving membrane invagination. Based on known biochemical functions of FH proteins, we propose that Diaphanous serves as a mediator between signaling molecules and actin organizers at specific phases of the cell cycle.     

Early embryonic development in the spider Achaearanea tepidariorum: Microinjection verifies that cellularization is complete before the blastoderm stage

The spider Achaearanea tepidariorum is emerging as a non-insect model for studying developmental biology. However, the availability of microinjection into early embryos of this spider has not been reported. We defined the early embryonic stages in A. tepidariorum and applied microinjection to its embryos. During the preblastoderm 16- and 32-nucleus stages, the energids were moving toward the egg periphery. When fluorochrome-conjugated dextran was microinjected into the peripheral region of 16-nucleus stage embryos, it was often incorporated into a single energid and inherited in the progeny without leaking out to surrounding energids. This suggested that 16-nucleus stage embryos consisted of compartments, each containing a single energid. These compartments were considered to be separate cells. Fluorochrome-conjugated dextran could be introduced into single cells of 16- to 128-nucleus stage embryos, allowing us to track cell fate and movement. Injection with mRNA encoding a nuclear localization signal/green fluorescent protein fusion construct demonstrated exogenous expression of the protein in live spider embryos. We propose that use of microinjection will facilitate studies of spider development. Furthermore, these data imply that in contrast to the Drosophila syncytial blastoderm embryo, the cell-based structure of the Achaearanea blastoderm embryo restricts diffusion of cytoplasmic gene products.     

Syntrophin-2 is required for eye development in Drosophila

Syntrophins are components of the dystrophin glycoprotein complex (DGC), which is encoded by causative genes of muscular dystrophies. The DGC is thought to play roles not only in linking the actin cytoskeleton to the extracellular matrix, providing stability to the cell membrane, but also in signal transduction. Because of their binding to a variety of different molecules, it has been suggested that syntrophins are adaptor proteins recruiting signaling proteins to membranes and the DGC. However, critical roles in vivo remain elusive. Drosophila Syntrophin-2 (Syn2) is an orthologue of human γ1/γ2-syntrophins. Western immunoblot analysis here showed Syn2 to be expressed throughout development, with especially high levels in the adult head. Morphological aberrations were observed in Syn2 knockdown adult flies, with lack of retinal elongation and malformation of rhabdomeres. Furthermore, Syn2 knockdown flies exhibited excessive apoptosis in third instar larvae and alterations in the actin localization in the pupal retinae. Genetic crosses with a collection of Drosophila deficiency stocks allowed us to identify seven genomic regions, deletions of which caused enhancement of the rough eye phenotype induced by Syn2 knockdown. This information should facilitate identification of Syn2 regulators in Drosophila and clarification of roles of Syn2 in eye development.     

Drosophila spectrin: the membrane skeleton during embryogenesis

The distribution of alpha-spectrin in Drosophila embryos was determined by immunofluorescence using affinity-purified polyclonal or monoclonal antibodies. During early development, spectrin is concentrated near the inner surface of the plasma membrane, in cytoplasmic islands around the syncytial nuclei, and, at lower concentrations, throughout the remainder of the cytoplasm of preblastoderm embryos. As embryogenesis proceeds, the distribution of spectrin shifts with the migrating nuclei toward the embryo surface so that, by nuclear cycle 9, a larger proportion of the spectrin is concentrated near the plasma membrane. During nuclear cycles 9 and 10, as the nuclei reach the cell surface, the plasma membrane-associated spectrin becomes concentrated into caps above the somatic nuclei. Concurrent with the mitotic events of the syncytial blastoderm period, the spectrin caps elongate at interphase and prophase, and divide as metaphase and anaphase progress. During cellularization, the regions of spectrin concentration appear to shift: spectrin increases near the growing furrow canal and concomitantly increases at the embryo surface. In the final phase of furrow growth, the shift in spectrin concentration is reversed: spectrin decreases near the furrow canal and concomitantly increases at the embryo surface. In gastrulae, spectrin accumulates near the embryo surface, especially at the forming amnioproctodeal invagination and cephalic furrow. During the germband elongation stage, the total amount of spectrin in the embryo increases significantly and becomes uniformly distributed at the plasma membrane of almost all cell types. The highest levels of spectrin are in the respiratory tract cells; the lowest levels are in parts of the forming gut. The spatial and temporal changes in spectrin localization suggest that this protein plays a role in stabilizing rather than initiating changes in structural organization in the embryo.