InteractiveFly: GeneBrief

sponge: Biological Overview | References

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. Quantitative analysis was performed of the effects of Sponge disruption on cortical cap (actin structures at the apical surface) growth, furrow formation, membrane trafficking, and cytoskeletal network regulation through live-imaging of the syncytial embryo. Membrane trafficking was found to be relatively unaffected by the defects in branched actin networks that occur after Sponge disruption, but Sponge was found to act 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, it is also suggested that Rac GTPase activity bridges Sponge regulation to the regulators of Arp2/3 function. Finally, the phasic requirements were demonstrated 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 (Henry, 2022).

This work demonstrates the F-actin networks that support the phased ingression of syncytial furrows, while also revealing that Sponge is critically required for the recruitment of several Arp2/3 regulators to the apical and furrow-supporting cortex in the early Drosophila blastoderm. Sponge knockdown causes the mislocalization as well as altered cortical levels of Arp2/3 subunits and Arp regulators such as Scar, Coronin, Pod1, and Cortactin (see Proposed model of Sponge activity in syncytial embryos). Sponge regulation of F-actin is essential for the transition of these regulators from caps onto apical regions of the growing furrows, as Cortactin, DPod1, Coronin, and Scar are absent on ingressing furrows in Sponge knockdown embryos. This leads to inadequate branched actin network function resulting in short, broad furrows that extend no longer than 2.1 ​μm in length through cycle 13, and small residual cap-like structures, as small as 33% the wild-type cap area. Based on these data, a mechanism is proposed in which Sponge regulates and recruits Scar, Coronin, and DPod1 to the apical caps, while antagonizing Cortactin localization. These proteins in turn modulate filamentous actin and activate Arp2/3 activity and/or stability. As the new apical actin cap forms and expands, Arp2/3 and its regulators remain associated with the branched Actin network and are present in the ring-like structure at the transition point from cap to furrow. This appears necessary for proper linear F-actin nucleation and polymerization by Diaphanous which enables the building of sufficiently extended furrows in each syncytial cycle (Henry, 2022).

Successful cell division relies on an ingressing plasma membrane furrow physically separating neighboring nuclei. In the early Drosophila blastoderm syncytium, as successive cell cycles progress and nuclei become more densely packed, the risk of chromosomal missegregation or mitotic collapse rises if furrows do not adequately segregate neighboring nuclei or provide appropriate anchoring points for spindles. Previous work has shown that furrow length is negatively correlated with mitotic defects. In Sponge embryos, where furrows do not ingress past ~2 ​μm, severe missegregation in the syncytial stages causes embryos to fail to survive past cellularization (cycle 14). In control embryos, segregation defects are avoided by building furrows in two phases, Ingression I and Ingression II, with a Stabilization period juxtaposed in between. Sponge knockdown does not affect the biphasic nature of furrow ingression, as each cycle maintains two separate ingression periods as well as a measurable stabilization phase that is not significantly different in duration than in control embryos. However, the rate of ingression that Sponge furrows reach in any given ingression phase is slower than in control embryos. This reveals that Sponge does not affect select portions of furrow ingression, but is acting on all phases that promote invagination of the plasma membrane. It also remains a possibility that defects in actin and/or early ingression events may indirectly affect the degree to which later phases such as Ingression II proceeds, though Sponge protein, and the actin and/or nucleator regulatory proteins (ANRP) proteins it regulates, are present at caps and furrows throughout the syncytial cycle. Whether direct or indirect, further evidence of the requirement for Sponge during both early and late furrow phases is observed in measurements of the width of syncytial furrows. As furrows transition from Ingression I to Ingression II, furrows change in morphology from a broad and diffuse appearance to very sharply delineated furrows. In Sponge embryos, furrows possess a broader morphology throughout cycles 10-13, but still partially sharpen as later cycles transition into Ingression II phasic behaviors. Together, these data show that furrow ingression after Sponge disruption occurs in a predictable phasic pattern but that the formation, efficiency, and organization of the furrow is compromised, suggesting that Sponge acts as a master regulator of the furrow ingression process. As the results suggest that Sponge primarily impacts cortical F-actin cap components, this further indicates the importance of the actin cortex in directing proper furrow ingression dynamics (Henry, 2022).

F-actin levels are dramatically reduced both on apical caps and on furrows in Sponge embryos. As these regions have different contributions from branched and unbranched actin populations (branched is more predominant in cap regions while linear is more strongly present at the furrow) this raises the possibility that Sponge could regulate both pathways of actin polymerization. Reducing linear F-actin populations through dia knockdown results in a shortened furrow phenotype reminiscent of what is seen in Sponge. However, several characteristics of dia furrows indicate they may be controlled by a separate mechanism than those in Sponge disrupted embryos. First, furrows are able to reach a significantly greater maximum depth of 3.5 ​μm, which also reduces the occurrence of mitotic defects. To achieve this greater length, dia furrows ingress at maximal rates closer to those in control during the early phases of furrow formation. In the Ingression I phases of cycles 12 and 13, Sponge furrows show reduced ingression rates, while dia maximal rates are slightly higher than or equal to control furrows, showing no significant difference. It is only in Ingression II phases that dia maximum ingression rates fall behind control. Similarly, dia furrow morphologies are thinner and sharper than those in Sponge throughout cycles 10-13, and are not significantly different from control furrows during Ingression I phases. It is only in Ingression II phases that dia furrows are significantly wider than control, although they do still transition to a sharper morphology than in respective Ingression I phases. In contrast to Sponge, which is needed for both Ingression I and Ingression II, Dia appears more important for Ingression II phases that are responsible for the bulk of a given cycles maximum furrow length. However, it should be noted that both shRNA-driven disruptions and a Dia genetic allele that was used previously, although similar in phenotype, may only be partial disruptions of function, and thus additional defects could be observed with amorphic loss-of-function approaches (Henry, 2022).

When branched F-actin networks are reduced through arpc4 knockdown, the resulting phenotype is more closely related to those observed in Sponge defective embryos. Furrows in arpc4 embryos reach a maximum length of 2.1 ​μm, the same as in Sponge. Maximum ingression rates of these furrows also mimic a Sponge phenotype. arpc4 furrows ingress at a maximum rate equal to that of Sponge furrows during the Ingression I phase of cycles 11-13, when dia maximum ingression rates are greater than or equal to wild-type. Consistent with the biphasic defects seen in Sponge, the maximum ingression rates during Ingression II phases when arpc4 is disrupted are also significantly slower than control and not significantly different than in Sponge embryos. These furrows also maintain a Sponge-like broad furrow morphology, and arpc4 furrows are significantly wider than wild-type in every syncytial ingression phase. As branched F-actin networks are primarily involved in apical F-actin caps, disrupting arpc4 also severely affects caps. While cap-like structures are produced with arpc4 shRNA, they are strongly reduced in both size and F-actin intensity, similar to the structures produced in Sponge embryos. Together, the many similarities between both furrows and caps in arpc4 and Sponge backgrounds suggest Sponge is likely to be a regulator of Arp2/3 function (Henry, 2022).

Many factors are involved in activating, stabilizing, and otherwise regulating Arp2/3 activity during the syncytial stage of embryogenesis, including Scar, Coronin, DPod1, and Cortactin. Each of these proteins can be found on the apical caps where Arp2/3 mediated branched F-actin is prominent (Xie, 2021). These regulators are all disrupted to varying degrees on caps when Sponge is knocked down. Scar, Coronin, and DPod1 are each reduced after Sponge disruption, with remaining protein mislocalized as random puncta throughout the cytoplasm. Within these cap-like structures, Scar is the most severely diminished; from cycle 10-13, Scar intensity is on average 70% reduced from control embryos. Coronin is the next most severely affected factor with Coronin levels at residual caps being 67% lower in intensity than on control caps. The Coronin-family member DPod1 also shows a 49% reduction over cycles 10-13. DPod1 has been shown to have the strongest impact on overall F-actin and Arp2/3 intensities at the cap, and thus its reduction is likely a major contributing factor to the observed loss of F-actin intensities in Sponge embryos, with changes in Coronin and Scar function also contributing to changes in cap growth rates and sizes (Xie, 2021). One regulator that does not appear to require Sponge activity for localization to the apical cap, however, is Cortactin. While Cortactin is only present on the small residual structures in Sponge embryos, it is present on these structures in significantly increased intensity levels, suggesting that Sponge and Cortactin may possess an antagonistic relationship. Prior work has demonstrated an inhibitory function of Coronin on Cortactin function, so it may be that Sponge regulation of Coronin in turn affects Cortactin levels at the cortical cap in an opposing fashion. Alternatively, it may be that Sponge affects Cortactin levels through its regulation of small GTPase activity or through its other scaffolding domains (Henry, 2022).

Interestingly, each of these regulators normally also localizes to the basal periphery of caps, where the cap structure meets the apical end of the ingressing furrows. While phenotypes of these proteins on the apical cap varies with Sponge disruption, the effect on their localization to the apical furrow is ubiquitous. When Sponge is knocked down, Scar, Coronin, DPod1, and Cortactin are all depleted from the furrow and Scar and Dpod1 become cytoplasmic while Cortactin and Coronin form punctate structures. Interestingly, Arp2/3 subunits are also found in a similar punctate fashion. Sponge embryos produce short furrows up to 2 μm in length after disruption, as Arp2/3 and its regulators are each mislocalized in these embryos. This suggests that a population of Arp2/3 branched F-actin is critical on the apical furrows to support Dia linear F-actin and membrane furrows extending beyond this point (Henry, 2022).

As a DOCK3/4 homologous protein, Sponge is predicted to be a Rac1 and/or Rap1 GEF. Indeed, there are several reports of Sponge acting as a GEF for both of these GTPases later in Drosophila development (Biersmith et al., 2011, 2015; Eguchi et al., 2013; Morishita et al., 2014). Prior work using a Rac1 GTP sensor suggested that levels of active Rac1 are greatly reduced in Sponge compromised embryos in the early embryo, and that Rac-GTP can be found in the transition zone between caps and ingressing furrows (Zhang, 2018). To examine if Rac1 had the potential to regulate ANRP recruitment to the apical cortex, Rac1 constructs were expressed that had altered binding preferences for GTP/GDP. Interestingly, each of the ANRPs tested (Scar, DPod1, Coronin, Cortactin) responded to altered Rac1 activity in a fashion that was strikingly similar to their behaviors to Sponge disruption. This would be consistent with Rac1-GTP serving as the bridging regulator between Sponge and the F-actin and Arp2/3 modifying ANRP proteins. In some instances, the changes in ANRP localization were not particularly robust, so it will be intriguing to examine if Rap1 plays a role in early syncytial furrowing in further experiments. Prior work has suggested the ELMO-Sponge complex regulates Rap1 activity necessary for Afadin/Canoe-based generation of a subapical domain at cycle 14, but whether earlier cycles are impacted is less clear. Lastly, Sponge is a very large protein (>2000 amino acids) and has several intriguing non-GEF domains including an SH3 domain, an Armadillo-like helical domain, and large regions of undefined natures whose function have been largely unexplored. These regions could be how Sponge is recruited for cortical actin function at cycles 10-14 or may provide a scaffolding function to coordinate higher order organization required for F-actin formation and structuring into cap and furrow supporting elements (Henry, 2022).

The Drosophila DOCK family protein Sponge is required for development of the air sac primordium

Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, this study examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway (Morishita, 2017).

Fine-tuning of the actin cytoskeleton and cell adhesion during Drosophila development by guanine nucleotide exchange factors Myoblast city and Sponge

The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to Engulfment and cell motility (ELMO) proteins, Dock-ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes. Both in vertebrate and invertebrate systems, the actin dynamics regulator Rac is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism to understand Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock-A) and Sponge (Spg; Dock-B). This study shows that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel (dv). Moreover, this study confirms the in vivo role of Mbc upstream of Rac and provides evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. These findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis (Biersmith, 2015).

Drosophila DOCK family protein sponge regulates the JNK pathway during thorax development

The dedicator of cytokinesis (DOCK) family proteins that are conserved in a wide variety of species are known as DOCK1-DOCK11 in mammals. The Sponge (Spg) is a Drosophila counterpart to the mammalian DOCK3. Specific knockdown of spg by pannir-GAL4 or apterous-GAL4 driver in wing discs induced split thorax phenotype in adults. Reduction of the Drosophila c-Jun N-terminal kinase (JNK), basket (bsk) gene dose enhanced the spg knockdown-induced phenotype. Conversely, overexpression of bsk suppressed the split thorax phenotype. Monitoring JNK activity in the wing imaginal discs by immunostaining with anti-phosphorylated JNK (anti-pJNK) antibody together with examination of lacZ expression in a puckered-lacZ enhancer trap line revealed the strong reduction of the JNK activity in the spg knockdown clones. This was further confirmed by Western immunoblot analysis of extracts from wing discs of spg knockdown fly with anti-pJNK antibody. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rac1 in the wing discs. Taken together, these results indicate Spg positively regulates JNK pathway that is required for thorax development and the regulation is mediated by interaction with Rac1 (Morishita, 2014).

The Drosophila DOCK family protein sponge is involved in differentiation of R7 photoreceptor cells

The Drosophila sponge (spg)/CG31048 gene belongs to the dedicator of cytokinesis (DOCK) family genes that are conserved in a wide variety of species. DOCK family members are known as DOCK1-DOCK11 in mammals. Although DOCK1 and DOCK2 involve neurite elongation and immunocyte differentiation, respectively, the functions of other DOCK family members are not fully understood. Spg is a Drosophila homolog of mammalian DOCK3 and DOCK4. Specific knockdown of spg by the GMR-GAL4 driver in eye imaginal discs induced abnormal eye morphology in adults. To mark the photoreceptor cells in eye imaginal discs, a set of enhancer trap strains was used that express lacZ in various sets of photoreceptor cells. Immunostaining with anti-Spg antibodies and anti-lacZ antibodies revealed that Spg is localized mainly in R7 photoreceptor cells. Knockdown of spg by the GMR-GAL4 driver reduced signals of R7 photoreceptor cells, suggesting involvement of Spg in R7 cell differentiation. Furthermore, immunostaining with anti-dpERK antibodies showed the level of activated ERK signal was reduced extensively by knockdown of spg in eye discs, and both the defects in eye morphology and dpERK signals were rescued by over-expression of the Drosophila raf gene, a component of the ERK signaling pathway. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rap1 in and around the plasma membrane of the eye disc cells. Together, these results indicate Spg positively regulates the ERK pathway that is required for R7 photoreceptor cell differentiation and the regulation is mediated by interaction with Rap1 during development of the compound eye (Eguchi, 2013).

The DOCK protein sponge binds to ELMO and functions in Drosophila embryonic CNS development

Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is essential to coordinate the development of tissues such as the musculature and nervous system during normal embryonic development. One class of signaling proteins that regulate actin cytoskeletal rearrangement is the evolutionarily conserved CDM (C. elegansCed-5, human DOCK180, DrosophilaMyoblast city, or Mbc) family of proteins, which function as unconventional guanine nucleotide exchange factors for the small GTPase Rac. This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We identified and characterized the role of Drosophila Sponge (Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein. Our analysis shows Spg mRNA and protein is expressed in the visceral musculature and developing nervous system, suggesting a role for Spg in later embryogenesis. As maternal null mutants of spg die early in development, we utilized genetic interaction analysis to uncover the role of Spg in central nervous system (CNS) development. Consistent with its role in ELMO-dependent pathways, we found genetic interactions with spg and elmo mutants exhibited aberrant axonal defects. In addition, our data suggests Ncad may be responsible for recruiting Spg to the membrane, possibly in CNS development. Our findings not only characterize the role of a new DOCK family member, but help to further understand the role of signaling downstream of N-cadherin in neuronal development (Biersmith, 2011)

Search PubMed for articles about Drosophila Sponge

Biersmith, B., Liu, Z. C., Bauman, K. and Geisbrecht, E. R. (2011). The DOCK protein sponge binds to ELMO and functions in Drosophila embryonic CNS development. PLoS One 6(1): e16120. PubMed ID: 21283588

Biersmith, B., Wang, Z. H. and Geisbrecht, E. R. (2015). Fine-tuning of the actin cytoskeleton and cell adhesion during Drosophila development by guanine nucleotide exchange factors Myoblast city and Sponge. Genetics 200(2):551-67. PubMed ID: 25908317

Eguchi, K., Yoshioka, Y., Yoshida, H., Morishita, K., Miyata, S., Hiai, H. and Yamaguchi, M. (2013). The Drosophila DOCK family protein sponge is involved in differentiation of R7 photoreceptor cells. Exp Cell Res 319(14): 2179-2195. PubMed ID: 23747680

Henry, S. M., Xie, Y., Rollins, K. R. and Blankenship, J. T. (2022). Sponge/DOCK-dependent regulation of F-actin networks directing cortical cap behaviors and syncytial furrow ingression. Dev Biol 491: 82-93. PubMed ID: 36067836

Morishita, K., Ozasa, F., Eguchi, K., Yoshioka, Y., Yoshida, H., Hiai, H. and Yamaguchi, M. (2014). Drosophila DOCK family protein sponge regulates the JNK pathway during thorax development. Cell Struct Funct 39(2): 113-124. PubMed ID: 25311449

Morishita, K., Anh Suong, D. N., Yoshida, H. and Yamaguchi, M. (2017). The Drosophila DOCK family protein Sponge is required for development of the air sac primordium. Exp Cell Res 354(2): 95-102. PubMed ID: 28341448

Xie, Y., Budhathoki, R. and Blankenship, J. T. (2021). Combinatorial deployment of F-actin regulators to build complex 3D actin structures in vivo. Elife 10. PubMed ID: 33949307

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