InteractiveFly: GeneBrief

steppke: Biological Overview | References

BIOLOGICAL OVERVIEW

Actin cytoskeletal networks push and pull the plasma membrane (PM) to control cell structure and behavior. Endocytosis also regulates the PM and can be promoted or inhibited by cytoskeletal networks. However, endocytic regulation of the general membrane cytoskeleton is undocumented. This study provides evidence for endocytic inhibition of actomyosin networks. Specifically, it was found that Steppke, a cytohesin Arf-guanine nucleotide exchange factor (GEF), controls initial PM furrow ingression during the syncytial nuclear divisions and cellularization of the Drosophila embryo. Acting at the tips of ingressing furrows, Steppke promotes local endocytic events through its Arf-GEF activity and in cooperation with the AP-2 clathrin adaptor complex. These Steppke activities appear to reduce local Rho1 protein levels and ultimately restrain actomyosin networks. Without Steppke, Rho1 pathways linked to actin polymerization and myosin activation abnormally expand the membrane cytoskeleton into taut sheets emanating perpendicularly from the furrow tips. These expansions lead to premature cellularization and abnormal expulsions of nuclei from the forming blastoderm. Finally, consistent with earlier reports, it was also found that actomyosin activity can act reciprocally to inhibit the endocytosis at furrow tips. It is proposed that Steppke-dependent endocytosis keeps the cytoskeleton in check as early PM furrows form. Specifically, a cytohesin Arf-GEF-Arf G protein-AP-2 endocytic axis appears to antagonize Rho1 cytoskeletal pathways to restrain the membrane cytoskeleton. However, as furrows lengthen during cellularization, the cytoskeleton gains strength, blocks the endocytic inhibition, and finally closes off the base of each cell to form the blastoderm (Lee, 2013).

Coupling actomyosin networks to the plasma membrane (PM) is essential for cells to migrate, interact, change shape, and divide. As examples, actin networks form and function at the leading edge of migratory cells, at cell-substrate adhesion complexes, and at cell-cell adhesion complexes in multicellular tissues. To assemble these complexes, receptors can physically engage the actin cytoskeleton and also induce cytoskeletal assembly via Rho- family guanosine triphosphate (GTP)ases and phosphoinositide signaling. Inversely, endocytosis can remove receptors from the PM promoting the turnover of adhesion and signaling complexes. More generally, the close links of both actin networks and endocytic machinery with the PM suggest possible crosstalk between these subsystems. Indeed, endocytic signaling nucleates local actin networks to help drive membrane invagination and scission (Mooren, 2012; Anitei, 2012). In contrast, more widespread membrane cytoskeleton activity can create tension that inhibits membrane invagination. Conceivably, endocytosis could also inhibit the membrane cytoskeleton, but such activity is undocumented (Lee, 2013).

The syncytial Drosophila embryo is a well-established model for studying actomyosin networks and membrane trafficking during PM furrow ingression. In the early syncytial embryo, nuclei divide synchronously just beneath the PM. At each division cycle, the activities of Rho-family GTPases, the Arp2/3 complex, and the formin Diaphanous (Dia) organize actomyosin-based PM ingressions (pseudocleavage furrows) that surround each nucleus to prevent nuclear collision and loss. Once ~6,000 nuclei form, similar mechanisms induce a final round of PM ingressions. These furrows persist and elongate through membrane trafficking to apical and lateral sites, and with support of actomyosin networks at their basal tips (the furrow canals). This massive PM growth cellularizes the first embryonic epithelium, a process completed with constriction of actomyosin rings formed at the base of each cell. Recently, endocytic events were detected at the tips of pseudocleavage furrows and early cellularization furrow canals by the presence of Amphiphysin (Amph)-positive tubules and the internalization of labeled PM (Sokac, 2008). These events have provided a model for studying how the actin cytoskeleton can both promote and inhibit endocytosis (Sokac, 2008; Yan, 2013). However, the role of this endocytosis is unclear, and paradoxically, it would appear to counteract membrane growth. This study examined how Arf G protein (Arf) activation might be involved. In other contexts, Arfs promote endocytosis by recruiting coat proteins, activating lipid signaling, and triggering actin polymerization. Like other G proteins, Arfs are activated by guanine nucleotide exchange factors (GEFs). Cytohesins are a major class of PM Arf-GEFs (Donaldson, 2011), and roles for cytohesin Arf-GEFs have been documented at migratory leading edges, focal adhesions, and adherens junctions in mammalian cell culture (Santy, 2005; Torii, 2010; Ikenouchi, 2010). Drosophila contains one cytohesin, called Steppke (Step). Step is known to function in postembryonic insulin and EGF signaling, which mammalian cytohesins do as well, but its contributions to the Drosophila embryo and to other cellular processes are unknown. This study shows that Step promotes endocytosis at pseudocleavage furrows and furrow canals to restrain actomyosin networks at these sites (Lee, 2013).

These data provide the first description of cytohesin function in a developing embryo. Drosophila Step promotes a subset of endocytic events at the tips of ingressing PM furrows during embryo cellularization. Endocytosis has been documented previously at these sites (Sokac, 2008), but its role has been unclear. By manipulating a conserved upstream activator of endocytosis, this study has identified an important role of endocytosis in controlling the membrane cytoskeleton. The data argue that Step acts at furrow tips to induce local Arf-dependent endocytosis, which in turn antagonizes Rho1-dependent actomyosin network assembly at these sites. It was also found that the cytoskeleton can inhibit endocytosis at the furrow tips, as has been previously shown in this system (Sokac, 2008; Yan, 2013) and in other contexts. An overall model is proposed in which this reciprocal relationship is one-sided at specific developmental stages. At newly forming PM furrows, Step dominates, promoting endocytosis that keeps cytoskeleton activity in check for proper pseudocleavage and cellularization furrow architecture and growth. During later cellularization, the cytoskeleton dominates. Zygotic expression of actin regulators such as Nullo normally increases actomyosin activity as cellularization proceeds and appears to work in conjunction with Dia to block endocytic events at the furrow tips (Sokac, 2008; Yan, 2013)]. By counteracting the inhibitory endocytosis, cytoskeletal activity would elevate further but at these later stages is locally restrained by a distinct mechanism requiring Bottleneck (Schejter, 1993). To form the blastoderm, this second restraint mechanism is removed, and contractile rings close off the base of each cell. In the absence of the initial step-mediated restraint mechanism, it is proposed that the cytoskeleton abnormally dominates the relationship at all early PM furrows. Without Step-based endocytic inhibition, it is speculated that actomyosin networks abnormally expand and inhibit other endocytic events leading to coexpansion of cytoskeletal polymers and PM from the furrow tips (Lee, 2013).

An important element of the model is the local induction of endocytic events. The data localize Step to the tips of ingressing PM furrows, and both the loss and overexpression of Step alter membrane organization specifically at these sites. This localized Step-regulated activity occurs in a dynamic global membrane trafficking system within each forming cell. During the peripheral nuclear divisions, each nucleus acquires its own endoplasmic reticulum and Golgi apparatus that function with recycling endosomes to direct exocytosis to growing PM furrows at cellularization. Simultaneously, endocytic events occur over the apical PM and at the furrow tips, with endocytosed material recycled to the growing furrows. Thus, the overall membrane system is in continual flux, and coordination by local regulation would be expected. The data identify a polarized endocytic activator required for the process. Step Arf-GEF activity is critical for restraining the membrane cytoskeleton at furrow tips, and a subset of AP-2 activities is involved as well (Lee, 2013).

How could endocytosis and actomyosin networks impact each other at the tips of PM furrows or elsewhere? This question can be considered from several levels of organization. First, a simple and direct connection could be endocytic removal of one or more PM actomyosin regulators. This work identifies Rho1 or an upstream regulator as a candidate. Intriguingly, membrane trafficking has been previously linked to the Rho1 pathway in this context. Specifically, recycling endosomes have been implicated in the trafficking of RhoGEF2 to the PM (Cao, 2008). It was hypothesized that RhoGEF2 might also be a target of Step for its removal from the PM but no difference was found in RhoGEF2 levels at furrow canals in step loss-of-function embryos. Thus, Rho1 may be a more specific target of Step, although a direct connection to the Rho1 pathway remains to be determined. Of note, a number of septins have also recently been observed on the Amph-positive tubules (Lee, 2013).

Second, interplay between different pools of actin is possible. For example, actin contributes to the invagination and scission of endocytic vesicles, and thus, endocytic actin and other PM actin networks could compete for regulators or components. Additionally, there could be signaling crosstalk between regulators of the different networks. For example, Arf signaling often elicits local Rac or Cdc42 activity, and this might trigger crosstalk affecting Rho activity. Interestingly, overexpression of Cdc42-interacting protein 4 (Cip4) appears to antagonize Dia at furrow canals, although Cip4 mutants have no cellularization phenotype on their own. Significantly, however, this study found that Step acts with AP-2 to control the membrane cytoskeleton. This Step-AP-2 cooperation suggests that clathrin-coated pits are involved in the antagonism, although it does not exclude the possibility of separate cytoskeletal crosstalk (Lee, 2013).

Third, larger scale interactions should be considered. Endocytosis could remove membrane in bulk that would otherwise support the membrane cytoskeleton, although observations of residual furrow canal endocytic activity with step loss of function suggest a more specific mechanism. Inversely, the membrane cytoskeleton could block endocytosis by elevating PM tensio or possibly by sterically blocking endocytic machinery from accessing the PM (Lee, 2013).

Endocytic-cytoskeletal crosstalk is relevant to many cellular processes. For example, receptor endocytosis occurs in proximity to actomyosin networks in various contexts, including migratory leading edges, focal adhesions, and adherens junctions. However, these endocytic events and actomyosin networks have mainly been studied independently, and thus their functional integration is not understood. This study highlights the possibility that endocytic activity at such assemblies could simultaneously remove receptors and antagonize local cytoskeletal networks, with both effects promoting complex turnover and cellular dynamics (Lee, 2013).

The Drosophila Arf GEF Steppke controls MAPK activation in EGFR signaling

Guanine nucleotide exchange factors (GEFs) of the cytohesin protein family are regulators of GDP/GTP exchange for members of the ADP ribosylation factor (Arf) of small GTPases. They have been identified as modulators of various receptor tyrosine kinase signaling pathways including the insulin, the vascular epidermal growth factor (VEGF) and the epidermal growth factor (EGF) pathways. These pathways control many cellular functions, including cell proliferation and differentiation, and their misregulation is often associated with cancerogenesis. In vivo studies on cytohesins using genetic loss of function alleles are lacking, however, since knockout mouse models are not available yet. Recently studies have identified mutants for the single cytohesin Steppke (Step) in Drosophila, and an essential role of Step in the insulin signaling cascade has been demonstrated. The present study provides in vivo evidence for a role of Step in EGFR signaling during wing and eye development. By analyzing step mutants, transgenic RNA interference (RNAi) and overexpression lines for tissue specific as well as clonal analysis, it was found that Step acts downstream of the EGFR and is required for the activation of mitogen-activated protein kinase (MAPK) and the induction of EGFR target genes. It was further demonstrated that step transcription is induced by EGFR signaling whereas it is negatively regulated by insulin signaling. Furthermore, genetic studies and biochemical analysis show that Step interacts with the Connector Enhancer of KSR (CNK). It is proposed that Step may be part of a larger signaling scaffold coordinating receptor tyrosine kinase-dependent MAPK activation (Hahn, 2013).

The proper development of multicellular organisms requires the coordination of proliferation and differentiation, which is a particular challenge during the formation of the tissues and organs of the body. Numerous studies have shown that receptor tyrosine kinases such as the vascular growth factor receptor (VEGFR) epidermal growth factor receptor (EGFR) and insulin/insulin-like growth factor receptors (InR/IGF-Rs) play prominent roles in signaling cell proliferation and differentiation. Misregulation of both pathways is often causative for tumor development and progression through their effects on uncontrolled cell growth, inhibition of apoptosis, angiogenesis, and tumor-associated inflammation. Determining how growth and differentiation are coordinated by these pathways is thus essential to understanding normal development, as well as disease states such as cancer (Hahn, 2013).

Steppke (Step) has been identified as a new and essential component of the insulin signaling pathway in Drosophila (Fuss, 2006). The insulin signaling cascade is conserved from flies to humans and was shown to regulate cell and organismal growth in response to extrinsic signals such as growth factors and nutrient availability. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes the Phosphatidylinositol-3-kinase (PI3K) and Protein Kinase B (PKB or AKT). AKT is involved in enhancement of glucose absorption and glycogen synthesis, and regulates the activity of the Forkhead box O (FoxO) transcription factor, a negative regulator of cell growth. Step acts downstream of the insulin receptor and upstream of PI3K in the insulin/IGF-like signaling (IIS) cascade (Fuss, 2006). Step is a member of the cytohesin family of guanine nucleotide exchange factors (GEFs) which regulate small GTPases of the ADP-ribosylation factor (ARF) family (Kolanus, 2007). Small ARF GTPases are involved in the regulation of many cellular processes including vesicle transport, cell adhesion and migration. Studies in mice have confirmed an evolutionary conserved role of cytohesin family members in IIS (Hafner, 2006; Hahn, 2013 and references therein).

Whereas previous studies focused on the role of Step in IIS-dependent larval growth control, this study examined its function in the Drosophila wing, which develops from an epithelial sheet during larval and pupal stages. The wing is an ectodermal structure formed by a dorsal and ventral epithelium, interspersed with cuticular ectodermal tubes, the so called wing veins. Stereotypical arrangement of wing veins is determined in the imaginal wing disc in late larval and pupal stages by several signaling pathways including the EGFR cascade. EGFR activation by EGF-like ligands Spitz or Vein results in the activation of the small GTPase RAS by its loading with guanosine triphosphate (GTP), which as a result triggers the activation of a number of downstream effector proteins including the Ser/Thr-kinase RAF [mitogen-activated protein kinase (MAPK) kinase kinase]. Once activated, RAF phosphorylates and activates MEK (MAPK kinase), which in turn phosphorylates and activates MAPK/ERK. Phosphorylated MAPK exerts its role in the cytoplasm as well as in the nucleus, where it controls expression of EGFR target genes like pointed (pnt), argos (aos), rhomboid (rho) and ventral nervous system defective (vnd). The scaffolding protein Connector enhancer of KSR (CNK) has been described to facilitate RAS/RAF/MAPK signaling by providing a protein scaffold at the plasma membrane that integrates Src and RAS activities to enhance RAF and MAPK activation (Claperon, 2007). EGFR/MAPK signaling is crucial quite early during wing vein differentiation, where phosphorylation of MAPK determines the positioning of proveins and later during development for maintenance of longitudinal veins. In addition to patterning, both EGFR/RAS/MAPK signaling and IIS control general cell proliferation and cell growth during wing development. Thus, EGFR/RAS/MAPK signaling controls both cell fate (vein versus intervein) and general cell proliferation along with IIS at similar times within the wing tissue (Hahn, 2013).

Recent studies in human lung and breast adenosarcoma cancer cell lines indicated a function of cytohesins in ErbB (EGFR) signaling, where they facilitate signaling by stabilizing an asymmetric ErbB receptor dimer (Bill, 2010). This study provides the first in vivo model that the cytohesin Step, in addition to its previously characterized function as component of IIS, regulates EGFR signaling dependent wing growth and vein differentiation. Genetic, immunohistochemical and biochemical experiments indicate that Step acts downstream of the EGF receptor in the EGFR signaling cascade and is necessary and sufficient for MAPK activation and the induction of EGFR target genes. Whereas step transcription is negatively regulated by IIS (Fuss, 2006), it is induced by EGFR signaling. Evidences are further provided that Step might directly interact with the Connector Enhancer of KSR (CNK) protein that is part of a protein scaffold known to coordinate RAS-dependent RAF and MAPK signaling from tyrosine kinase receptors (Hahn, 2013).

This study demonstrates an in vivo function of the Arf GEF Step as an essential component of the EGFR signaling pathway which acts downstream of the EGFR. Step is necessary and sufficient for activation of MAPK and the induction of EGFR target genes in the Drosophila wing. Based on biochemical, immunohistochemical and the genetic data a mechanistic model is proposed in which Step and dCNK interaction is important for EGFR signaling. dCNK is the single member of the CNK protein family in Drosophila. CNK proteins are scaffolding proteins that have been linked with RAS, Rho, Rac, Ral and Arf GTPases and are proposed to act as general regulators of GTPase-mediated events downstream of receptor tyrosine kinases, including EGFR and InR/insulin-like growth factor receptors (Claperon, 2007). Together with the kinase suppressor of RAS (KSR), CNK was shown to assemble a signaling complex including RAF and MEK which promotes RAS-dependent RAF activation and the subsequent phosphorylation of MAPK. It is suggested that Step is a functional part of this scaffolding complex via its direct interaction with CNK. This is also consistent with recent data in HeLa and 393T cells showing that human CNK1 directly interacts with cytohesin-2 to coordinate PI3K/AKT signaling downstream of InR/IGF-R (Lim, 2010). It was proposed that CNK1 recruits cytohesin-2 to the plasma membrane, where activity of plasma membrane bound GTPases leads to a PIP2 rich microenvironment, which enhances IRS1 recruitment and hence facilitates PI3K/AKT signaling (Lim, 2010). Similarly, Drosophila cytohesin Step was shown to be required for PI3K activation (Fuss, 2006). Together, several lines of evidence support a role of cytohesins and CNK in similar signaling contexts (RAS/RAF/MAPK and PI3K/AKT signaling), where a direct interaction of both proteins as part of a signaling platform might promote downstream signaling events like MAPK phosphorylation and PI3K activation. This does not exclude other functions of cytohesins, e.g. the stabilization of asymmetric ErbB (EGFR) dimers, as shown recently in human lung and breast adenosarcoma cancer cell lines (Bill, 2010). The data indicate, however, that a major function of the Drosophila cytohesin Step in EGFR signaling resides downstream of the EGFR and upstream of MAPK (Hahn, 2013).

Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo

Cytohesin Arf-GEFs are conserved plasma membrane regulators. The sole Drosophila cytohesin, Steppke, restrains Rho1-dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the. By mass spectrometry, a single major Steppke-interacting protein from syncytial embryos was identified that was named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homolog of the mammalian cytohesin adaptor FRMD4A. Experiments supported this relationship. Specifically, heterophilic coiled-coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C-terminal region was required for Sstn localization to furrows. Sstn mutant and RNAi embryos displayed abnormal, Rho1-dependent membrane cytoskeleton expansion from the base of pseudocleavage and cellularization furrows, closely mimicking Steppke loss-of-function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting Steppke acts downstream of Sstn, and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled-coil domain of Steppke was required for Sstn binding but additionally homo-dimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall, it is proposed that Sstn acts as a cytohesin adaptor that promotes Steppke activity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo (Liu, 2014).

Small G proteins are binary switches that control a wide range of cellular processes. Conversion to their active GTP-bound state is regulated in space and time by guanine nucleotide exchange factors (GEFs). A major question is how the GEFs are controlled. GEFs are typically multi-domain proteins and their recruitment by scaffold/adaptor proteins provides one regulatory mechanism. This mechanism is exemplified by Son-of-sevenless (Sos), a Ras-GEF that is recruited to receptor tyrosine kinases by the SH2-SH3 protein Grb2 (Liu, 2014).

Another example is that of G-protein coupled receptors, which, through the same polypeptide chain, connect reception of a ligand with GEF activity. Other than these paradigms, however, there is only limited knowledge of scaffold/adaptor proteins that promote GEF activity (Liu, 2014).

Different small G protein families are regulated by structurally distinct families of GEFs. Previous work focused on Steppke (Step), the sole cytohesin family member in Drosophila. Cytohesins are composed of multiple domains (a coiled-coil (CC) domain, a Sec7 GEF domain, a PH domain and a poly-basic region) and activate plasma membrane Arf small G proteins. Plasma membrane Arf small G proteins are major inducers of endocytosis, lipid signaling and actin remodeling affecting a range of membrane complexes (Liu, 2014).

Thus far, several scaffold/adaptor proteins have been identified to link cytohesins to specific complexes. Connector Enhancer of KSR 1 (CNK1) binds cytohesins through their CC domain and recruits them to the plasma membrane in response to insulin signaling (Liu, 2014).

Myeloid-differentiation factor 88 (MYD-88) forms a complex with cytohesins for interleukin-1 β signaling. Tamalin/GRP1-associated scaffold protein (GRASP) can recruit cytohesins to the plasma membrane and mediates interactions with group 1 metabotropic glutamate receptors at synapses. Paxillin binds to cytohesins via their polybasic region and links them to focal adhesions at the leading edge of migratory cells. FERM domain containing 4A (FRMD4A) and GRP1-binding partner (GRSP-1) each bind to cytohesins through CC domain interactions and link them to Par-3 at adherens junctions (Liu, 2014).

These mammalian cell culture studies suggest that adaptor proteins couple cytohesins to specific complexes. To test the generality and evolution of cytohesin adaptors, it is important to examine them in other animal models. For example, in Drosophila, Step regulates both insulin and epidermal growth factor signaling. Significantly, Step can bind Drosophila CNK and has been shown to interact genetically with the adaptor during epidermal growth factor-dependent patterning of the wing. Thus, CNK may be commonly used to link cytohesins with receptor tyrosine kinase signaling pathways (Liu, 2014).

Additionally, Step functions to control plasma membrane growth in the early embryo. The early Drosophila embryo is a syncytium in which plasma membrane furrows transiently separate dividing peripheral nuclei and then cellularize ~6000 nuclei to form the cellular blastoderm. In this model of cell division, furrows normally extend straight down from the embryo surface plasma membrane and form a matrix of lateral membranes to separate nuclei. Without Step activity, the furrows extend into the embryo but then abnormally expand perpendicularly at their basal tips. Normally, these basal tips are maintained by actomyosin networks organized by Rho1 pathways. Without Step, these networks become over-active and drive the abnormal membrane expansion. As a result, basal cell membranes form prematurely and physically expel nuclei from the forming blastoderm (Liu, 2014).

Normally, Step localizes at the basal tips of the furrows and uses its Arf-GEF activity to keep the membrane cytoskeleton in check. It is hypothesized that a specific cytohesin adaptor might aid Step for the restraint of the membrane cytoskeleton in the syncytial embryo. Of the known cytohesin adaptors, Myd88, CNK and Paxillin have annotated Drosophila homologs that are expressed in the syncytial embryo; Tamalin/GRASP has no annotated homolog and the most similar Drosophila protein from BLAST searches (Short spindle 6) in not expressed in the syncytial embryo; and FRMD4A and GRSP-1 have no significant sequence similarities with Drosophila proteins with the exception of their FERM domains that most closely resemble the FERM domain of Moesin. With these candidates in mind, this study took a non-biased approach to identify Step complex components of the syncytial embryo by liquid chromatography mass spectrometry (LC-MS). These analyses identified one major interacting protein that was named Stepping stone (Sstn). Despite substantial sequence divergence, Sstn appears to be a structural and functional homolog of FRMD4A, and aids Step in the restraint of the membrane cytoskeleton (Liu, 2014).

The data argue that an adaptor-Arf-GEF pair first identified in mouse cell culture is conserved and functional in Drosophila. Although the sequence similarity between mammalian FRMD4A and Drosophila Sstn is low, their relationship is supported by a range of data: (1) They share the same unique homologs in other species, (2) They lack similarity with any other proteins in mammals or Drosophila, (3) With the exception of the FERM domain, they share a common domain organization, (4) They both interact directly with cytohesins through CC domain hetero-dimerization, (5) They both colocalize with cytohesins in cells, and (6) They both promote cytohesin-dependent cellular processes (Liu, 2014).

In the early Drosophila embryo, the data suggests that Sstn supports the activity of the cytohesin homolog Step for the local control of plasma membrane growth. Specifically, Sstn localizes at the base of both pseudocleavage and cellularization furrows where it appears to engage Step through direct interactions to keep the membrane cytoskeleton in check. Without Sstn, the basal tips of the furrows expand perpendicularly in a Rho1-dependent process that leads to abnormal plasma membrane encroachment into space normally occupied by nuclei of the forming cells (Liu, 2014).

This misregulation is strikingly similar to that of step loss-of-function embryos. Moreover, it could be overcome by Step overexpression, suggesting that Sstn normally acts by enhancing the activity of a limited supply of endogenous Step. Thus, a model is proposed in which a Sstn-Step-Arf small G protein axis acts at the base of furrows to control their growth. Within this axis, Sstn and Step interact directly through their CC domains, but each also has independent interactions with other membrane components, mediated by the CR in the case of Sstn. The data suggest that the Sstn-Step interaction may stabilize Step furrow localization, where Step has been shown to use its Arf-GEF activity to control membrane growth (Lee, 2013; Liu, 2014 and references therein).

It is proposed that Sstn acts as an adaptor to link Step to specific targets for the local endocytic regulation of furrow tips. Sstn structure-function analysis shows that its C-terminal CR is critical for recruiting it to the plasma membrane, whereas its N-terminal CC domain is responsible for interacting with Step. It is proposed that the CR interacts with targets linked to the membrane cytoskeleton, and that Sstn recruits Step for Arf small G protein activation, the induction of local endocytosis, and downstream antagonism of the membrane cytoskeleton. However, mass spectrometry experiments did not identify any cytoskeletal proteins as partners of Sstn (Liu, 2014).

Thus, in this model of Sstn as a cytohesin adaptor, it is unknown what targets Sstn bridges to Step. It is speculated that the failure to identify these proteins may be related to their transient associations with Sstn as part of the proposed endocytosis pathway. For mouse FRMD4A, an interaction with cytohesins at its CC domain is coupled to an interaction with Par-3 at its C-terminal region to link cyt ohesin signaling to epithelial cell-cell junctions (Ikenouchi, 2010), but Drosophila Par-3 (Bazooka) has no apparent effect on the basal tips of furrows in the early Drosophila embryo (Liu, 2014).

In addition to Sstn, the data suggest that other positive regulators of Step localization and function are active in the early embryo. Specifically, despite high-level expression of three distinct shRNA constructs that reduced Sstn-GFP levels to below background levels, over-expressed Step-GFP could still localize to the membrane, and the severity of membrane expansion with the loss of Sstn was never as strong at that with Step loss. A number of known cytohesin interactions can be considered as additional contributors to Step activities. First, the PH domains of cytohesins, and of Step, bind and respond to phosphoinostides. However, manipulations of PIP2 and PIP3 levels have no apparent effect on Drosophila pseudocleavage or early cellularization furrows, despite a strong effect on later cellularization furrows (Reversi, 2014). Thus, Step-PIP3 interactions may be dispensable or non-existent at the furrow tips where Step acts. Second, the PH domains of cytohesins also bind and respond to GTP-bound Arf-like 4 (Arl4) and Arf small G proteins forming recruitment pathways and positive feedback loops, but these have not yet been examined for Step. Third, the Step CC domain mediates direct interactions with the adaptor CNK (Hahn, 2013). CNK has not been examined in the early embryo, but it is expressed (FlyBase). Although it was not detected with Step in an IP LC-MS analyses, it is possible that Step-CNK interactions might occur with the loss of Sstn (Liu, 2014).

Fourth, the cytohesin CC domain can also mediate cytohesin homo-dimerization, an interaction that was detected for Step. Thus, a lack of Step homo-dimerization with loss of its CC domain might explain why the Step CC domain deletion had greater effects on Step localization and function compared to Sstn loss. With homo-dimerization capability, endogenous Step may be able to use additional activation mechanisms to elicit partial effects with Sstn loss, whereas with over-expression Step can achieve greater localization and activity to make Sstn unnecessary. Although over-expressed Step can overcome the need for Sstn, it does have negative consequences (sporadic furrow loss). Thus, Sstn may be needed to aid Step when it is expressed at lower, optimal, normal levels. A recent study has shown that FRMD4A is up-regulated in human squamous cell carcinoma and that it contributes to tumour growth and metastasis (Goldie, 2012). Similarly, elevated cytohesin and Arf small G protein activities have also been shown to promote cancer progression (Liu, 2014).

Thus, elucidation of the roles and regulation of the Sstn (FRMD4A)-Step (cytohesin)-Arf small G protein axis during normal development should increase understanding of such disease states. Specifically, improper pathway activity may lead to mis-regulation of the membrane cytoskeleton in other contexts, with potential downstream consequences for cell division, cell-cell adhesion or cell migration (Liu, 2014).

An Actomyosin-Arf-GEF negative feedback loop for tissue elongation under stress

In response to a pulling force, a material can elongate, hold fast, or fracture. During animal development, multi-cellular contraction of one region often stretches neighboring tissue. Such local contraction occurs by induced actomyosin activity, but molecular mechanisms are unknown for regulating the physical properties of connected tissue for elongation under stress. This study shows that cytohesins, and their Arf small G protein guanine nucleotide exchange activity, are required for tissues to elongate under stress during both Drosophila dorsal closure (DC) and zebrafish epiboly. In Drosophila, protein localization, laser ablation, and genetic interaction studies indicate that the cytohesin Steppke reduces tissue tension by inhibiting actomyosin activity at adherens junctions. Without Steppke, embryogenesis fails, with epidermal distortions and tears resulting from myosin misregulation. Remarkably, actomyosin network assembly is necessary and sufficient for local Steppke accumulation, where live imaging shows Steppke recruitment within minutes. This rapid negative feedback loop provides a molecular mechanism for attenuating the main tension generator of animal tissues. Such attenuation relaxes tissues and allows orderly elongation under stress (West, 2017).

The results show that Step antagonizes actomyosin networks and that these networks recruit Step, forming a negative feedback loop. This negative feedback loop seems to act in close proximity to AJs, and actomyosin accumulations can recruit Step within minutes. It is proposed that continual action of this actomyosin-Step negative feedback loop relaxes cell-cell junctions, prevents tissue distortions, and allows orderly tissue elongation under stress (West, 2017).

A central consideration for tissue stretching is epithelial viscoelasticity, a physical property recently demonstrated in vivo for the Drosophila lateral ectoderm during gastrulation and for the Drosophila amnioserosa during DC. Consistent with the lateral epidermis retaining viscoelasticity at DC, cell and tissue elongation reverses when the leading edge of the tissue is torn or ablated. As a viscoelastic material, the response of the lateral epidermis to deforming stress can be viewed as a combination of elastic springs and viscous dashpots. Genetic, imaging, and ablation data suggest that the actomyosin-Step negative feedback loop relaxes the elastic spring component of the standard linear model of viscoelasticity that applies to the Drosophila epidermis. First, the distortions of the step mutant epidermis are accompanied by junctional accumulation of actomyosin networks, the major contributors to cell and tissue contractility and elasticity. Second, the distortions are reduced by decreasing myosin levels. Third, sites of actomyosin activity recruit Step within minutes, providing a mechanism for local junctional relaxation in a timescale relevant to the tissue elongation. Fourth, the response of step mutant tissue to laser ablation-abnormally fast instantaneous recoil followed by an unaltered contraction rate-is most easily explained by local increases to contractile force, rather than a general change to viscosity. Thus, the actomyosin-Step negative feedback loop seems to allow the spring component of a viscoelastic epithelium to relax itself, thereby facilitating the elongation of the material under stress. Exactly how actomyosin recruits Step remains to be determined, but the mechanism does not rely solely on AJs as Step also accumulates at cytokinetic rings. Step localization to cleavage furrows of the early embryo involves coiled-coil domain interaction with the adaptor protein Stepping stone and pleckstrin homology PH domain interaction with Arf small G proteins. At DC, the PH domain of Step is recruited to the epidermal leading edge by phosphatidylinositol-3,4,5-P3. One or more of these mechanisms might link Step to actomyosin (West, 2017).

The data highlight a role for an actomyosin-Step negative feedback loop in tissue relaxation and elongation, but what prevents such actomyosin inhibition when tissue contractility is needed? For example, Step inhibits actomyosin activity at cleavage furrows of the syncytial embryo, but, at the end of cellularization, full cytokinesis is required to form individual cells of the blastoderm. Following cellularization, the ventral and lateral ectoderm become highly contractile to drive ventral furrow formation and convergent extension of the long body axis. Notably, endogenous Step protein seems to localize to the supracellular actomyosin cables that pull cells into multi-cellular rosettes for axis elongation. Similarly, Step localizes to cytokinetic rings that fully constrict. In such contexts, actomyosin activity might surpass a threshold, making it resistant to more limited Step signaling, as evident during the division of primordial germ cells from the syncytial embryo. Alternatively, Step or downstream Arf small G protein signaling might be inhibited post-translationally (West, 2017).

Step downregulates actomyosin for syncytial cleavage of the early Drosophila embryo, the C. elegans cytohesin GRP-1 localizes to cytokinetic rings and regulates asymmetric cell division, and the human cytohesin ARNO locally downregulates actomyosin networks to allow podosome formation. This study shows that cytohesin Arf-GEF activity also promotes tissue relaxation and elongation in both Drosophila and zebrafish embryos. Thus, cytohesins seem to regulate a variety of actomyosin-based processes from invertebrates to vertebrates. In some contexts the regulatory mechanism tempers stress generation, and in others it facilitates the stretching of cells and tissues (West, 2017).

The cytohesin Steppke is essential for insulin signalling in Drosophila

In metazoans, the insulin signalling pathway has a key function in regulating energy metabolism and organismal growth. Its activation stimulates a highly conserved downstream kinase cascade that includes phosphatidylinositol-3-OH kinase (PI(3)K) and the serine-threonine protein kinase Akt. This study identifies a new component of insulin signalling in Drosophila, the steppke gene (step). step encodes a member of the cytohesin family of guanine nucleotide exchange factors (GEFs), which have been characterized as activators for ADP-ribosylation factor (ARF) GTPases. In step mutant animals both cell size and cell number are reduced, resulting in decreased body size and body weight in larvae, pupae and adults. step acts upstream of PI(3)K and is required for the proper regulation of Akt and the transcription factor FOXO. Temporally controlled interference with the GEF activity of the Step protein by feeding the chemical inhibitor SecinH3 causes a block of insulin signalling and a phenocopy of the step mutant growth defect. Step represses its own expression and the synthesis of growth inhibitors such as the translational repressor 4E-BP. These findings indicate a crucial role of an ARF-GEF in insulin signalling that has implications for understanding insulin-related disorders, such as diabetes and obesity (Fuss, 2006).

All animals coordinate growth to reach their final size and shape. The insulin–insulin-like growth factor signalling pathway, which is genetically conserved from flies to humans, has been identified as a key regulator of cell growth in response to extrinsic signals such as growth factors and nutrient availability. In mammals, loss of the ability to respond to insulin, a phenomenon known as insulin resistance, is associated with pathological manifestations such as type 2 diabetes. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes PI(3)K and Akt. This signalling cascade controls organismal growth directly by regulating cell size and cell number (Fuss, 2006).

In a search for genes controlling larval growth in Drosophila, a genetic locus was identified that was named steppke (step). Molecular analysis and genetic rescue experiments show that the lethality of the P element alleles is linked to the step gene function. The step gene encodes a protein that belongs to the highly conserved cytohesin protein family of GEFs that consists of four family members in humans and one family member in invertebrates such as the nematode, mosquito and fly. GEFs mediate the exchange of GDP for GTP on the ARFs, which belong to the Ras superfamily of small GTPases. Like other Ras-related GTP-binding proteins, the ARF proteins cycle between their active GTP-bound and inactive GDP-bound conformations. In concert with ARFs, cytohesin proteins regulate vesicle trafficking, cell adhesion, migration and structural organization at the cell surface (Fuss, 2006).

Cytohesin proteins contain two characteristic motifs: a Sec7 domain responsible for the GEF activity, and a pleckstrin homology domain (PH) required for plasma membrane recruitment as a result of specific binding to phosphatidylinositol-3,4,5-trisphosphate, the second messenger generated by class I PI(3)Ks. The Sec7 and PH domains of Step are highly conserved compared with the corresponding protein domains of mammalian cytohesins (Fuss, 2006).

Phenotypic analysis of homozygous step^k08110 and step^SH0323 mutants and transheterozygous allelic combinations indicate an essential role of step in regulating growth and body size at all stages of the Drosophila life cycle. Both males and females of step^k08110/step^SH0323 transheterozygous adults are significantly smaller than control animals; however, the body proportions of these animals are not changed. Consistently, larval and pupal development are also slowed down in step mutants and body size is reduced. The observed growth defects mimic a starvation phenotype that is not caused by a failure of food intake, as verified by feeding coloured yeast and by the analysis of a metabolic marker gene (Fuss, 2006).

It is known that larval growth is largely based on an increase in cell size in all terminally differentiated tissues that is accomplished by endoreplication, a modified cell cycle, consisting of successive rounds of DNA synthesis without intervening mitoses. To examine the cause for the growth defects of step mutant larvae, cell cycle activity was investigated in the midgut and the salivary glands, which are representative endoreplicating tissues in the larval stage. A general decrease in endoreplication activity was found, indicated by a slowing down of the S phase of the cell cycle. Both the size and the total number of salivary gland cells are decreased, resulting in a smaller organ (Fuss, 2006).

Because embryonic lethality was observed in a small proportion of the homozygous step^k08110 mutants, it was important to exclude the possibility that the growth defects observed in the mutant larvae derive from a defect laid down during embryogenesis. For this purpose an assay was established to analyse step function exclusively in the larval stage, in which the growth rate is maximal. Use was made of the small molecule SecinH3, which was recently identified as an inhibitor of the Drosophila Step protein and the vertebrate cytohesin family members (Hafner, 2006). SecinH3 binds to the Sec7 domain of Step, thereby inhibiting the guanine nucleotide exchange of interacting ARF proteins (Hafner, 2006). Feeding SecinH3 to wild-type larvae induced a phenocopy of the growth defects observed in step mutants and led to a marked decrease in body size. It is concluded from the phenotypic analysis of step mutants and from the experiments inhibiting Step protein function directly by using the chemical inhibitor SecinH3 that Step is essential for organismal growth of Drosophila larvae, pupae and adults (Fuss, 2006).

In step mutants, organismal growth is strongly reduced and development is delayed, which is also a hallmark of mutants affecting the insulin signalling pathway. To investigate whether step has a function in insulin signalling, the expression of two known target genes of the pathway was analysed in step mutants, namely 4E-BP, encoding a translational repressor, and InR, encoding the insulin receptor, by using quantitative reverse-transcriptase-mediated polymerase chain reaction (RT–PCR); both 4E-BP and InR transcription are upregulated in response to repressed insulin signalling. Lipase3 (Lip3) expression was used as a starvation marker in these experiments. In step mutant larvae and also in wild-type larvae treated with the Step inhibitor SecinH3, 4E-BP and InR transcription is activated, whereas Lip3 expression is unaffected. This indicates that the growth phenotype observed in step mutant larvae is not caused by a complete block of nutrition but is associated with a specific downregulation of insulin signalling activity. Similarly, interfering with Step function by feeding SecinH3 to transheterozygous step mutant flies or applying SecinH3 in S2 tissue culture cells also results in an activation of 4E-BP and InR transcription (Fuss, 2006).

It has been shown previously that 4E-BP and InR are target genes of the transcription factor FOXO (forkhead box, sub-group ‘O’). In Drosophila cells, insulin receptor signalling results in a high activity of PI(3)K and phosphorylation of Akt. Akt phosphorylates FOXO and causes cytoplasmic retention of FOXO, whereas low activities of PI(3)K and Akt allow FOXO to enter the nucleus, where it promotes the expression of factors such as 4E-BP that retard cell growth and proliferation. In step mutant larvae or in S2 tissue culture cells in which Step protein function is inhibited with SecinH3, a nuclear localization of FOXO was found, indicating that step is required for insulin-signalling-dependent cytoplasmic localization of FOXO. Because this is regulated by phosphorylation by means of Akt, whether step is necessary for Akt phosphorylation was tested, and it was found that under conditions in which the step function is affected, the amount of phosphorylated Akt protein is significantly decreased (Fuss, 2006).

It has been shown that activation of Akt during growth in Drosophila is regulated by the class I PI(3)K Dp110. Overexpression of Dp110-CAAX, a constitutively active form of PI(3)K, in wing or eye imaginal discs enhances cellular growth, resulting in enlarged cells and organs, whereas mutations in Dp110 are lethal and result in a larval growth arrest in the third instar. It has been shown previously that Dp110 interacts with key components of the insulin signalling pathway including Chico, PTEN and Akt to control insulin-signalling-dependent cell and organ growth in Drosophila. To test whether step acts together with PI(3)K in a common pathway involved in Akt and FOXO regulation and, if so, to address whether step is genetically upstream or downstream of PI(3)K in the insulin pathway, Dp110-CAAX was expressed in heterozygous and transheterozygous step mutant animals (Fuss, 2006).

step mutant adults are greatly decreased in size and weight in comparison with wild-type animals. In control flies in which Dp110-CAAX has been overexpressed, body size and weight are greatly increased in comparison with wild-type flies. If step were positioned downstream of PI(3)K, the oversize phenotype induced by the expression of Dp110-CAAX should be suppressed or at least strongly reduced, whereas if step were positioned upstream of PI(3)K, Dp110-CAAX expression would rescue the growth phenotype of step mutants. The latter was found, providing in vivo evidence that the cytohesin family member step is upstream of PI(3)K (Fuss, 2006).

Tight regulation of insulin signalling activity has been shown to be crucial for cell and organ growth in Drosophila and for numerous growth-related and homeostasis-related diseases such as cancer and type 2 diabetes in humans. It is known from recent studies in Drosophila that InR represses its own synthesis by a feedback mechanism directed by the transcription factor FOXO. To test whether step is also part of a negative feedback control mechanism, step transcription was analysed at different levels of insulin signalling activity in vivo by using quantitative RT–PCR experiments. Similarly to the 4E-BP and InR genes, step transcription was found to be upregulated under conditions promoting FOXO activity such as starvation or in mutants of the insulin signalling pathway, such as chico mutants. Consistently, step transcription is induced 24-fold in response to a brief pulse of ectopic FOXO expression during larval development. These results indicate a FOXO-dependent transcription of step, which may be direct, presumably through several FOXO consensus binding motifs present in the step promoter, or indirect (Fuss, 2006).

It is therefore proposed that Step is a previously unrecognized and essential component of the insulin signalling cascade in Drosophila that regulates organismal growth. These results are consistent with the findings of a parallel study (Hafner, 2006) on the role of mammalian cytohesins. Both papers provide independent evidence for the central involvement of cytohesins in the insulin pathway upstream of PI(3)K and show a functional conservation of these proteins for at least 900 million years (Fuss, 2006).

Search PubMed for articles about Drosophila Steppke

Anitei, M. and Hoflack, B. (2012). Bridging membrane and cytoskeleton dynamics in the secretory and endocytic pathways. Nat Cell Biol 14: 11-19. PubMed ID: 22193159

Bill, A., Schmitz, A., Albertoni, B., Song, J. N., Heukamp, L. C., Walrafen, D., Thorwirth, F., Verveer, P. J., Zimmer, S., Meffert, L., Schreiber, A., Chatterjee, S., Thomas, R. K., Ullrich, R. T., Lang, T. and Famulok, M. (2010). Cytohesins are cytoplasmic ErbB receptor activators. Cell 143: 201-211. PubMed ID: 20946980

Cao, J., Albertson, R., Riggs, B., Field, C. M. and Sullivan, W. (2008). Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization. J Cell Biol 182: 301-313. PubMed ID: 18644888

Claperon, A. and Therrien, M. (2007). KSR and CNK: two scaffolds regulating RAS-mediated RAF activation. Oncogene 26: 3143-3158. PubMed ID: 17496912

Donaldson, J. G. and Jackson, C. L. (2011). ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol 12: 362-375. PubMed ID: 21587297

Fuss, B., Becker, T., Zinke, I. and Hoch, M. (2006). The cytohesin Steppke is essential for insulin signalling in Drosophila. Nature 444: 945-948. PubMed Citation: 17167488

Goldie, S. J., Mulder, K. W., Tan, D. W., Lyons, S. K., Sims, A. H. and Watt, F. M. (2012). FRMD4A upregulation in human squamous cell carcinoma promotes tumor growth and metastasis and is associated with poor prognosis. Cancer Res 72: 3424-3436. PubMed ID: 22564525

Hafner, M., Schmitz, A., Grune, I., Srivatsan, S. G., Paul, B., Kolanus, W., Quast, T., Kremmer, E., Bauer, I. and Famulok, M. (2006). Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance. Nature 444: 941-944. PubMed ID: 17167487

Hahn, I., Fuss, B., Peters, A., Werner, T., Sieberg, A., Gosejacob, D. and Hoch, M. (2013). The Drosophila Arf GEF Steppke controls MAPK activation in EGFR signaling. J Cell Sci 126: 2470-2479. PubMed ID: 23549788

Ikenouchi, J. and Umeda, M. (2010). FRMD4A regulates epithelial polarity by connecting Arf6 activation with the PAR complex. Proc Natl Acad Sci U S A 107: 748-753. PubMed ID: 20080746

Kolanus, W. (2007). Guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction. Immunol Rev 218: 102-113. PubMed ID: 17624947

Lee, D. M. and Harris, T. J. (2013). An Arf-GEF Regulates Antagonism between Endocytosis and the Cytoskeleton for Drosophila Blastoderm Development. Curr Biol 23: 2110-2120. PubMed ID: 24120639

Lim, J., Zhou, M., Veenstra, T. D. and Morrison, D. K. (2010). The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling. Genes Dev 24: 1496-1506. PubMed ID: 20634316

Liu, J., Lee, D. M., Yu, C. G., Angers, S. and Harris, T. J. (2014). Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo. Mol Biol Cell 26(4): 711-25. PubMed ID: 25540427

Mooren, O. L., Galletta, B. J. and Cooper, J. A. (2012). Roles for actin assembly in endocytosis. Annu Rev Biochem 81: 661-686. PubMed ID: 22663081

Reversi, A., Loeser, E., Subramanian, D., Schultz, C. and De Renzis, S. (2014). Plasma membrane phosphoinositide balance regulates cell shape during Drosophila embryo morphogenesis. J Cell Biol 205: 395-408. PubMed ID: 24798734

Santy, L. C., Ravichandran, K. S. and Casanova, J. E. (2005). The DOCK180/Elmo complex couples ARNO-mediated Arf6 activation to the downstream activation of Rac1. Curr Biol 15: 1749-1754. PubMed ID: 16213822

Schejter, E. D. and Wieschaus, E. (1993). bottleneck acts as a regulator of the microfilament network governing cellularization of the Drosophila embryo. Cell 75: 373-385. PubMed ID: 8402919

Sokac, A. M. and Wieschaus, E. (2008). Local actin-dependent endocytosis is zygotically controlled to initiate Drosophila cellularization. Dev Cell 14: 775-786. PubMed ID: 18477459

Torii, T., Miyamoto, Y., Sanbe, A., Nishimura, K., Yamauchi, J. and Tanoue, A. (2010). Cytohesin-2/ARNO, through its interaction with focal adhesion adaptor protein paxillin, regulates preadipocyte migration via the downstream activation of Arf6. J Biol Chem 285: 24270-24281. PubMed ID: 20525696

West, J. J., Zulueta-Coarasa, T., Maier, J. A., Lee, D. M., Bruce, A. E. E., Fernandez-Gonzalez, R. and Harris, T. J. C. (2017). An Actomyosin-Arf-GEF negative feedback loop for tissue elongation under stress. Curr Biol 27(15): 2260-2270. PubMed ID: 28736167

Yan, S., Lv, Z., Winterhoff, M., Wenzl, C., Zobel, T., Faix, J., Bogdan, S. and Grosshans, J. (2013). The F-BAR protein Cip4/Toca-1 antagonizes the formin Diaphanous in membrane stabilization and compartmentalization. J Cell Sci 126: 1796-1805. PubMed ID: 23424199

date revised: 15 September 2022

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