InteractiveFly: GeneBrief

Shroom: Biological Overview | References

Gene name - Shroom

Synonyms -

Cytological map position - 50F4-50F4

Function - signaling, scaffold protein

Keywords - an asymmetrically localized actin- and Rho-kinase-binding protein - amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling, apical constriction, convergent extension, adherens junction

Symbol - Shrm

FlyBase ID: FBgn0085408

Genetic map position - chr2R:14,308,144-14,351,669

NCBI classification - N-terminal PDZ domain, a centrally located Shroom domain 1 (SD1), and the C-terminally located SD2

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Shroom orthologs: Biolitmine

Regulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, Drosophila melanogaster was used to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, several cytoskeletal and signaling protein were identified that may cooperate with Shroom. Two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology (Hildebrand, 2021).

Tissue architecture is typically defined during specific stages of embryonic development and errors in these processes can result in human disease. One example is formation of the vertebrate neural tube. The neural tube is formed via the concerted effort of many cellular pathways that functionally convert a plate of neural ectoderm into a closed tube. Errors in this process can result in birth defects such as spina bifida, exencephaly, or craniorachischisis. One cellular pathway that controls this process is regulated by the Shroom3 cytoskeletal adaptor protein (Haigo, 2003; Hildebrand, 1999). Shroom3 controls neural tube morphogenesis via the formation of apically positioned contractile networks of actomyosin and these networks facilitate neural tube closure by inducing apical constriction and the anisotropic contraction of actin filaments (Haigo, 2003; Hildebrand, 2005; McGreevy, 2015). This is accomplished via the modular nature of Shroom3. Shroom3 localizes to the apical compartment of epithelial adherens junctions via a direct interaction with F-actin (Hildebrand, 1999). This interaction is mediated by the Shroom Domain (SD) 1, a unique actin-binding motif present in most Shroom proteins characterized to date. Shroom3 function is also dependent on Rho-kinase (Rock), such that Shroom3 directly binds to Rock and regulates both its localization and catalytic activity (Das, 2014; Nishimura, 2008; Zalewski, 2016). The interaction between Shroom and Rock has been elucidated at the molecular level and is mediated by the conserved SD2 region of Shroom and a conserved coiled-coil region of Rock (Mohan, 2013, 2012; Zalewski, 2016). The interaction between Shroom and Rock results in the localized activation of non-muscle myosin II (myosin II) contractility, which provides the mechanical force needed to facilitate neural tube morphogenesis. The regulation of myosin II activity by Rock and other cellular pathways has been well described. Rock modulates myosin II activity in two ways. First, Rock can directly phosphorylate the associated regulatory light chain (RLC), which modulates the actin-associated ATPase activity and the conformation of myosin II. Secondly, Rock negatively regulates the phosphatase that dephosphorylates the RLC, thus preventing the inactivation of myosin II (Hildebrand, 2021 and references therein).

Shroom proteins are required for numerous biological processes and are associated with several human diseases. In mammals, there are three definitive Shroom proteins, Shroom2, Shroom3, and Shroom4, each of which contains an N-terminal PDZ domain, the centrally located SD1, and the C-terminally located SD2. All three proteins can directly interact with F-actin and regulate cell morphology via Rock. In humans, SHROOM2 has been linked to neural tube morphogenesis, colorectal cancer, and medulloblastoma, while in vitro studies indicate it is important for cell migration, vasculogenesis, metastasis, and melanosome biogenesis. SHROOM3 mutations have been implicated in chronic kidney disease, heart morphogenesis, and neural tube closure in humans. Using model organisms or cell culture, Shroom3 has been shown to control neural tube closure, axon growth, intestine architecture, eye morphogenesis, thyroid budding, and kidney development. Finally, SHROOM4 mutations have been associated with X-linked mental defects (Hildebrand, 2021).

The Shroom gene is conserved in Drosophila and encodes multiple protein isoforms that have different subcellular distributions and activities in vivo (Bolinger, 2010). The most highly conserved region of Drosophila Shroom is the SD2, the region that binds to Drosophila Rho-kinase (Rok) (Mohan, 2012). Drosophila Shroom also contains a divergent SD1 motif and this appears to mediate localization to adherens junctions in polarized epithelia (Bolinger, 2010). Consistent with the known activities of mammalian Shroom3, expression of Drosophila Shroom in epithelial cells induces apical constriction in a Rok and myosin II dependent manner (Bolinger, 2010). While Shroom3 is essential for mouse and human development, Shroom is not absolutely essential for Drosophila viability, as Shroom null flies can be recovered, albeit with significantly reduced frequency (Simoesr if the co-expression of Shroom, Ena, and Shot is controlled pre- or post-transcriptionally. It is possible that the expression of Shroom, Ena, and Shot is coordinately rSimoes, 2014). These elegant studies showing the role of Shroom in regulating directional contractility are supported by observations that Shroom proteins can be polarly distributed in mammalian tissues and cells (Hildebrand, 2021).

To better understand the mechanisms that control Shroom-regulated changes in cell and tissue morphology, this study has established tools to perform genetic screens for modifiers of Shroom activity in Drosophila. Shroom gain-of-function phenotypes in the eye and wing can be suppressed or enhanced by known components of the Shroom pathway. Using a candidate approach, several cytoskeletal regulators were identified, including Short stop and Enabled, as participants in Shroom-mediated changes in cell morphology. Shroom regulates the distribution of Ena and this is likely mediated by conserved proline-rich sequences in Shroom and the EVH1 domain of Ena. This study further shows that while Ena is required for the Shroom gain-of-function phenotypes, apical recruitment of Ena is not sufficient to cause changes in cell morphology. Additionally, by using an isoform of Shroom that does not bind Ena, but still engages Rok, this study showed that apical constriction can be modulated by different cellular pathways depending on the context (Hildebrand, 2021).

This study describes a genetic approach to identify cellular pathways that participate in tissue morphogenesis. This method takes advantage of the observation that ectopic Shroom protein can utilize the endogenous contractile machinery within epithelial cells to induce apical constriction and disrupt normal tissue morphology. While this work focuses on candidate genes that encode known regulators of epithelial and tissue architecture, it is predicted these tools can be used to perform unbiased, genome-wide screens to identify novel participants in Shroom-mediated cellular processes. Two different tissues, eye and wing imaginal discs, were used for these studies, and these screens can identify factors that are used in a wide range of tissues and cells to control cell dynamics. This is based on the observations that ShroomA, the isoform most similar to mammalian Shroom3, induces similar cellular phenotypes in both types of imaginal discs, and the phenotypes can be modified in both tissues. A powerful aspect of this screen is that these processes are functionally conserved in vertebrate cells and tissues. Additionally, the simplified nature of the Drosophila genome makes these screens possible. Due to genetic and functional redundancy, it is predicted that the analysis performed in this study would be more complicated using vertebrate or cell culture model systems. Drosophila have single genes for Shroom, Rok, myosin II, and Ena while mammals possess gene families for these factors. In support of this, previous work has shown that both Rock1 and Rock2 must be inhibited to prevent Shroom3-mediated apical constriction in cell culture. This screening approach should allow for the identification of novel genetic interactions in Drosophila that can be further verified in mammalian model systems to define their potential role in human disease (Hildebrand, 2021).

Most of the modifiers identified in this study participate in defining actin or microtubule architecture. Of these, several regulate actin dynamics at the level of polymerization or stability, including Ena, Diaphanous, Chickadee, and Slingshot. Interestingly, three of these proteins can be linked, directly or indirectly, to neural tube formation in mice. It should be noted that several classes of actin regulators did not appear to modify the Shroom phenotypes, including nucleators, binding proteins, or adaptors, suggesting that specific types of actin organization are required for Shroom-induced perturbation of cell architecture. This is further supported by the observation that Tropomyosin was also identified in the screen. Tropomyosin regulates the structure of actin filaments and the binding of other proteins, including myosin II and cofilin, that in turn modulate cell architecture or behavior. It is particularly intriguing to note that Tropomyosin mutations can suppress phenotypes caused by the loss of Flapwing, presumably caused by increased myosin II activity. In addition to the actin cytoskeleton, these studies also support a role for microtubules in Shroom-induced phenotypes. This is consistent with the role of microtubules in apical constriction in Drosophila. Recent evidence indicates that apical-medial microtubules play an important role in ventral furrow invagination and this is mediated by Patronin, a protein known to interact with Shot. These studies show that microtubules stabilize the connection of contractile networks to cell junctions to facilitate tissue morphogenesis. These studies are consistent with the current results in relation to Shroom function and Shot distribution in the wing epithelium. It will be interesting to determine if the identified proteins act upstream or downstream of Shroom. While the data suggest Ena acts downstream of Shroom, proteins such as Tropomyosin could function upstream by regulating the amount of Shroom that can bind to F-actin or downstream by modulating the amount of myosin II that can be recruited or activated by the Shroom-Rok complex. It was surprising that determinants of cell adhesion or polarity, such as cadherins or Par complex proteins, were not identified in this screen. It is possible that these proteins are present in sufficient quantity and reducing the dosage is unable to modify the Shroom overexpression phenotype and thus other genetic approaches will be needed to assess the role of these pathways (Hildebrand, 2021).

The data show that endogenous Shroom protein is expressed in epithelial cells during wing and eye development, suggesting it functions in these tissues under normal circumstances. Shroom null flies that survive to adults do not exhibit significant defects in the eyes or wings, although null embryos do exhibit defects in convergent extension (Simoes, 2014) and perhaps this could contribute to the observed reduction in viability. In embryos, Shroom is important for the polarized distribution of contractile myosin II needed for convergent extension. It is possible that Shroom activity in disc epithelial cells is redundant to other pathways that regulate Rok and myosin II and Shroom normally functions to make these pathways more robust or function with higher fidelity. Uncovering these subtle interactions will require additional genetic approaches. The localization of Shroom in the eye and wing disc appears to be highly regulated and is reminiscent of that exhibited by myosin II and phosphorylated Sqh, particularly in the eye imaginal disc. A dramatic increase was observed in Shroom protein in cells that are exiting the morphogenetic furrow and forming the pre-clusters that will give rise to the ommatidia. As the ommatidia form, Shroom expression becomes restricted to the R3/4 cells and eventually is lost from these cells. This distribution is essentially the inverse to that of E-cadherin, which is highest in the radial junctions and lower in the circumferential junctions. This could reflect differences in adhesive interactions between the ommatidia pre-clusters and the inter-ommatidia cells, which facilitates rotation of the ommatidia. This hypothesis is supported by previous studies demonstrating that differential adhesion generates specific cellular organization and compartmentalization in the developing eye. Interestingly, the PCP protein Flamingo is also expressed in R3 and R4 and previous studies have identified interactions between the Shroom3 and PCP pathways in the neural tube (Ho, 2010; McGreevy, 2015). As eye development continues, this study observed Shroom expression in the pigment cells of the pupal retina. In both the imaginal disc and the retina, Shroom distribution is restricted to specific cell junctions, suggesting there are differential adhesive or contractile forces associated with these membranes (Hildebrand, 2021).

In the wing imaginal disc, expression of Shroom protein was observed in rows of cells that border the anterior half of the wing margin. Consistent with the genetic interactions, a similar expression pattern was observed for both Ena and Shot in these cells. It is currently unclear if the co-expression of Shroom, Ena, and Shot is controlled pre- or post-transcriptionally. It is possible that the expression of Shroom, Ena, and Shot is coordinately regulated in a gene network. Alternatively, the stability or apical localization of these proteins may be interdependent or closely orchestrated. This expression pattern in the anterior wing margin is similar to members of the Irre cell Recognition Module (IRM), including cell surface receptors Roughest, Hibris, and Kirre, which help position the sensory organs. This is particularly interesting in light of the fact that the vertebrate orthologs of these genes, Neph and Nephrin-1, and Shroom3 are all involved in formation of podocytes in the glomerulus of the mammalian kidney. It will be exciting to apply genetic analysis to investigate if these pathways cooperate to regulate tissue morphology (Hildebrand, 2021).

Ena and Shroom show extensive co-expression and colocalization in both the wing and eye imaginal disc, although Ena is more widely expressed than Shroom. In both the wing and eye imaginal disc, Ena is expressed in most cells and is localized primarily in the tricellular junctions with lower expression in the adherens junctions. However, as seen in the wing margin and the morphogenetic furrow, cells that express Shroom protein also exhibit high levels of Ena in the cell junctions. Importantly, reducing the amount of Shroom protein perturbs the localization of Ena in the anterior wing margin. The relationship between Ena, Shroom, Rok, and myosin II in defining cell shape is likely to be complicated. This stems from the observations that these factors could be placed both upstream and downstream of Shroom. For example, it has been previously shown that Shroom distribution to the apical adherens junctions is mediated, at least in part, by direct binding to F-actin. However, it has also been established that RhoA and Rok regulate F-actin architecture to influence Shroom distribution, which then facilitates the polarized distribution of Rok and myosin II (Simoes, 2014). Ena has been shown to have multiple roles in Drosophila development, including axon guidance, collective cell migration, and epithelial morphogenesis. The role Ena plays in Shroom-mediated apical constriction is unclear. The current data suggest that Ena functions downstream of Shroom and is recruited to adherens junctions via an LPPPP-EVH1 interaction. Ena is primarily defined as a modulator of F-actin dynamics that facilitates the formation of long filaments by competing with barbed-end capping and promoting the addition of actin monomers to the barbed end. This activity may be important for providing the substrate for activated myosin II to drive cell contraction. This is consistent with studies in vertebrate cells showing that Diaphanous 1, is also required for contractility in adherens junctions and that this study has also identified Dia as a potential modifier of Shroom activity (Hildebrand, 2021).

Elegant studies from several groups have identified many other signaling pathways that control the distribution of contractile myosin II networks during Drosophila development, including the Fog, PCP, HH, Dpp, EGF, Toll, and integrin signaling pathway. How all these signaling pathways are orchestrated and converge on myosin II at the cellular and tissue level is a fascinating question. It has been shown that the above processes use a variety of methods to regulate the small GTPase RhoA, which activates Rok, including several GTP exchange factors or GTPase Activating Proteins. It should be noted that other GTPases such as Rap1 or CDC42 also regulate apical constriction. This work has shown that Shroom3 may activate Rock independent of RhoA, suggesting that there as mechanisms to bypass small GTPases in the activation of myosin II. It will be informative to utilize this screening approach to further test how these pathways might work with ShroomA to control cell morphology (Hildebrand, 2021).

Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension

Actomyosin contraction generates mechanical forces that influence cell and tissue structure. During convergent extension in Drosophila, the spatially regulated activity of the myosin activator Rho-kinase promotes actomyosin contraction at specific planar cell boundaries to produce polarized cell rearrangement. The mechanisms that direct localized Rho-kinase activity are not well understood. This study shows that Rho GTPase recruits Rho-kinase to adherens junctions and is required for Rho-kinase planar polarity. Shroom, an asymmetrically localized actin- and Rho-kinase-binding protein, amplifies Rho-kinase and myosin II planar polarity and junctional localization downstream of Rho signaling. In Shroom mutants, Rho-kinase and myosin II achieve reduced levels of planar polarity, resulting in decreased junctional tension, a disruption of multicellular rosette formation, and defective convergent extension. These results indicate that Rho GTPase activity is required to establish a planar polarized actomyosin network, and the Shroom actin-binding protein enhances myosin contractility locally to generate robust mechanical forces during axis elongation (Simoes, 2014).

Rho-kinase is an essential regulator of actomyosin contractility, but the mechanisms that generate Rho-kinase asymmetry to produce spatially regulated forces during development are not well understood. This study shows that Rho GTPase signaling is required for the planar polarized localization of Rho-kinase and myosin II during Drosophila axis elongation. Direct interaction between Rho and Rho-kinase recruits Rho-kinase to adherens junctions but is not sufficient for full Rho-kinase planar polarity, suggesting that other mechanisms amplify the effects of Rho signaling. This study provides evidence that the actin-binding protein Shroom regulates Rho-kinase localization and planar polarized actomyosin contractility to promote sustained cell rearrangements during axis elongation. Shroom is present in a planar polarized distribution at adherens junctions in intercalating cells, consistent with a direct and localized function. Shroom planar polarity requires Rho activity, indicating that Shroom is an effector of Rho signaling. In Shroom mutants, Rho-kinase and myosin II junctional localization and planar polarity initiate normally but fail to be amplified and maintained during axis elongation. Consequently, planar polarized contractile forces and multicellular rosette rearrangements are reduced in Shroom mutants, resulting in decreased convergent extension. These results support a role for Shroom in regulating planar polarized actomyosin contractility and junctional remodeling during convergent extension, expanding the morphogenetic functions of this highly conserved protein beyond its known role in apical constriction (Simoes, 2014).

The data support a model in which Rho GTPase and Shroom have distinct functions in regulating Rho-kinase localization and planar polarized myosin contractility during convergent extension. Rho GTPase recruits Rho-kinase to adherens junctions and initiates planar polarity, and Shroom plays a modulatory role in enhancing and maintaining planar polarized myosin contractility downstream of Rho signaling. Rho GTPase binds to Rho-kinase and could regulate its localization directly. Rho does not bind to Shroom but may regulate Shroom planar polarity indirectly through its effect on the actin cytoskeleton. Rho-kinase, usually viewed as a downstream effector of Shroom, feeds back to maintain Shroom planar polarity and its own planar polarized localization. Rho-kinase could directly phosphorylate Shroom to reinforce planar cell polarity. Alternatively, Rho-kinase could promote Shroom localization through remodeling of the actin cytoskeleton, as the Shroom actin-binding domain is necessary and sufficient for targeting to planar junctions, and Rho-kinase can phosphorylate known regulators of actin (Simoes, 2014).

These findings may be relevant to neural tube development in vertebrates, which involves a combination of apical constriction, polarized junctional remodeling, and cell shape changes. Shroom3 is required for neural tube closure in the mouse, frog, and chick, and disrupting the interaction between Shroom and Rho-kinase reduces the number of rosettes in the chick neural plate. Unlike mutants that have disrupted rosette-based movements caused by defects in cell adhesion, the defects in Shroom mutants are likely a result of reduced myosin II activity. Rosette behaviors in Drosophila predominate midway through elongation at stage 8, coinciding with the stage when myosin becomes mislocalized in Shroom mutants. A failure to reinforce actomyosin contractility during elongation in Shroom mutants could selectively disrupt later-onset, higher-order cell rearrangements, with no effect on local neighbor exchange events that are more frequent at earlier stages. Alternatively, rosette formation may require more force, as rosettes form through the contraction of multicellular actomyosin cables that are under a higher level of tension and accumulate more myosin. In Shroom mutants, defects in myosin junctional localization may prevent contractile forces from reaching the levels necessary to produce rosette-based convergent extension movements. It will be interesting to explore whether planar polarized Shroom activity plays a general role in promoting junctional remodeling and enhancing mechanical force generation in processes that require strong actomyosin contractility during development (Simoes, 2014).

Rho GTPase signaling is an excellent candidate to break planar symmetry, as a small fraction of active Rho protein can trigger rapid and dramatic changes in the actin cytoskeleton. In one model, a subtle increase in Rho activity at AP cell boundaries could provide an instructive cue, guiding planar cell polarity by recruiting Rho-kinase, modifying the actin cytoskeleton, and facilitating the cortical association of the Rho-kinase regulator Shroom. Alternatively, Rho could regulate Rho-kinase planar polarity indirectly through its role in promoting Rho-kinase apical localization. Although it is challenging to visualize a small and highly dynamic population of active Rho protein in vivo, several findings support the idea that localized Rho activity could play an instructive role in planar polarity. First, myosin planar polarity and directional cell rearrangements occur normally at early stages in Shroom mutants, suggesting that other signals are able to generate localized myosin activity. The partial planar asymmetry of a fragment containing the RB domain of Rho-kinase, which is predicted to interact with the active pool of Rho GTPase, suggests that Rho could contribute to this asymmetry. Second, Rho is required for the planar polarized localization of Shroom, raising the possibility that Rho signaling could provide an essential source of Shroom asymmetry. Third, the upstream Rho activator RhoGEF2 in Drosophila and PDZ-RhoGEF in the chick display a subtle planar asymmetry during epithelial bending and elongation. Multiple activators and inhibitors of Rho could act together to generate a spatially localized pattern of Rho activity, as is the case for apical constriction. Notably, although Rho GTPase activity is necessary to establish Rho-kinase and myosin planar polarity, it is not sufficient to maintain their activity at high enough levels to allow sustained force generation and rosette rearrangements in Shroom mutants. It is proposed that Rho promotes the recruitment of Shroom as part of a positive feed-forward mechanism that reinforces planar polarized actomyosin contractility during convergent extension (Simoes, 2014).

Planar polarized cell rearrangements require the active maintenance of cell polarity in large populations of dynamically moving cells. This study shows that Shroom and Rho GTPase signaling play distinct roles in the establishment and maintenance of polarized actomyosin contractility during convergent extension. The upstream spatial cues that localize actomyosin contractility to specific planar cellular domains are not known. An asymmetry in the organization of the actin cytoskeleton is the earliest evidence of planar polarity in the Drosophila embryo. Distinct actin-binding domains in different Shroom isoforms have been proposed to target Shroom protein and its effectors to different regions of the cell. Moreover, the actin-binding domain is critical for Shroom planar polarity. These findings support the idea that an asymmetry in the actin cytoskeleton is an essential spatial input that regulates the localization of Shroom, the contractile machinery, and ultimately the forces that control cell rearrangement and tissue structure. The upstream spatial cues that generate these asymmetries could involve an asymmetry in Rho signaling, perhaps through the local activation of upstream signaling proteins that regulate Rho GTPase activity. Alternatively, the critical event in the establishment of planar cell polarity could be a Rho-independent reorganization of the actin cytoskeleton that biases the activity of Shroom, Rho-kinase, and myosin, which in turn modify the cytoskeleton to allow robust and sustained cell polarization. Elucidation of the upstream spatial cues that regulate actomyosin localization and dynamics will provide insight into the mechanisms that direct polarized cell behavior (Simoes, 2014).

Specific isoforms of Drosophila shroom define spatial requirements for the induction of apical constriction

Vertebrate Shroom proteins define cytoskeletal organization and cellular architecture by binding directly to F-actin and Rho-kinase and spatially regulating the activity of nonmuscle myosin II (myosin II). This study reports characterization and gain-of-function analysis of Drosophila Shroom. The dShrm locus expresses at least two protein isoforms, dShrmA and dShrmB, which localize to adherens junctions and the apical membrane, respectively. dShrmA and dShrmB exhibit differing abilities to induce apical constriction that are based on their subcellular distribution and the subsequent assembly of spatially and organizationally distinct actomyosin networks that are dependent on the ability to engage Rho-kinase and the activity of myosin II. These data show that the differential subcellular distribution of naturally occurring isoforms of Shroom proteins can define both the position and organization of actomyosin networks in vivo. It is further hypothesized that differentially positioned contractile arrays have distinct effects on cellular morphologies and behaviors (Bolinger, 2010).

Previously, it was not clear how well the basic characteristics and functions of Shrm proteins were conserved in invertebrates. This was particularly true in Drosophila as the lone identifiable Shrm-related protein exhibited only limited sequence homology. While dShrm has been identified in genetic screens as a potential regulator of longevity and architecture of the neuromuscular junction, no studies on dShrm function in flies has been performed. This study shows that the dShrm locus encodes at least two protein isoforms that have different activities in vivo. It is predicted that dShrmA is the most functionally similar to Shrm3 as it is expressed in epithelia, is spatial restricted to the same relative position in polarized cells, directly interacts with F-actin and dRok, and can induce robust apical constriction (Bolinger, 2010).

The results show that dShrmA and dShrmB are not functionally equivalent when expressed in the same cellular contexts, such that dShrmA triggers the assembly of a circumferential contractile network while dShrmB induces the formation of a more disorganized actin network at the apical surface. Importantly, these alternate contractile networks seem to exhibit profoundly different activities in vivo. In a side-by-side comparison, the circumferential network appears to be more efficient at causing apical constriction than the apically positioned network. This is an interesting observation in light of the elegant studies showing that contractile actomyosin networks that span the apical plasma membrane are more critical to the apical constriction that drives ventral furrow formation. It is suggested that dShrmA and dShrmB may participate in distinct morphogenic processes and their function may be dependent on the cellular context in which they are expressed (Bolinger, 2010).

The apically targeted dShrmB isoform appears to be specific to Drosophila and its localization seems to be mediated by a unique mechanism. In contrast to dShrmA, the mechanism by which dShrmB is targeted to the apical plasma membrane is currently unknown, but is independent of both the actin and microtubule cytoskeletons. However, the molecular basis for this localization is conserved because dShrmB is apically targeted in vertebrate epithelia. Sequence analysis of dShrmB does not indicate the presence of any signal peptides, transmembrane segments, or lipid modifications that could target it to the plasma membrane. These results indicate that dShrmB may directly interact with a protein or lipid constituent of the apical plasma membrane (Bolinger, 2010).

There are several explanations as to why dShrmA and dShrmB have different activity in vivo. First, it is possible that the SD2-dRok interaction is either inhibited or less efficient in the apical plasma membrane as opposed to the AJ. This could be achieved by a currently unknown mechanism, such as phosphorylation, which regulates the interaction specifically within the apical compartment. Alternatively, the ability to assemble a robust apical actomyosin network could be inhibited by another pathway. This concept is supported by the recent characterization of Jak signaling as a negative regulator of apical actomyosin networks in the lateral ectoderm. Alternatively, there may be more total Rok or active Rok available in the AJ than at the apical plasma membrane. Of interest, only dShrmB causes constriction in MDCK cells. This is likely due to the intrinsic differences in the organization of the cell-cell junctions between the species, where in vertebrate cells the TJs occupy the same relative position along the apical-basal axis as AJs in Drosophila cells. This is particularly intriguing in light of previous observations that mShrm3 is specifically localized to TJs and can induce apical constriction in MDCK cells (Haigo,2003; Hildebrand, 2005). In contrast, dShrmA localizes to the AJ and does not cause changes in cell morphology in MDCK cells, suggesting that there are strict requirements for where Shrm proteins can be localized and still induce the formation of actomyosin networks (Bolinger, 2010).

Initial sequence analysis indicated that the SD2 was the only motif conserved between vertebrate and Drosophila Shrm proteins. However, analysis of dShrmA localization and actin-binding activity identified sequences that are highly conserved in other insect species and have low homology to the actin-binding region of Shrm3. It appears that this functional motif is conserved across animal phyla and is probably the primary mechanism by which both dShrmA and Shrm3 are targeted to the correct cellular locale. While these proteins share a common actin binding activity, the characteristic that is more stringently conserved between these proteins is their spatial distribution within polarized epithelial cells. It is predicted that there may have been selective pressure to retain the localization of mShrm3 and dShrmA proteins to the same relative position along the apical-basal axis. This has occurred even though the apical compartment of the lateral membrane is comprised of TJ in vertebrate cells and AJ in Drosophila cells. The current results indicate that, whereas a conserved actin-binding activity targets both dShrmA and mShrm3 to their respective domains in the cell, this property has been modified to convey specific localization to distinct actin populations in vivo (Bolinger, 2010).

In summary, the data suggest that the Shrm-Rock pathway has been conserved during animal evolution. It is also proposed that naturally occurring protein isoforms that exhibit differential subcellular localization are important for determining the spatial distribution of contractile networks and this specifies developmentally regulated changes in cell morphology (Bolinger, 2010).

Functions of Shroom orthologs in other species

The Ajuba family protein Wtip regulates actomyosin contractility during vertebrate neural tube closure

Ajuba family proteins are implicated in the assembly of cell junctions and have been reported to antagonize Hippo signaling in response to cytoskeletal tension. To assess the role of these proteins in actomyosin contractility, the localization and function of Wtip, a member of the Ajuba family, was examined in Xenopus early embryos. Targeted in vivo depletion of Wtip inhibited apical constriction in neuroepithelial cells and elicited neural tube defects. Fluorescent protein-tagged Wtip showed predominant punctate localization along the cell junctions in the epidermis and a linear junctional pattern in the neuroectoderm. In cells undergoing Shroom3-induced apical constriction, the punctate distribution was reorganized into a linear pattern. Conversely, the linear junctional pattern of Wtip in neuroectoderm changed to a more punctate distribution in cells with reduced myosin II activity. The C-terminal fragment of Wtip physically associated with Shroom3 and interfered with Shroom3 activity and neural fold formation. It is therefore proposed that Wtip is a tension-sensitive cytoskeletal adaptor that regulates apical constriction during vertebrate neurulation (Chu, 2018).

Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity

Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. This study presents the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, the first direct evidence that Shroom can function as a Rock activator is provided. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization (Zalewski, 2016).

Shroom3 functions downstream of planar cell polarity to regulate myosin II distribution and cellular organization during neural tube closure

Neural tube closure is a critical developmental event that relies on actomyosin contractility to facilitate specific processes such as apical constriction, tissue bending, and directional cell rearrangements. These complicated processes require the coordinated activities of Rho-Kinase (Rock), to regulate cytoskeletal dynamics and actomyosin contractility, and the Planar Cell Polarity (PCP) pathway, to direct the polarized cellular behaviors that drive convergent extension (CE) movements. This study investigated the role of Shroom3 as a direct linker between PCP and actomyosin contractility during mouse neural tube morphogenesis. In embryos, simultaneous depletion of Shroom3 and the PCP components Vangl2 or Wnt5a results in an increased liability to NTDs and CE failure. It was further shown that these pathways intersect at Dishevelled, as Shroom3 and Dishevelled 2 co-distribute and form a physical complex in cells. It was observed that multiple components of the Shroom3 pathway are planar polarized along mediolateral cell junctions in the neural plate of E8.5 embryos in a Shroom3 and PCP-dependent manner. Finally, it was demonstrated that Shroom3 mutant embryos exhibit defects in planar cell arrangement during neural tube closure, suggesting a role for Shroom3 activity in CE. These findings support a model in which the Shroom3 and PCP pathways interact to control CE and polarized bending of the neural plate and provide a clear illustration of the complex genetic basis of NTDs (McGreevy, 2015).

Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation

Core planar cell polarity (PCP) proteins are well known to regulate polarity in Drosophila and vertebrate epithelia; however, their functions in vertebrate morphogenesis remain poorly understood. This study describes a role for PCP signaling in the process of apical constriction during Xenopus gastrulation. The core PCP protein Vangl2 (Drosophila homolog: Strabismis) is detected at the apical surfaces of cells at the blastopore lip, and it functions during blastopore formation and closure. Further experiments show that Vangl2, as well as Daam1 and Rho-associated kinase (Rock; see Drosophila Rok), regulate apical constriction of bottle cells at the blastopore and ectopic constriction of ectoderm cells triggered by the actin-binding protein Shroom3. At the blastopore lip, Vangl2 is required for the apical accumulation of the recycling endosome marker Rab11 (see Drosophila ). Rab11 and the associated motor protein Myosin V (see Drosophila Didum) play essential roles in both endogenous and ectopic apical constriction, and might be involved in Vangl2 trafficking to the cell surface. Overexpression of Rab11 RNA was sufficient to partly restore normal blastopore formation in Vangl2-deficient embryos. These observations suggest that Vangl2 affects Rab11 to regulate apical constriction during blastopore formation (Ossipova, 2015).

The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice

Shroom3 is an actin-associated regulator of cell morphology that is required for neural tube closure, formation of the lens placode, and gut morphogenesis in mice and has been linked to chronic kidney disease and directional heart looping in humans. Numerous studies have shown that Shroom3 likely regulates these developmental processes by directly binding to Rho-kinase and facilitating the assembly of apically positioned contractile actomyosin networks. This study has characterized the molecular basis for the neural tube defects caused by an ENU-induced mutation that results in an arginine-to-cysteine amino acid substitution at position 1838 of mouse Shroom3. This substitution has no effect on Shroom3 expression or localization but ablates Rock binding and renders Shroom3 non-functional for the ability to regulate cell morphology. These results indicate that Rock is the major downstream effector of Shroom3 in the process of neural tube morphogenesis. Based on sequence conservation and biochemical analysis, it is predictd that the Shroom-Rock interaction is highly conserved across animal evolution and represents a signaling module that is utilized in a variety of biological processes (Das, 2014).

Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, this study has solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, this study has identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, it was shown that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology (Mohan, 2013)

Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction

Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho-logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm-Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm-Rock interaction is regulated at the molecular level. To begin investigating this issue, the structure is presented of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and conserved residues on the surface and within the dimerization interface were identified that are required for the Rock-Shrm interaction and Shrm activity in vivo. It was further shown that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm-Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks (Mohan, 2012).

Pax6-dependent Shroom3 expression regulates apical constriction during lens placode invagination

Embryonic development requires a complex series of relative cellular movements and shape changes that are generally referred to as morphogenesis. Although some of the mechanisms underlying morphogenesis have been identified, the process is still poorly understood. This study addresses mechanisms of epithelial morphogenesis using the vertebrate lens as a model system. The apical constriction of lens epithelial cells that accompanies invagination of the lens placode is dependent on cytoskeletal protein Shroom3, a molecule associated with apical constriction during morphogenesis of the neural plate. Shroom3 is required for the apical localization of F-actin and myosin II, both crucial components of the contractile complexes required for apical constriction, and for the apical localization of Vasp, a Mena family protein with F-actin anti-capping function that is also required for morphogenesis. Finally, the expression of Shroom3 is shown to be dependent on the crucial lens-induction transcription factor Pax6. This provides a previously missing link between lens-induction pathways and the morphogenesis machinery and partly explains the absence of lens morphogenesis in Pax6-deficient mutants (Plageman, 2010).

Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling

Remodeling of epithelial sheets plays important roles in animal morphogenesis. Shroom3 is known to regulate the apical constriction of epithelial cells. This study shows that Shroom3 binds ROCKs and recruits them to the epithelial apical junctions. The Shroom3-binding site (RII-C1) on ROCKs was identified, and it was found that RII-C1 could antagonize the Shroom3-ROCK interaction, interfering with the action of Shroom3 on cell morphology. In the invaginating neural plate/tube, Shroom3 colocalized with ROCKs at the apical junctions; Shroom3 depletion or RII-C1 expression in the tube removed these apically localized ROCKs, and concomitantly blocked neural tube closure. Closing neural plate exhibited peculiar cell assemblies, including rosette formation, as well as a planar-polarized distribution of phosphorylated myosin regulatory light chain, but these were abolished by ROCK inhibition or RII-C1 expression. These results demonstrate that the Shroom3-ROCK interaction is crucial for the regulation of epithelial and neuroepithelial cell arrangement and remodeling (Nishimura, 2008).

Shroom family proteins regulate gamma-tubulin distribution and microtubule architecture during epithelial cell shape change

Cell shape changes require the coordination of actin and microtubule cytoskeletons. The molecular mechanisms by which such coordination is achieved remain obscure, particularly in the context of epithelial cells within developing vertebrate embryos. A role has been identified for the novel actin-binding protein Shroom3 as a regulator of the microtubule cytoskeleton during epithelial morphogenesis. Shroom3 is sufficient and also necessary to induce a redistribution of the microtubule regulator gamma-tubulin. Moreover, this change in gamma-tubulin distribution underlies the assembly of aligned arrays of microtubules that drive apicobasal cell elongation. Finally, experiments with the related protein, Shroom1, demonstrate that gamma-tubulin regulation is a conserved feature of this protein family. Together, the data demonstrate that Shroom family proteins govern epithelial cell behaviors by coordinating the assembly of both microtubule and actin cytoskeletons (Lee, 2007).

Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network

The actin-binding protein Shroom is essential for neural tube morphogenesis in multiple vertebrate organisms, indicating its function is evolutionarily conserved. Shroom facilitates neurulation by regulating the morphology of neurepithelial cells. Shroom localizes to the apical tip of adherens junctions of neural ectoderm cells in vivo and to the apical junctional complex (AJC) in MDCK cells. Induced expression of Shroom in polarized epithelia elicits apical constriction and dramatic reorganization of the apical arrangement and packing of cells without altering apical-basal polarity. These events likely mimic the cell shape changes and cellular movements required for neurulation in vivo. The observed phenotypes depend on the ability of Shroom to alter F-actin distribution and regulate the formation of a previously uncharacterized contractile actomyosin network associated with the AJC. Targeting the C-terminal domain of Shroom to the apical plasma membrane elicits constriction and reorganization of the actomyosin network, indicting that this domain mediates Shroom's activity. In vivo, Shroom-mutant neural epithelia show a marked reduction in apically positioned myosin. Thus, Shroom likely facilitates neural tube closure by regulating cell shape changes via the apical positioning of an actomyosin network in the neurepithelium (Hildebrand, 2005).

Shroom induces apical constriction and is required for hingepoint formation during neural tube closure

The morphogenetic events of early vertebrate development generally involve the combined actions of several populations of cells, each engaged in a distinct behavior. Neural tube closure, for instance, involves apicobasal cell heightening, apical constriction at hingepoints, convergent extension of the midline, and pushing by the epidermis. Although a large number of genes are known to be required for neural tube closure, in only a very few cases has the affected cell behavior been identified. For example, neural tube closure requires the actin binding protein Shroom, but the cellular basis of Shroom function and how it influences neural tube closure remain to be elucidated. This study shows that expression of Shroom is sufficient to organize apical constriction in transcriptionally quiescent, naive epithelial cells but not in non-polarized cells. Shroom-induced apical constriction was associated with enrichment of apically localized actin filaments and required the small GTPase Rap1 but not Rho. Endogenous Xenopus shroom was found to be expressed in cells engaged in apical constriction. Consistent with a role for Shroom in organizing apical constriction, disrupting Shroom function resulted in a specific failure of hingepoint formation, defective neuroepithelial sheet-bending, and failure of neural tube closure. These data demonstrate that Shroom is an essential regulator of apical constriction during neurulation. The finding that a single protein can initiate this process in epithelial cells establishes that bending of epithelial sheets may be patterned during development by the regulation of expression of single genes (Haigo, 2003).

Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice

Using gene trap mutagenesis, this study has identified a mutation in mice that causes exencephaly, acrania, facial clefting, and spina bifida, all of which can be attributed to failed neural tube closure. This mutation is designated shroom (shrm) because the neural folds "mushroom" outward and do not converge at the dorsal midline. shrm encodes a PDZ domain protein that is involved at several levels in regulating aspects of cytoarchitecture. First, endogenous Shrm localizes to adherens junctions and the cytoskeleton. Second, ectopically expressed Shrm alters the subcellular distribution of F-actin. Third, Shrm directly binds F-actin. Finally, cytoskeletal polarity within the neuroepithelium is perturbed in mutant embryos. In concert, these observations suggest that Shrm is a critical determinant of the cellular architecture required for proper neurulation (Hildebrand, 1999).


Search PubMed for articles about Drosophila Shroom

Bolinger, C., Zasadil, L., Rizaldy, R. and Hildebrand, J. D. (2010). Specific isoforms of Drosophila Shroom define spatial requirements for the induction of apical constriction. Dev Dyn 239(7): 2078-2093. PubMed ID: 20549743

Chu, C. W., Xiang, B., Ossipova, O., Ioannou, A. and Sokol, S. Y. (2018). The Ajuba family protein Wtip regulates actomyosin contractility during vertebrate neural tube closure. J Cell Sci 131(10). PubMed ID: 29661847

Das, D., Zalewski, J. K., Mohan, S., Plageman, T. F., VanDemark, A. P. and Hildebrand, J. D. (2014). The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice. Biol Open 3(9): 850-860. PubMed ID: 25171888

Haigo, S. L., Hildebrand, J. D., Harland, R. M. and Wallingford, J. B. (2003). Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. Curr Biol 13(24): 2125-2137. PubMed ID: 14680628

Hildebrand, J. D. and Soriano, P. (1999). Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice. Cell 99(5): 485-497. PubMed ID: 10589677

Hildebrand, J. D. (2005). Shroom regulates epithelial cell shape via the apical positioning of an actomyosin network. J Cell Sci 118(Pt 22): 5191-5203. PubMed ID: 16249236

Hildebrand, J. D., Leventry, A. D., Aideyman, O. P., Majewski, J. C., Haddad, J. A., Bisi, D. C. and Kaufmann, N. (2021). A modifier screen identifies regulators of cytoskeletal architecture as mediators of Shroom-dependent changes in tissue morphology. Biol Open 10(2). PubMed ID: 33504488

Ho, Y. H., Lien, M. T., Lin, C. M., Wei, S. Y., Chang, L. H. and Hsu, J. C. (2010). Echinoid regulates Flamingo endocytosis to control ommatidial rotation in the Drosophila eye. Development 137(5): 745-754. PubMed ID: 20110316

Lee, C., Scherr, H. M. and Wallingford, J. B. (2007). Shroom family proteins regulate gamma-tubulin distribution and microtubule architecture during epithelial cell shape change. Development 134: 1431-1441. Medline abstract: 17329357

McGreevy, E. M., Vijayraghavan, D., Davidson, L. A. and Hildebrand, J. D. (2015). Shroom3 functions downstream of planar cell polarity to regulate myosin II distribution and cellular organization during neural tube closure. Biol Open 4(2): 186-196. PubMed ID: 25596276

Mohan, S., Rizaldy, R., Das, D., Bauer, R. J., Heroux, A., Trakselis, M. A., Hildebrand, J. D. and VanDemark, A. P. (2012). Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction. Mol Biol Cell 23(11): 2131-2142. PubMed ID: 22493320

Mohan, S., Das, D., Bauer, R. J., Heroux, A., Zalewski, J. K., Heber, S., Dosunmu-Ogunbi, A. M., Trakselis, M. A., Hildebrand, J. D. and Vandemark, A. P. (2013). Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology. PLoS One 8(12): e81075. PubMed ID: 24349032

Nishimura, T. and Takeichi, M. (2008). Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling. Development 135(8): 1493-1502. PubMed ID: 18339671

Ossipova, O., Chuykin, I., Chu, C. W. and Sokol, S. Y. (2015). Vangl2 cooperates with Rab11 and Myosin V to regulate apical constriction during vertebrate gastrulation. Development 142: 99-107. PubMed ID: 25480917

Plageman, T. F., Jr., Chung, M. I., Lou, M., Smith, A. N., Hildebrand, J. D., Wallingford, J. B. and Lang, R. A. (2010). Pax6-dependent Shroom3 expression regulates apical constriction during lens placode invagination. Development 137(3): 405-415. PubMed ID: 20081189

Simoes, S., Mainieri, A. and Zallen, J. A. (2014). Rho GTPase and Shroom direct planar polarized actomyosin contractility during convergent extension. J Cell Biol 204: 575-589. PubMed ID: 24535826

Zalewski, J. K., Mo, J. H., Heber, S., Heroux, A., Gardner, R. G., Hildebrand, J. D. and VanDemark, A. P. (2016). Structure of the Shroom-Rho Kinase Complex Reveals a Binding Interface with Monomeric Shroom That Regulates Cell Morphology and Stimulates Kinase Activity. J Biol Chem 291(49): 25364-25374. PubMed ID: 27758857

Biological Overview

date revised: 21 May 2021

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