InteractiveFly: GeneBrief

Girdin: Biological Overview | References

Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. This study shows that Drosophila Girdin (girders of actin filaments) and its human ortholog GIRDIN or GIV (Galpha-interacting vesicle associated protein) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, cell dissemination was observed from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, this study discovered a core mechanism contributing to epithelial cell polarization from flies to humans. These data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination (Biehler, 2020).

The ability of epithelia to form physical barriers is provided by specialized cell-cell junctions, including the zonula adherens (ZA). The latter is a belt-like adherens junction composed primarily of the transmembrane homotypic receptor E-cadherin, which is linked indirectly to circumferential F-actin bundles through adaptor proteins such as β-catenin and α-catenin. In Drosophila embryonic epithelia, the protein Girdin stabilizes the ZA by reinforcing the association of the cadherin-catenin complex with the actin cytoskeleton (Ha, 2015). This function in cell-cell adhesion is preserved in mammals, and supports collective cell migration (Wang, 2018; Wang, 2015). Fly and human Girdin also contribute to the coordinated movement of epithelial cells through the organization of supracellular actin cables (Biehler, 2020).

In addition to creating barriers, epithelial tissues generate vectorial transport and spatially oriented secretion. The unidirectional nature of these functions requires the polarization of epithelial cells along the apical-basal axis. In Drosophila, the scaffold protein Bazooka (Baz) is crucial to the early steps of epithelial cell polarization, and for proper assembly of the ZA. Baz recruits atypical Protein Kinase C (aPKC) together with its regulator Partitioning defective protein 6 (Par-6) to the apical membrane. The small GTPase Cdc42 contributes to the activation of aPKC and p21-activated kinase (Pak1), thereby acting as a key regulator of cell polarity. Baz also contributes to apical positioning of the Crumbs (Crb) complex, which is composed mainly of Crb, Stardust (Sdt), and PALS1-associated Tight Junction protein (Patj). Once properly localized, the aPKC-Par-6 and Crb complexes promote the apical exclusion of Baz, which is then restricted to the ZA. The apical exclusion of Baz is essential to the positioning of the ZA along the apical-basal axis, and for full aPKC activation (Biehler, 2020).

The function of aPKC is evolutionarily preserved, and human atypical PKCι (PKClambda in other mammals) and PKCζ PKCzeta) contribute to epithelial cell polarization. aPKC maintains the identity of the apical domain through phospho-dependent exclusion of lateral polarity proteins such as Yurt (Yrt) and Lethal (2) giant larvae (Lgl). In return, these proteins antagonize the Crb- and aPKC-containing apical machinery to prevent the spread of apical characteristics to the lateral domain. In combination with the function of Baz, these feedback mechanisms provide a fine-tuning of aPKC activity in addition to specifying its subcellular localization. This is crucial, as both over- and under-activation of aPKC is deleterious to epithelial polarity in fly and mammalian cells, and ectopic activation of aPKC can lead to cell transformation (Biehler, 2020).

Cell culture work has established that mammalian GIRDIN interacts physically with PAR3 -the ortholog of Baz-and PKCλ (Ohara, 2012; Sasaki, 2015). Depletion of GIRDIN in Madin-Darby Canine Kidney (MDCK) epithelial cells delays the formation of tight junctions in Ca2+ switch experiments. GIRDIN is also an effector of AMP-activated protein kinase (AMPK) in the maintenance of tight junction integrity under energetic stress (Sasaki, 2015). Moreover, mammalian GIRDIN is required for the formation of epithelial cell cysts with a single lumen, supporting a role for this protein in epithelial morphogenesis as reported in flies. As cyst morphogenesis is linked to epithelial cell polarity, these studies suggest that GIRDIN is involved in establishing the apical-basal axis. However, further studies are required to clarify the role of GIRDIN in apical-basal polarity per se, as other cellular processes could explain the phenotype associated with altered GIRDIN expression. For instance, spindle orientation defects impair the formation of epithelial cysts. Of note, PAR3, aPKC, and AMPK are all required for proper spindle positioning in dividing epithelial cells. The molecular mechanisms sustaining the putative role of GIRDIN in epithelial cell polarity also need to be better deciphered. This study investigated the role of fly and human Girdin proteins in the regulation of epithelial cell polarity, and showed that these proteins are part of the lateral polarity protein network. One crucial function of Girdin proteins is to repress aPKC function. It was also discovered that loss of Girdin proteins promotes overgrowth of cell cysts, and cell dissemination from these multicellular structures. Consistent with these data, it was found that low GIRDIN expression correlates with poor overall survival in subtypes of breast and lung cancers (Biehler, 2020).

Using classical genetics in flies, this study has shown that mutation in Girdin exacerbates the polarity defects in zygotic lgl or yrt mutant embryos and concludes that Girdin is part of the lateral polarity network. It was also found that Girdin opposes the function of aPKC, which plays a crucial role in the establishment and maintenance of the apical domain by antagonizing lateral proteins such as Lgl and Yrt. Thus a model is proposed in which Girdin supports the activity of Yrt and Lgl by restricting the activity of aPKC. This work demonstrates that the role of Girdin in restricting aPKC activity is evolutionarily conserved. This function confers on human GIRDIN the ability to maintain apical-basal polarity in Caco-2 cells, and to support epithelial cyst morphogenesis. These results are in line with previous studies suggesting a role for GIRDIN in polarity and cystogenesis in MDCK and MCF10A epithelial cells. It was shown that PKCλ enhances GIRDIN expression in MDCK cells. Moreover, knockdown of aPKC or GIRDIN gives a similar phenotype characterized by defects in tight junction integrity and cyst formation. It was thus proposed that GIRDIN is an effector of PKCλ. Although cell-type-specific mechanisms may exist, the current data suggest that this hypothesis needs to be revisited in favor of a model in which the induction of GIRDIN expression by PKClambda in MDCK cells initiates a negative feedback loop instead of cooperation between these proteins. The fact that both overactivation of aPKC or inhibition of its activity is deleterious to epithelial cell polarity and cyst morphogenesis may underlie the conflicting interpretations of the data in the literature. GIRDIN is also known to modulate heterotrimeric G protein signaling-a role that seems to contribute to the formation of normal cysts by MDCK cells (Sasaki, 2015). In addition, it was demonstrated recently that GIRDIN acts as an effector of AMP-activated protein kinase (AMPK) under energetic stress to maintain tight junction function (Aznar, 2016). Of note, these two functions are not shared by fly Girdin (Ghosh, 2017; Garcia-Marcos, 2009; Ghosh, 2017), and were thus acquired by GIRDIN during evolution to fulfill specialized functions. In contrast, the discovery in this study of the Girdin-dependent inhibition of aPKC reveals a core mechanism contributing to epithelial cell polarization from flies to humans (Biehler, 2020).

GIRDIN is considered to be an interesting target in cancer due to its role in cell motility, and high levels of GIRDIN have been reported to correlate with a poor prognosis in some human cancers. Notwithstanding that GIRDIN may favor tumor cell migration, the current study indicates that inhibition of GIRDIN function in the context of cancer would be a double-edged sword for many reasons. Indeed, this study showed that knockdown of GIRDIN exacerbates the impact of aPKC overexpression, and leads to overgrowth and lumen filling of Caco-2 cell cysts. Of note, overexpression of aPKC can lead to cell transformation, and was associated with a poor outcome in several epithelial cancers. This study thus establishes that inhibiting GIRDIN in patients showing increased aPKC expression levels could worsen their prognosis. According to the data, abolishing GIRDIN function in tumor cells with decreased levels of the human Lgl protein LLGL1, as reported in many cancers, could also support the progression of the disease by altering the polarity phenotype. Cell detachment and dissemination was observed from GIRDIN knockdown cysts, thus showing that GIRDIN is required for the cohesion of multicellular epithelial structures. Of note, cells, either individually or as clusters, detaching from cysts are alive and some of them remain viable. This is analogous to what was reported in Girdin mutant Drosophila embryos in which cell cysts detach from the ectoderm and survive outside of it. Other phenotypes in Girdin mutant embryos are consistent with a role for Girdin in epithelial tissue cohesion, including rupture of the ventral midline and fragmentation of the dorsal trunk of the trachea. Mechanistically, Girdin strengthens cell-cell adhesion by promoting the association of core adherens junction components with the actin cytoskeleton (Ha, 2015). A recent study established that this molecular function is evolutionarily conserved, and that GIRDIN favors the association of β-CATENIN with F-ACTIN (Wang, 2018). Since knockdown of GIRDIN results in cell dispersion from Caco-2 cell cysts, and since weakening of E-CADHERIN-mediated cell-cell adhesion contributes to cancer cell dissemination and metastasis, it is plausible that reduced GIRDIN expression contribute to the formation of secondary tumors and cancer progression. This may explain why this study found that low mRNA expression levels of GIRDIN correlates with decreased survival in more aggressive breast cancer subtypes and lung adenocarcinoma. Future studies using xenograft in mice, and investigating the expression of GIRDIN protein in cancer patients will help validating whether GIRDIN can repress the progression of certain types of epithelial cancers (Biehler, 2020).

In conclusion, using a sophisticated experimental scheme combining in vivo approaches in D. melanogaster with 3D culture of human cells, this study defined a conserved core mechanism of epithelial cell polarity regulation. Specifically, Girdin was shown to repress the activity of aPKC to support the function of Lgl and Yrt, and ensure stability of the lateral domain. This is of broad interest in cell biology, as proper epithelial cell polarization is crucial for the morphogenesis and physiology of most organs. In addition, the maintenance of a polarized epithelial architecture is crucial to prevent various pathological conditions such as cancer progression. Importantly, this study showed that normal GIRDIN function potentially impairs the progression of epithelial cancers by preserving cell polarity whilst restricting cell growth and cell dissemination. Thus, these results place a caveat on the idea that GIRDIN could be an interesting target to limit cancer cell migration, and indicate that inhibition of GIRDIN in the context of cancer could be precarious. Potential drugs targeting GIRDIN would thus be usable only in the context of precision medicine where a careful analysis of aPKC, LLGL1, and E-CAD expression, as well as the polarity status of tumor cells would be analyzed prior to treatment. Inhibition of GIRDIN in patients carrying tumors with altered expression of these proteins would likely worsen the prognosis (Biehler, 2020).

Drosophila Hook-Related Protein (Girdin) is essential for sensory dendrite formation

The dendrite of the sensory neuron is surrounded by support cells and is composed of two specialized compartments: the inner segment and the sensory cilium. How the sensory dendrite is formed and maintained is not well understood. Hook-related proteins (HkRP) like Girdin, DAPLE, and Gipie are actin-binding proteins, implicated in actin organization and in cell motility. This study shows that the Drosophila melanogaster single member of the Hook-related protein family, Girdin, is essential for sensory dendrite formation and function. Mutations in girdin were identified during a screen for fly mutants with no mechanosensory function. Physiological, morphological, and ultra-structural studies of girdin mutant flies indicate that the mechanosensory neurons innervating external sensory organs (bristles) initially form a ciliated dendrite that degenerates shortly after, followed by the clustering of their cell bodies. Importantly, it was observed that Girdin is expressed transiently during dendrite morphogenesis in three previously unidentified actin-based structures surrounding the inner segment tip and the sensory cilium. These actin structures are largely missing in girdin. Defects in cilia are observed in other sensory organs such as those mediating olfaction and taste, suggesting that Girdin has a general role in forming sensory dendrites in Drosophila. These suggest that Girdin functions temporarily within the sensory organ and that this function is essential for the formation of the sensory dendrites via actin structures (Ha, 2015).

This study reports the identification and characterization of girdin mutants and their role in dendrite formation. This study shows a novel function of Girdin during sensory neuron development in Drosophila, suggesting that Girdin is essential for morphogenesis of sensory dendrites. This conclusion stems from several lines of evidence. First, girdin¹⁰¹ exhibits complete unc-type uncoordination, a phenotype that is present only in flies with mutations in the sensory neuron or its attachment to the bristle. Second, girdin¹⁰¹ flies have no mechanoreceptor currents (MRC) but a normal transepithelial potentials (TEP), the socket and the bristle appear normal, and the dendrite sheath is present, suggesting that, although the support cells of the sensory organ are present, the neuron itself is defective. Third, in girdin¹⁰¹, GFP-tubulin labeling exhibits dramatic defects in dendrite and cilium morphogenesis but a normal morphology of the cell body and axon. Finally, Girdin-GFP is present in relatively high intensity in the sensory organ at the general location of the dendritic tip during its morphogenesis (Ha, 2015).

This finding suggests a role for Girdin in dendrite formation and neuron cell body positioning. During the early stages of mechanosensory neuron development, the morphology of the cell body, dendrite, and cilium all appear normal. Only later in development, the ciliated dendrite begins shortening. This degeneration continues until the dendrites are fully absent and the cell bodies become clustered. Since the cell bodies in girdin¹⁰¹ tend to form a cluster after their dendrites degenerate, it is possible that the dendrite is essential for positioning of the sensory neuron cell body. Alternatively, since Girdin is important in cell migration in many cell types (Enomoto, 2005; Ghosh, 2008), it is possible that the mislocalization of the cell body is due to a function of Girdin in cell migration. One possible explanation for the clustering of the cell bodies is that cell bodies migrate along their axons until they bundle. In C. elegans, dendrites of ciliated sensory cells are extended in a retrograde fashion where the cell body migrates away from the dendritic tip, which remains anchored in place. A similar mechanism that regulates cell-body positioning during mechanosensory neuronal development may also occur in Drosophila (Ha, 2015).

Girdin shares some similarities with NompA: both are essential for fly viability, coordinated behavior, and MRC, while both are not essential for bristle or socket formation and TEP. Both have a similar dendrite formation defect in which the tip of the sensory dendrite terminates at a significant distance from the bristle base and socket. Finally, both NompA and Girdin localize inside the sensory organ at the vicinity of the dendritic tip. Taking together, these phenotypes and localization suggest that Girdin is associated with the function of NompA to attach the dendritic tip to the base of the bristle for the mechanical transduction of mechanosensory signals. However, since Girdin is expressed only transiently in the sensory organ, it is likely to have only a developmental role in forming the mechanosensory organ rather than a direct role in mechanosensation as is proposed for NompA. Also, since girdin¹⁰¹ sensory neurons showed a more severe defect in dendrite morphology than nompA, Girdin might have additional functions beyond temporarily connecting the dendrite to the bristle shaft. One such additional function of Girdin may be to stabilize the dendrite as it differentiates (Ha, 2015).

Girdin is an intracellular, actin-binding protein that associates with the plasma membrane or cytoplasm of epithelial cells (Enomoto, 2005; Puseenam, 2009). In the sensory organ cells, Girdin-GFP is present in three actin structures surrounding the tip of the inner segment and the sensory cilium. Phalloidin staining relative to neurons expressing GFP-tubulin suggests that the cap-like and loop-like structures of actin are found outside the sensory neuron and in the support cells. Since actin colocalizes with Girdin-GFP, this suggests that Girdin-GFP is a component of the support cells at these two locations. On the other hand, the tube-like structure of actin and Girdin-GFP appears to localize near the inner segment and may be part of the neuron or sheath cell (Ha, 2015).

The Girdin-GFP-labeled tube-like structure is found at the tip of the inner segment, a specialized location that has cell-cell contacts with the sheath cell. The Girdin-GFP-labeled cap-like structure is found at the tip of the cilium where the dendritic sheath forms during neuron differentiation. The Girdin-GFP-labeled loop-like structure is found away from the neuronal tip as determined by neurons expressing GFP-tubulin, and the loop-like structure diameter is too large to fit within the dimensions of either the sensory neuron or the sheath cell. Therefore, it is likely that the loop-like structure is inside either the shaft or the socket support cells, which are bigger and are found at that location. Which of these three Girdin-GFP/actin structures is essential for normal dendrite formation is currently not known (Ha, 2015).

Based on the current findings and known functions of Girdin, a model for Girdin function in sensory organs during sensory neuron development is proposed (see A model for Girdin function during sensory neuron development). This study shows that Girdin functions near the tip of the dendrite at three locations. In these locations, Girdin regulates the formation of actin structures that may temporarily stabilize the dendritic tip and is essential for its connection to the dendritic sheath. In girdin mutants, dendrite degeneration is likely due to a failure in complete development of the dendrite after its initiation. Once the tip of the dendrite is destabilized and disappears, the neuron cell body recedes toward the axon until it meets other sensory cell bodies and forms clusters where the individual axon meets to form a bundle. This model can serve to direct further studies of Girdin (Ha, 2015).

Because of the similarities between fly sensory neurons and human photoreceptor cells, Girdin may have a similar function in the formation of rod and cone dendrites. Rod and cone photoreceptors are capable of surviving throughout a life span, yet they are sensitive to various insults that result in their degeneration, leading to gradual loss of visual acuity and blindness. Since RPE cells surround the tip of the photoreceptors and are essential for forming and maintaining the morphology of the photoreceptors, studying the role of Girdin in RPE cell lines may provide more insights into understanding how RPE cells regulate the morphology of photoreceptor cells. By better understanding the mechanisms of how ciliated a sensory neuron functions and forms its structure in Drosophila, it is hoped to assist in confronting retinal diseases and provide insight to treat them (Ha, 2015).

Girdin shares some similarities with NompA: both are essential for fly viability, coordinated behavior, and MRC, while both are not essential for bristle or socket formation and TEP. Both have a similar dendrite formation defect in which the tip of the sensory dendrite terminates at a significant distance from the bristle base and socket. Finally, both NompA and Girdin localize inside the sensory organ at the vicinity of the dendritic tip. Taking together, these phenotypes and localization suggest that Girdin is associated with the function of NompA to attach the dendritic tip to the base of the bristle for the mechanical transduction of mechanosensory signals. However, since Girdin is expressed only transiently in the sensory organ, it is likely to have only a developmental role in forming the mechanosensory organ rather than a direct role in mechanosensation as is proposed for NompA. Also, since girdin¹⁰¹ sensory neurons showed a more severe defect in dendrite morphology than nompA, Girdin might have additional functions beyond temporarily connecting the dendrite to the bristle shaft. One such additional function of Girdin may be to stabilize the dendrite as it differentiates (Ha, 2015).

Girdin-mediated interactions between cadherin and the actin cytoskeleton are required for epithelial morphogenesis in Drosophila

E-cadherin-mediated cell-cell adhesion is fundamental for epithelial tissue morphogenesis, physiology and repair. E-cadherin is a core transmembrane constituent of the zonula adherens (ZA), a belt-like adherens junction located at the apicolateral border in epithelial cells. The anchorage of ZA components to cortical actin filaments strengthens cell-cell cohesion and allows for junction contractility, which shapes epithelial tissues during development. This study reports that the cytoskeletal adaptor protein Girdin physically and functionally interacts with components of the cadherin-catenin complex during Drosophila embryogenesis. Fly Girdin is broadly expressed throughout embryonic development and enriched at the ZA in epithelial tissues. Girdin associates with the cytoskeleton and co-precipitates with the cadherin-catenin complex protein alpha-Catenin (alpha-Cat). Girdin mutations strongly enhance adhesion defects associated with reduced DE-cadherin (DE-Cad) expression. Moreover, the fraction of DE-Cad molecules associated with the cytoskeleton decreases in the absence of Girdin, thereby identifying Girdin as a positive regulator of adherens junction function. Girdin mutant embryos display isolated epithelial cell cysts and rupture of the ventral midline, consistent with defects in cell-cell cohesion. In addition, loss of Girdin impairs the collective migration of epithelial cells, resulting in dorsal closure defects. It is proposed that Girdin stabilizes epithelial cell adhesion and promotes morphogenesis by regulating the linkage of the cadherin-catenin complex to the cytoskeleton (Houssin, 2015).

This study suggests that Girdin is required for epithelial tissue morphogenesis and integrity. Specifically, Girdin coordinates collective cell migration, a function that probably depends on the ability of Girdin to organize the actin cytoskeleton. Moreover, the data indicate that Girdin strengthens cell-cell adhesion by promoting anchorage of the cadherin-catenin complex to the cytoskeleton. Girdin might realize this function by favoring the polymerization and organization of the cortical F-actin ring associated with the ZA. Alternatively, Girdin might be directly involved in the bridging of the cadherin-catenin complex to microfilaments, an intriguing possibility suggested by the association of Girdin with α-Cat. Multiple α-Cat interaction partners have actin-binding activity, and so do Girdin and mammalian Girdin. The data therefore contribute to the understanding of adherens junction regulation, which is crucial for epithelial tissue morphogenesis, physiology and homeostasis. It is likely that the function of Girdin in epithelial tissue cohesion and morphogenesis is evolutionarily conserved, as mammalian Girdin interacts with Par-3 that sustains cell-cell cohesion, and controls epithelial cyst formation in three-dimensional (3D) cell culture. In line with a putative role for mammalian Girdin in cell-cell adhesion, neuroblasts show cohesion defects in Girdin knockout mice. Thus, these data put into perspective the emerging idea that human Girdin is an interesting target to limit cell invasion in cancer. Girdin inhibition might exacerbate loss of cell-cell adhesion and cell dissemination in tumor cells with altered E-cadherin functions, as suggested by the strong enhancement of the shg zygotic mutant phenotype by loss of Girdin. A better understanding of Girdin functions will help to uncover whether this protein is an attractive target for therapeutic intervention (Houssin, 2015).

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. This study identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, it is concluded that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway (Puseenam, 2009).

Akt/PKB regulates actin organization and cell motility via Girdin/APE

The serine/threonine kinase Akt is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. This study identifies an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Database searches reveal homologs in mouse, rat, and Drosophila, but no apparent matches in Caenorhabditis elegans and Dictyostelium. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 is replaced with alanine, form abnormal elongated shapes and exhibit limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility (Enomoto, 2005).

The structure of Girdin predicted by the COILS algorithm showed a tendency to assume an alpha-helical coiled-coil conformation in its middle domain, between Ala-253 and Lys-1375, with a high coiled-coil probability of 1.0. The predicted coiled-coil domain contains 135 continuous heptad repeats ([abcdefg]135) that are typical of alpha-helical coiled-coils. The 9.5 kb Girdin transcript was found to be expressed ubiquitously in various human tissues by high-stringency Northern blot analysis (Enomoto, 2005).

Four different regions can be distinguished in the Girdin molecule based on the sequences of its subunits, subcellular localization, and functions: an N-terminal region that seems to facilitate the formation of a dimer (NT), an extremely long coiled-coil region, a region that binds to the plasma membrane through the interaction with phosphoinositides (CT1), and a C-terminal region that encompasses an actin binding site (CT2). The amino acid sequence of the CT2 domain shows no homology with the calponin homology (CH) domain, a common actin binding domain that is present in most actin binding proteins such as alpha-actinin, filamins, fimbrin, spectrins, cortexillins, and dystrophin, suggesting that Girdin represents a novel class of actin binding proteins (Enomoto, 2005).

Analysis of the sequence of Girdin reveals that it includes 135 heptad repeats, (abcdefg)135, between Leu-253 and Lys-1375 that correspond to a central rod domain. Within the repeats, positions a and d are preferentially occupied by hydrophobic residues like Leu, Ile, Met, or Val; this is consistent with the signature of canonical coiled-coil structures that wind around each other in a superhelix. The oligomerization properties of coiled-coil sequences are determined by the distribution of alpha-branched residues in the a or d positions. Val and Ile in position a favor dimerization, they favor tetramerization in position d, and their presence in both a and d positions facilitate the formation of trimers. In the coiled-coil sequence of Girdin, 22 repeats have Val or Ile in the a position, whereas they are present in the d position of only 9 repeats, suggesting that the coiled-coil domain of Girdin tends to form a dimer. This is consistent with the findings suggested by gel filtration that the NT domain of Girdin forms a dimer (Enomoto, 2005).

The possession of two actin binding sites enables crosslinking or bundling proteins to link filaments and to stabilize higher-order assemblies of actin filaments. Possessing two actin binding CT2 domains in juxtaposition, the dimeric Girdin molecules seem to be designed to gather actin filaments together into bundles or a meshwork. Consistent with this possibility are the findings of immunofluorescent staining and electron microscopy that the depletion of Girdin interfers with actin networks, leading to the disruption of stress fibers, cortical actin filaments, and actin meshwork at the leading edge. During migration, the Girdin knockdown cells produce multiple protrusions, resulting in limited directional migration. These observations indicate that Girdin fulfils an essential function in determining the stability and integrity of actin bundles and meshwork. These mediate a variety of important biological processes. Eukaryotic cells have a fail-safe mechanism in the multiple actin crosslinking proteins that share overlapping functions. The phenotypic consequences of the depletion of Girdin indicate that the presence of other proteins cannot completely compensate for its loss. Because the speculated primary structure, molecular size, and putative function of Girdin are reminiscent of those of filamin, it is important to clarify the functional difference and synergism between the two (Enomoto, 2005).

The CT1 domain of Girdin associates with the plasma membrane through the cluster of basic amino acid residues Arg-1389 to Lys-1407. This positively charged sequence is related to a consensus sequence for PI(4,5)P2 binding, which has been found in gelsolin, villin, profilin, vinculin, and other various cytoskeletal proteins. Unexpectedly, the basic amino acid cluster in Girdin does not bind to PI(4,5)P2, but binds to PI(4)P and binds weakly to PI(3)P. Considering that PI(4)P, but not PI(3)P, is abundant in mammalian cells, it is plausible to conclude that Girdin binds to PI(4)P, which resides in the membranes of mammalian cells in an amount equal to that of PI(4,5)P2. It is speculated that it stabilizes the cortical actin filaments by anchoring them at the plasma membrane (Enomoto, 2005).

Akt phosphorylates Girdin in vitro and in intact cells. The phosphorylation of Girdin is induced by EGF and during cell migration, suggesting a significance for phosphorylation in physiological cellular events. In migrating Vero fibroblasts, the phosphorylated Girdin preferentially localizes to lamellipodia at the leading edge, which is in line with observations that activated Akt is also localized at the leading edge during migration in mammalian cells. It is plausible that Akt, activated downstream of PI3K, translocates from the cytosol to the leading edge through its PH domain, and subsequently phosphorylates Girdin on the actin filaments at the front of the cells (Enomoto, 2005). How does Akt regulate the function of Girdin by phosphorylation? Insight into this issue comes from the observation that the phosphorylation of the CT domain of Girdin affects in vitro binding to PI(4)P. Because the phosphorylation site is present in the neighborhood of the phosphoinositide binding site, it is speculated that phosphorylation induces a conformational change around these sites, and this change in turn alters affinity for the phosphoinositide. It was further found that the phosphorylated CT domain retains the property of actin binding, and its affinity for F-actin is comparable to that of the nonphosphorylated form. Based on these observations, it is speculated that phosphorylation by Akt releases Girdin from PI(4)P and allows it to localize at the leading edge in order to crosslink the newly generated actin filaments in the lamellipodium network (Enomoto, 2005).

Search PubMed for articles about Drosophila Girdin

Aznar, N., Patel, A., Rohena, C. C., Dunkel, Y., Joosen, L. P., Taupin, V., Kufareva, I., Farquhar, M. G. and Ghosh, P. (2016). AMP-activated protein kinase fortifies epithelial tight junctions during energetic stress via its effector GIV/Girdin. Elife 5. PubMed ID: 27813479

Biehler, C., Wang, L. T., Sevigny, M., Jette, A., Gamblin, C. L., Catterall, R., Houssin, E., McCaffrey, L. and Laprise, P. (2020). Girdin is a component of the lateral polarity protein network restricting cell dissemination. PLoS Genet 16(3): e1008674. PubMed ID: 32196494

Chen, C., Enomoto, A., Weng, L., Taki, T., Shiraki, Y., Mii, S., Ichihara, R., Kanda, M., Koike, M., Kodera, Y. and Takahashi, M. (2020). Complex roles of the actin-binding protein Girdin/GIV in DNA damage-induced apoptosis of cancer cells. Cancer Sci. PubMed ID: 32875699

Enomoto, A., Murakami, H., Asai, N., Morone, N., Watanabe, T., Kawai, K., Murakumo, Y., Usukura, J., Kaibuchi, K. and Takahashi, M. (2005). Akt/PKB regulates actin organization and cell motility via Girdin/APE. Dev Cell 9(3): 389-402. PubMed ID: 16139227

Garcia-Marcos, M., Ghosh, P. and Farquhar, M. G. (2009). GIV is a nonreceptor GEF for G alpha i with a unique motif that regulates Akt signaling. Proc Natl Acad Sci U S A 106(9): 3178-3183. PubMed ID: 19211784

Ghosh, P., Garcia-Marcos, M., Bornheimer, S. J. and Farquhar, M. G. (2008). Activation of Galphai3 triggers cell migration via regulation of GIV. J Cell Biol 182(2): 381-393. PubMed ID: 18663145

Ghosh, P. (2017). The stress polarity pathway: AMPK 'GIV'-es protection against metabolic insults. Aging (Albany NY) 9(2): 303-314. PubMed ID: 28209925

Ha, A.1, Polyanovsky, A. and Avidor-Reiss, T. (2015). Drosophila Hook-Related Protein (Girdin) is essential for sensory dendrite formation. Genetics 200(4): 1149-1159. PubMed ID: 26058848

Hartung, A., Ordelheide, A. M., Staiger, H., Melzer, M., Haring, H. U. and Lammers, R. (2013). The Akt substrate Girdin is a regulator of insulin signaling in myoblast cells. Biochim Biophys Acta 1833(12): 2803-2811. PubMed ID: 23886629

Houssin, E., Tepass, U. and Laprise, P. (2015). Girdin-mediated interactions between cadherin and the actin cytoskeleton are required for epithelial morphogenesis in Drosophila. Development 142(10): 1777-1784. PubMed ID: 25968313

Ohara, K., Enomoto, A., Kato, T., Hashimoto, T., Isotani-Sakakibara, M., Asai, N., Ishida-Takagishi, M., Weng, L., Nakayama, M., Watanabe, T., Kato, K., Kaibuchi, K., Murakumo, Y., Hirooka, Y., Goto, H. and Takahashi, M. (2012). Involvement of Girdin in the determination of cell polarity during cell migration. PLoS One 7(5): e36681. PubMed ID: 22574214

Puseenam, A., Yoshioka, Y., Nagai, R., Hashimoto, R., Suyari, O., Itoh, M., Enomoto, A., Takahashi, M. and Yamaguchi, M. (2009). A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size. Exp Cell Res 315(19): 3370-3380. PubMed ID: 19560458

Sasaki, K., Kakuwa, T., Akimoto, K., Koga, H. and Ohno, S. (2015). Regulation of epithelial cell polarity by PAR-3 depends on Girdin transcription and Girdin-Galphai3 signaling. J Cell Sci 128(13): 2244-2258. PubMed ID: 25977476

Swanson, L., Katkar, G. D., Tam, J., Pranadinata, R. F., Chareddy, Y., Coates, J., Anandachar, M. S., Castillo, V., Olson, J., Nizet, V., Kufareva, I., Das, S. and Ghosh, P. (2020). TLR4 signaling and macrophage inflammatory responses are dampened by GIV/Girdin. Proc Natl Acad Sci U S A 117(43): 26895-26906. PubMed ID: 33055214

Wang, X., Enomoto, A., Weng, L., Mizutani, Y., Abudureyimu, S., Esaki, N., Tsuyuki, Y., Chen, C., Mii, S., Asai, N., Haga, H., Ishida, S., Yokota, K., Akiyama, M. and Takahashi, M. (2018). Girdin/GIV regulates collective cancer cell migration by controlling cell adhesion and cytoskeletal organization. Cancer Sci 109(11): 3643-3656. PubMed ID: 30194792

Wang, Y., Kaneko, N., Asai, N., Enomoto, A., Isotani-Sakakibara, M., Kato, T., Asai, M., Murakumo, Y., Ota, H., Hikita, T., Namba, T., Kuroda, K., Kaibuchi, K., Ming, G. L., Song, H., Sawamoto, K. and Takahashi, M. (2011). Girdin is an intrinsic regulator of neuroblast chain migration in the rostral migratory stream of the postnatal brain. J Neurosci 31(22): 8109-8122. PubMed ID: 21632933

date revised: 18 November 2020

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.