InteractiveFly: GeneBrief

Chromosome segregation 1: Biological Overview | References

Members of the Cas family of Src homology 3 (SH3)-domain-containing cytosolic signaling proteins are crucial regulators of actin cytoskeletal dynamics in non-neuronal cells; however, their neuronal functions are poorly understood. A Drosophila Cas (Crk-associated substrate: DCas) is highly expressed in neurons: DCas is required for correct axon guidance during development. Functional analyses reveal that Cas specifies axon guidance by regulating the degree of fasciculation among axons. These guidance defects are similar to those observed in integrin mutants, and genetic analysis shows that integrins function together with Cas to facilitate axonal defasciculation. These results strongly support Cas proteins working together with integrins in vivo to direct axon guidance events (Huang, 2007).

The identification of molecular cues that guide neuronal processes to their targets in vertebrates and invertebrates reveals fundamental mechanisms underlying the development of neural networks. These guidance cues include both membrane-bound and secreted proteins that instruct the navigation of axons and dendrites through both attraction and repulsion. Cell surface receptors for these guidance cues initiate the signal transduction pathways essential for steering neuronal processes. However, the signaling pathways initiated by guidance cue receptors, and the molecular mechanisms through which these signaling cascades alter axonal and dendritic cytoskeletal dynamics to direct attractive or repulsive steering events, are still poorly defined (Huang, 2007).

Some of the mechanisms that direct growing axons are similar to those that promote cell migration. Proteins originally characterized as important for cell migration through their effects on cytoskeletal dynamics have more recently been shown to play roles in axon guidance during development. Integrins are a large family of receptors crucial for promoting cell migration, supporting cellular adhesion to extracellular matrix (ECM) components, and regulating intracellular actin filament dynamics. Integrins also participate in directing growing axons to their targets. Genetic analyses in C. elegans, Drosophila and mice reveal that axon extension in integrin mutants in vivo is not compromised during development; however, defects in axon guidance result from loss of integrin function. In combination with in vitro and in vivo observations addressing the effects integrins and their ligands exert on growing axons, these studies define functions for integrin signaling in guiding growing axons, strongly suggesting that integrins are essential for axon guidance (Huang, 2007).

The molecular mechanisms by which integrins guide neuronal processes during development remain to be determined. Integrin receptors are heterodimers composed of a ligand-binding αα-subunit and a β-subunit that together signal through their cytoplasmic domains. Numerous molecules function downstream of integrin receptor activation to mediate cell migration, including tyrosine kinases such as focal adhesion kinase (FAK) and Src, Rho GTPases, focal adhesion proteins including Paxillin, and proteins in the Crk-associated substrate (Cas) family (Wiesner, 2005). Cas proteins are intriguing integrin signaling components because they are Src homology 3 (SH3)-domain-containing proteins that physically link integrins to kinases, phosphatases, guanine nucleotide exchange factors and proteins that nucleate actin filaments (Chodniewicz, 2004; O'Neill, 2000). Furthermore, Cas proteins transduce intracellular signals that stimulate actin filament assembly (Bouton, 2001) and act as force sensors during cell motility (Sawada, 2006). Do Cas proteins provide a crucial link between integrin activation and actin dynamics essential for guiding growing axons to their targets in vivo? This study shows that Cas proteins are highly expressed in the nervous system and together with integrins direct discrete axonal steering events during development (Huang, 2007).

Cas proteins play central roles in mediating cell migration and actin cytoskeletal reorganization, but their contributions to the establishment of nervous system connectivity remain to be determined. Therefore, attempts were made to understand how Cas proteins function during neural development using Drosophila as a model system. Several overlapping Drosophila EST sequences were identified with a high degree of similarity to human CAS (BCAR1-Human Gene Nomenclature Database), and these ESTs were localized to a single gene on the third chromosome that was named Drosophila Cas (DCas, CG1212; not to be confused with the Drosophila genes CAS/CSE1 segregation protein and castor). The DCas locus covers ~13 kb of genomic sequence and includes at least four exons. The longest DCas cDNA encodes a protein of 793 amino acids. In addition, a smaller DCas EST was identified that when translated is missing the DCas SH3 domain (Huang, 2007).

The Cas family includes the mammalian p130Cas (Cas, Bcar1), Cas-L (Hef1, Nedd9) and Efs (Sin) proteins, all of which contain, along with several additional conserved motifs, a highly conserved SH3 domain that is important for Cas-dependent cell migration. DCas, like vertebrate Cas proteins, includes this N-terminal SH3 domain and is 70% identical at the amino acid level to the SH3 domain of human CAS. Cas proteins are also characterized by their 'substrate' and 'serine-rich (Ser)' regions, which constitute a major portion of the Cas protein and contain several conserved tyrosine, serine and threonine phosphorylation sites. Many of these amino acid residues are conserved between DCas and human CAS, suggesting they are important for Cas function. Among these conserved tyrosine residues are five YxxP motifs that, when phosphorylated, serve as binding sites for proteins with Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains. These proteins include the adaptor proteins Crk and Nck (Dock) and also the phosphatase Ship2 (Inppl1). DCas also contains a stretch of conserved amino acids found in all vertebrate Cas proteins, YDYV, that serves as an Src tyrosine kinase-binding site. At its C-terminus, DCas includes residues, conserved in all mammalian Cas proteins, that are important for dimerization, suggesting that DCas might also exist as a dimer. Human CAS and EFS, but not CAS-L, also contain a proline-rich (PxxP) region immediately C-terminal to their SH3 domains. However, although DCas contains conserved proline residues both within this region and C-terminal to the serine-rich region, it is most similar to CAS-L. Therefore, Drosophila, unlike mammals, has only one Cas family member, eliminating issues of functional redundancy in assessing Cas contributions to neural development (Huang, 2007).

DCas expression during embryonic development was examined. In situ hybridization analysis using DCas antisense and sense probes showed that DCas mRNA is deposited maternally and is also highly expressed during embryonic development. During embryonic stages that include the period of axon pathfinding, high levels of DCas are observed in most, if not all, CNS neurons within the developing brain and nerve cord. DCas mRNA was also found at low levels in embryonic peripheral sensory neurons, including chordotonal organs, and in the developing musculature (Huang, 2007).

The distribution of endogenous DCas protein was examined. Attempts to generate a DCas antibody were unsuccessful. However, several different antibodies generated against Cas proteins were tested and an antibody against an epitope highly conserved between mammalian and Drosophila Cas (Fonseca, 2004) was found to recognizes DCas. Western analysis using this polyclonal antibody revealed that this vertebrate Cas antibody recognizes bands at ~105 kDa and ~140 kDa in Drosophila embryos, and that these bands are absent in embryos harboring a deletion of the DCas gene. In Drosophila embryos, DCas immunostaining was observed during neural development in a pattern consistent with that of DCas mRNA and with the expression of a ^MycDCas transgene under the control of a GAL4 enhancer trap insertion in DCas. DCas protein first appeared in motor and CNS projections during initial stages of axon outgrowth. At later embryonic stages, DCas immunostaining was present in neuronal cell bodies and in axons that contribute to all motor axon pathways. DCas immunostaining was also observed in the chordotonal sensory organs and in muscle attachment sites. Importantly, no Cas immunostaining was observed in embryos harboring a small deficiency that includes the DCas locus, showing that this Cas antibody generated against vertebrate Cas specifically recognizes DCas (Huang, 2007).

To further address the role of Cas proteins in neurons, it was asked whether any of the mammalian Cas proteins is also expressed in axons during development. p130Cas and Cas-L transcripts are found in the developing and postnatal brain, and p130Cas localizes to axons in early development and into adulthood in the spinal cord, cerebellum and cerebral cortex. Several antibodies that specifically recognize Cas proteins were used and their patterns of immunostaining in the developing rat spinal cord was tested. It was observed that mammalian Cas family members are indeed highly expressed in neurons during development (Huang, 2007).

To determine whether Cas proteins serve axon guidance functions during development, DCas mutant Drosophila embryos were examined. A search of public Drosophila stock collections identified two potential DCas loss-of-function (LOF) mutants as well as a large deficiency that removes DCas (Df(3L)ED201). One of these potential DCas LOF mutants, DCas^P1, is the GAL4-containing P-element transposon situated in the intron downstream from the start of DCas translation. Another potential DCas LOF mutant is a small deficiency (Df(3L)Exel6083) that removes DCas and five putative adjacent genes. No DCas mRNA transcript or immunostaining were observed in embryos homozygous for the DCas deficiencies, and very little DCas transcript or immunostaining was present in homozygous DCas^P1 embryos. These expression data, in combination with genetic experiments described below, show that Df(3L)Exel6083 is a DCas-null allele and that DCas^P1 is a DCas hypomorphic LOF allele (Huang, 2007).

DCas function was assessed in the development of the stereotypic Drosophila embryonic neuromuscular connectivity pattern as this is an excellent system to study axon guidance events, including motor axon fasciculation and defasciculation and target recognition. All motor axon trajectories can be easily observed in late stage 16 to early stage 17 embryos using the mAb 1D4 (anti-Fasciclin II), and individual motor axons can be followed to their targets. All five motor axon pathways, and also the transverse nerve, were found to be defective in DCas mutant embryos. The ISNb pathway in wild-type embryos, and also in DCas heterozygotes, is formed correctly by motor axons defasciculating from the ISN and extending dorsally through the ventral musculature to innervate muscles 6, 7, 12 and 13. In DCas mutants, ISNb axons exhibited highly penetrant axon guidance phenotypes, often failing to innervate muscles 6 and 7 and muscles 12 and 13. These guidance phenotypes include the failure of ISNb axons to defasciculate from the ISN, ISNb axons stalling within the ISNb following defasciculation from the ISN, ISNb axons bypassing their target muscles, and abnormal ISNb axonal trajectories to their targets. Characterization and quantification of these phenotypes reveal that these defects are indicative of increased motor axon fasciculation, as has been observed in the absence of several different guidance-cue signaling cascades that regulate fasciculation of developing Drosophila motor projections. In DCas mutant embryos, SNa motor axons and CNS axons also exhibit highly penetrant guidance defects consistent with increased axonal fasciculation. Phenotypes characteristic of decreased fasciculation are not observed to any appreciable extent in DCas LOF mutants. Importantly, no defects were observed in muscle integrity or neuronal cell fate determination in DCas LOF mutants (Huang, 2007).

To confirm that DCas axon guidance phenotypes result from decreased DCas function, DCas expression was restored in homozygous DCas mutant embryos using the GAL4-UAS system. The DCas^P1 allele was used to express ^MycDCas under control of what is likely to be the endogenous DCas promoter, as DCas^P1 is a GAL4-containing P-element insertion located downstream of the DCas ATG and drives gene expression in a pattern similar to that detected with a Cas antibody. Indeed, expression of the ^MycDCas transgene in a DCas mutant background that includes the DCas^P1 allele significantly rescued the embryonic ISNb axon guidance phenotypes. To confirm that this rescue results from DCas expression in neurons, UAS-^MycDCas was placed under the transcriptional control of the neuron-specific driver ELAV-GAL4, which drives robust expression of ^MycDCas in motor and CNS neurons. Neuronal ^MycDCas expression rescued ISNb, SNa and CNS DCas embryonic axon guidance defects. Therefore, axon guidance phenotypes observed in DCas mutant embryos result from a lack of neuronal DCas, demonstrating that DCas plays a key role in the regulation of motor axon defasciculation (Huang, 2007).

To further characterize DCas neuronal functions, it was asked whether neuronal overexpression of DCas affects axon pathfinding. Overexpression of ^MycDCas in all neurons in a wild-type background led to highly penetrant motor axon guidance phenotypes. Interestingly, most of these DCas gain-of-function (GOF) axon guidance defects are similar to those observed in DCas LOF mutants: reductions in motor axon defasciculation at characteristic choice points. For example, when one copy of the ^MycDCas transgene was expressed in all neurons with one copy of the ELAV-GAL4 driver in a wild-type background (designated '+'), ISNb axons often failed to innervate their muscle targets and instead remained fasciculated in the ISNb nerve. Increasing the levels of neuronal DCas (++) resulted in more-severe motor axon pathfinding defects, including the inability of ISNb axons to defasciculate from the ISN nerve, and extensive stalling and aberrant bundling of ISNb axons along their trajectory. Increasing neuronal DCas levels still further (+++) produced dramatic motor axon stalling within their nerve roots, just after their exit from the ventral nerve cord, resulting in noticeably fewer motor axons extending into the periphery. SNa motor axon guidance phenotypes in DCas neuronal GOF mutants were also similar to those observed in DCas LOF mutants and exhibited a similar dosage-dependent increase in the severity of abnormal fasciculation and stalling defects. It was also found that DCas GOF dramatically affects CNS axonal trajectories, producing phenotypes reminiscent of those observed in the absence of the Slit receptor Roundabout (Robo) or by increased Netrin attractive signaling. Increased fasciculation and stalling of CNS axons was also observed. As was the case for DCas LOF mutants, no defects were observed in neuronal cell fate in these neuronal DCas GOF mutants (Huang, 2007).

To better characterize mechanisms underlying these DCas GOF axon guidance defects, the DCas^P1-GAL4 driver was used to overexpress DCas in its endogenous temporal and spatial pattern. DCas overexpression using the DCas^P1-GAL4 driver resulted in axon guidance defects qualitatively and quantitatively similar to those observed using the ELAV-GAL4 driver. These results show that overexpression of DCas results in phenotypes strikingly similar to those observed in DCas LOF mutants: in both cases highly penetrant reductions were observed in the ability of motor axons to separate from their original trajectories, resulting in increased fasciculation of these axons. Taken together, these neuronal DCas LOF and GOF phenotypes show that although DCas is necessary for axon defasciculation, it is not sufficient, as DCas neuronal overexpression does not cause an increase in axonal defasciculation (Huang, 2007).

Next, attempts were made to better define the signaling pathway through which Cas regulates axon fasciculation. In mammals, Cas proteins function downstream of several different receptors in non-neuronal cells, including growth factor receptors, G-protein-coupled receptors, T-cell receptors, B-cell receptors and integrins. Interestingly, integrin receptor subunit mutations in Drosophila give rise to CNS and motor axon guidance defects that are strikingly similar to those observed in DCas mutants, suggesting that Cas might function together with integrin receptors to guide axons (Huang, 2007).

Drosophila integrins, like vertebrate integrins, are composed of an α-subunit and a ß-subunit. In Drosophila, there is one gene encoding a typical laminin-binding-type α-subunit (α1, called mew), one encoding an RGD-binding-type α-subunit (α2, called if), and a single ß-subunit gene (ß1, called mys) very similar to the prototype vertebrate ß1 receptor. To investigate the connection between integrin and Cas signaling, the role of integrin receptor function in embryonic motor axon pathfinding was revisited, and it was found that integrin-null mutant embryos exhibit defects that are qualitatively and quantitatively similar to DCas mutants. Embryos harboring null alleles for either α1 (mew^M6) or α2 (if^K27E) integrin genes exhibited ISNb and SNa axon guidance defects very similar to those observed in DCas mutants, including increased fasciculation resulting in the absence of muscle innervation. CNS axon guidance defects were also observed in both α1 and α2 integrin mutants that were similar to those observed in DCas mutants (Huang, 2007).

To further address DCas involvement in integrin-mediated axon guidance, dominant genetic interactions between DCas and integrin subunit LOF mutations were sought. Such transheterozygous interactions provide genetic support for two proteins functioning together in the same signaling pathway. It was asked whether removal of a single copy of DCas dominantly enhances heterozygosity at the α1, α2 or ß1 integrin loci. In α1, α2, ß1 or DCas heterozygotes, motor axon trajectories were not significantly different from wild type. However, removal of a single copy of DCas together with a single copy of α1, α2 or ß1 integrin resulted in highly penetrant axon guidance defects, suggesting that these three genes function in the same signaling pathway. Importantly, the phenotypes resulting from dominant enhancement by DCas are indicative of increased fasciculation, similar to those observed in DCas, α1 or α2 integrin LOF embryos (Huang, 2007).

To further assess the role integrins play in DCas-mediated axon guidance, it was asked whether ß1integrin LOF mutants dominantly suppress DCas GOF motor axon guidance phenotypes. The Drosophila ß1 integrin (encoded by mys¹) is the predominant neuronal ß1 integrin and therefore likely to mediate most, if not all, nervous system integrin signaling. If integrins are indeed necessary for activating DCas signaling, removing a single copy of ß1 integrin should suppress DCas GOF phenotypes. When low levels of DCas were expressed in all neurons in an otherwise wild-type background, moderate guidance defects were observed involving axons of the ISNb, SNa and CNS third longitudinal. Removing a single copy of the ß1 integrin gene in this same neuronal DCas GOF genetic background significantly rescued axon guidance phenotypes resulting from DCas GOF. Importantly, these results also show that neuronal overexpression of DCas does not simply function in a dominant-negative fashion to block integrin, or other, signaling pathways (Huang, 2007).

Taken together, analysis of integrin LOF mutations and the observation of robust genetic interactions between DCas and α1, α2 and ß1integrin mutants strongly support DCas serving a crucial role in transducing in vivo integrin-mediated signaling events essential for directing axon guidance. Furthermore, these results show that integrin/Cas signaling is necessary for axonal defasciculation events during neural development (Huang, 2007).

Therefore, the genetic analyses suggest that DCas functions with integrin receptors in vivo to mediate correct motor and CNS axon guidance, providing mechanistic insight into how integrins direct axon guidance events during development. In vitro studies define roles for Cas proteins in promoting cell migration, and Cas proteins have also been implicated as mediators of axon outgrowth and turning (Huang, 2006; Liu, 2007; Yang, 2002). Integrin effects on axon outgrowth have been studied extensively in vitro, but certain integrin functions suggested by these experiments have thus far not been reflected in the integrin mutant phenotypes observed in vivo. Examination of integrin mutant mice, flies and worms does not reveal a decrease in axon outgrowth during development. Indeed, in vivo functional analysis shows that one role of integrin/Cas signaling in neurons during development is to regulate the degree of fasciculation, or adhesion, among axons. Drosophila embryonic motor axons have proven useful for modeling these types of fasciculation defects and identifying the genes that govern them. Loss of DCas or integrin receptors from Drosophila motor neurons results in axons remaining abnormally fasciculated, growing past their normal defasciculation choice points, or sometimes stalling at these choice points. DCas neuronal overexpression produces similar hyperfasciculation defects, dependent upon integrins, including abnormal bundling and stalling of motor axons. Although other interpretations are possible, these results suggest a model in which integrin/Cas signaling normally serves to slow growing axons at choice points in vivo: experimentally decreasing integrin/Cas signaling attenuates adhesive functions that serve to slow growing axons, whereas increasing integrin/Cas signaling results in excessive adhesion among axons, or between axons and other substrates upon which they extend (Huang, 2007).

Supporting this model for how integrins and Cas regulate axonal fasciculation and pathfinding is extensive work on neuronal integrin functions in vitro. Growing axons and migrating cells preferentially elongate on surfaces to which they adhere most strongly, including integrin ligands. How might adhesive interactions influence axonal guidance decisions in vivo? Neuronal growth cones tend to form extensive lamellae, which are indicative of strong adhesive interactions, when cultured on highly adhesive substrata containing integrin ligands. These adhesive interactions stabilize elongating nerve fibers by promoting filopodial extension and expansion of growth cone surfaces. Disruption of axon-substrate attachment in vitro with integrin function-blocking antibodies encourages axon-axon adhesive interactions (fasciculation) in place of axon-substrate adhesion. Furthermore, contact with integrin ligands can slow axon elongation, as axons encountering an increasing gradient of laminin peptide exhibit reduced velocity, but growth cone velocity returns to previous rates when axons turn down the gradient. This in vitro observation resembles in vivo situations in which growth cones slow at a choice point, exhibit increased morphological complexity and then extend along distinct pathways. Drosophila motor axon growth cones also exhibit similar changes in morphological complexity upon contacting different substrates in vivo, suggesting that similar processes function to generate motor axon trajectories. Different combinations of integrin ligands might be responsible for these effects. When vertebrate growth cones in vitro contact either the α1 or α2 integrin ligands, laminin and fibronectin respectively, they decelerate, pause and exhibit short-term growth arrest. Interestingly, in vivo observations show that DCas functions with both α1 (laminin-binding) and α2 [RGD (e.g. Tiggrin)-binding] integrins to mediate correct axon navigation by regulating motor axon fasciculation at choice points, suggesting that integrin/Cas-mediated spatial regulation of growth cone extension underlies correct navigation at these choice points (Huang, 2007).

The molecular mechanisms underlying integrin-mediated axon guidance remain to be completely defined. However, results derived from analysis of integrin/Cas signaling on cell migration shed light on how Cas and integrins might specify axonal defasciculation events in vivo. During cell migration, Cas proteins serve to establish linkage between migrating cells and the ECM. Cas plays an important role in regulating cytoskeletal organization, cell adhesion and force sensing, and fibroblasts isolated from p130Cas-null mutant mouse embryos exhibit disorganized and short actin filaments and decreased cell migration (Cho, 2000; Honda, 1999; Honda, 1998). In non-neuronal cells, Cas becomes phosphorylated in response to integrin engagement by many ECM components, including fibronectin and laminin (Defilippi, 2006). FAK and Src family kinases have been implicated in integrin-dependent phosphorylation of Cas. Interestingly, recent in vitro observations reveal that FAK signaling at sites of integrin-mediated adhesion controls axon pathfinding. Furthermore, pharmacological inhibitors of Src family kinases decrease the level of neuronal phosphorylated Cas in vitro (Huang, 2006; Liu, 2007), supporting a role for Src kinases in regulating Cas proteins in neurons. Finally, the activity of Rho-family small GTPases is also regulated by Cas interactions with the guanine nucleotide exchange factor Dock180 (Dock1) (Defilippi, 2006). Taken together, these links between Cas signaling components and cytoskeletal reorganization suggest that some of these signaling proteins might also influence axon guidance in vivo during development (Huang, 2007).

The results demonstrate that integrin/Cas-mediated signaling is necessary but not sufficient for axonal defasciculation, revealing that integrin/Cas-mediated axon guidance must be integrated with other axon guidance signaling cascades to regulate axon defasciculation events during development. The identity of these other axon guidance pathways is not known. The attractive/permissive guidance cue Netrin binds to integrins, and functions with integrins in non-neuronal cells. The Netrin receptor Deleted in colorectal cancer (DCC) has been found to utilize the integrin effector FAK and recently p130Cas, to mediate Netrin-dependent attractive growth cone steering (Li, 2004; Liu, 2004; Liu, 2007; Ren, 2004). Ephrins, best known for their role as repulsive axon guidance cues, also induce cell adhesion and actin cytoskeletal changes in fibroblasts in a p130Cas-dependent manner (Carter, 2002). Repulsive axon guidance cues may also regulate integrin/Cas-dependent axon guidance during development. The axonal repellent Slit genetically interacts with integrins and their ligands to guide commissural axons in Drosophila. Semaphorin and Ephrin-mediated repulsive effects on non-neuronal cells also appear to involve inhibition of integrin signaling events. Interestingly, a crucial component of semaphorin-dependent repulsive axon guidance, a member of the molecule interacting with Cas-L (MICAL) family, physically associates with Cas-L (Suzuki, 2002; Terman, 2002) and preliminary data suggest that these interactions are functionally important for axon guidance. The observation that Cas functions with integrins to mediate axon guidance during development suggests new directions to better understand how integrin/Cas signaling modulates neuronal guidance through interactions with distinct axon guidance signaling pathways (Huang, 2007).

Dcas supports cell polarization and cell-cell adhesion complexes in development

Mammalian Cas proteins regulate cell migration, division and survival, and are often deregulated in cancer. However, the presence of four paralogous Cas family members in mammals (BCAR1/p130Cas, EFS/Sin1, NEDD9/HEF1/Cas-L, and CASS4/HEPL) has limited their analysis in development. The single Drosophila Cas gene, Dcas, was deleted to probe the developmental function of Dcas. Loss of Dcas had limited effect on embryonal development. However, Dcas is an important modulator of the severity of the developmental phenotypes of mutations affecting integrins (If and mew) and their downstream effectors Fak56D or Src42A. Strikingly, embryonic lethal Fak56D-Dcas double mutant embryos had extensive cell polarity defects, including mislocalization and reduced expression of E-cadherin. Further genetic analysis established that loss of Dcas modified the embryonal lethal phenotypes of embryos with mutations in E-cadherin (Shg) or its signaling partners p120- and beta-catenin (Arm). These results support an important role for Cas proteins in cell-cell adhesion signaling in development (Tikhmyanova, 2010).

This work identifies a strong interaction between the Dcas, and integrin pathway genes, including integrins and their effector kinases Fak56D and Src42A, during early embryonal development in Drosophila. The synthetic lethal phenotypes found in double mutants of Dcas and Src or FAK56D were marked by defects in dorsal closure and in some cases by the appearance of anterior cuticle holes that suggested head involution defects. These defects were commonly accompanied by abnormalities in epithelial function, including failure to appropriately localize shg/E-cadherin to cell junctions, and reduced shg expression. The data are compatible with the idea that either Fak56D or Dcas is sufficient to support shg/E-cadherin localization and cell polarization during morphogenetic movements in Drosophila embryos, but the absence of both cannot be sustained (Tikhmyanova, 2010).

Building from these observations, a novel synthetic lethal relationship was established between DCas, shg, and arm. As with crosses to alleles of Fak56D and Src42A, the point of lethality was at the time of dorsal closure, at embryonal stages 15-16, and associated with defective cuticle formation. One way to integrate these observations is to hypothesize that the DCas, Fak56D, and Shg protein products are normally in dynamic balance, with Dcas regulating Shg cycling. The fact that Crb and Dlg1, a mammalian homolog of Dlg, have been reported to support Shg localization to adherens junctions, suggests that Dcas/Fak56/Src42A specifically interact to support this cell polarity/cell junctional control system. In this context, it is suggestive that the Crb family protein CRB3 has been described as part of a complex including CRB3, Pals1, and PatJ that becomes tightly associated with Src kinase during reorganization of cell polarity. In the absence of DCas and Fak56D, Shg cannot localize properly; the moderately elevated levels of Shg proteins found in these embryos most likely arises as part of a cellular compensatory mechanism in response to decreased functional Shg signaling complexes. In further indirect support of the idea that this is a specific Dcas action, the fact that genetic interactions were not observed between Dcas¹ and Aur or Dock indicates that Dcas does not promiscuously interact with other genetic lesions to reduce viability (Tikhmyanova, 2010).

A previous study demonstrated a role for Dcas in axonal guidance in the development of the nervous system of adult flies (Huang, 2007). That work analyzed the hypomorphic Dcas mutant allele Dcas^P1, and the small deficiency Df(3L)Exel6083, including Dcas and five adjacent genes, which were also used in this study. The earlier study focused exclusively on analyzing the contribution of Dcas to axonal guidance in late (stage 16/17) embryos: in that analysis, Dcas functioned similarly to integrins, and genetically interacted with integrins (if, mew, and mys) in regulating axon guidance and axonemal defasciculation. In this context, it is intriguing that the mammalian Cas family NEDD9 gene is abundant during neuronal development, has been proposed as a candidate locus for oral cleft defects in humans based on its chromosomal location near the OFC-1 locus. Together these findings raise the possibility that this specific Dcas paralog has a specific role in human neuronal migration and morphogenesis of the head. As with the current data using the new Dcas¹ allele, homozygous deletion of Dcas in conjunction with integrins had moderate effect on viability of adult flies, although this work for the first time demonstrates an interaction between Dcas and if and mew, and also between Dcas and Src, in regulation of wing development (Tikhmyanova, 2010).

Generation of the first null allele of Dcas provides a useful new tool to study the role of this protein in Drosophila development. This work illuminates the evolutionary conservation of Dcas function within the integrin and receptor tyrosine kinase network, including FAK, Src, and integrins genes. The finding that a low percentage of embryos with mutant Dcas and all embryos with double mutations in Dcas and Fak56D, have perturbed localization of polarity markers, including Shg, indicates a novel function for Cas family in regulation of cell polarity. To date, the evidence directly connecting Cas proteins to a known mechanism for control of cell polarity is sparse. Although NEDD9 was in fact discovered in a functional genomics screen for cell cycle and polarity modifiers in budding yeast (leading to its designation as HEF1, Human Enhancer of Filamentation 1), the mechanism involved was not established, and given the great evolutionary distance involved, may not be relevant to a role in metazoans. Both BCAR1 and NEDD9 interact physically with proteins that influence cell polarity controls during pseudopod extension and other actin polarization processes: these include the GTP exchange factor AND-34 (Tikhmyanova, 2010).

The data in the present study indicating genetic interactions with cell-cell junction regulatory proteins Shg, Arm and p120-catenin may have considerable significance in the sphere of cancer research, as it implies that overexpression of Cas proteins may promote cancer progression by influencing the polarized movement of cells and influencing lateral attachments. The fact that one report has indicated interactions between BCAR1 and nephrocystins at cell-cell junctions in polarized epithelial cells implies that a potentially direct interaction of Cas proteins in these structures is conserved through mammals. However, given the known interactions of Cas proteins with FAK and SRC at focal adhesions, another possibility is that Cas may additionally or alternatively impact Shg function through indirect signaling emanating from these structures. Notably, it has been reported that NEDD9 overexpression induced by dioxin caused downregulation of E-cadherin, and it will be of great interest to study the consequences of overexpressing Dcas on Drosophila development. Consequences for loss of NEDD9 expression on E-cadherin expression or localization are not yet known. Resolving these questions will provide intriguing directions for future studies (Tikhmyanova, 2010).

Dcas is required for importin-α3 nuclear export and mechano-sensory organ cell fate specification in Drosophila

The in vivo function and tissue specificity of Dcas, the Drosophila ortholog of CAS, the importin β-like export receptor for importin α. While dcas mRNA is specifically expressed in the embryonic central nervous system, Dcas protein is maternally supplied to all embryonic cells and its nuclear/cytoplasmic distribution varies in different tissues and times in development. Unexpectedly, hypomorphic alleles of dcas show specific transformations in mechano-sensory organ cell identity, characteristic of mutations that increase Notch signaling. Dcas is essential for efficient importin-α3 nuclear export in mechano-sensory cells and the surrounding epidermal cells and is indirectly required for the import of one component of the Notch pathway, but not others tested. The specificity of the dcas phenotype is interpreted as indicating that one or more Notch signaling components are particularly sensitive to a disruption in nuclear protein import. It is proposed that mutations in house keeping genes often cause specific developmental phenotypes, such as those observed in many human genetic disorders (Tekotte, 2002).

Nucleocytoplasmic transport is essential in all eukaryotic cells. Considerable progress in understanding the mechanism of nuclear import and export has been made, largely through in vivo analyses in yeast and in vitro assays in permeabilized cells. The transport of different protein cargo is mediated by members of the importin β (impβ)/karyopherin β family of transport receptors, which function as importins and exportins. This family of proteins share a highly homologous Ran binding domain, first identified in impβ, which mediates nuclear localization signal (nls)-dependent import. Other members of the family include, CRM1, the export receptor for nuclear export signal (NES) containing proteins and transportin, the import receptor for proteins containing the M9 shuttling signal (Tekotte, 2002).

Cargo proteins that carry NES or M9 domains bind directly to impβ family members, whereas proteins with a 'classical' nuclear localization signal (NLS) bind indirectly to impβ via an association with an adaptor protein, importin β (impβ)/karyopherinβ. The impα family is highly conserved with one member in S. cerevisiae, Srp1p, and at least six in humans, which show some tissue specificity. The impα adaptors themselves require CAS {cellular apoptosis susceptibility factor, (Brinkmann, 1995), subsequently referred to as human CAS}, an impβ-like exportin, for their re-export to the cytoplasm following protein import (Kutay, 1997). The directionality of transport of cargo and recycling of transport receptors and adaptors is ensured in most cases through the action of the small GTPase Ran. RanGTP concentrations are high in the nucleoplasm, where it promotes the dissociation of importins from their cargo by binding to the Ran binding domain of the recepto. Exportins in contrast only export their cargo when bound to RanGTP in the nucleus. Ran GDP is found at high levels in the cytoplasm, where it promotes the dissociation of exportins from their cargo after export is achieved (Tekotte, 2002).

While it is clear that most aspects of nucleocytoplasmic transport are essential for cell viability in multicellular organisms, the extent to which particular receptors display tissue specificity or have regulatory roles is poorly understood. It is also not clear to what extent different tissues and developmental processes display varying requirements for transport components. This study addressed these issues by studying the expression patterns of a number of transport receptors and one importin-α during Drosophila melanogaster embryonic development. It was found that while Drosophila CRM1/embargo (emb), Transportin (Trn) and importin-α3 (imp-α3) mRNA are largely ubiquitously expressed, Drosophila dcas mRNA shows distinct tissue specificity. dcas mRNA is maternally supplied and disappears at the midblastoderm stage. Later dcas is zygotically expressed in the embryonic central nervous system (CNS). Strong dcas mutations abolish the embryonic nervous system specific dcas mRNA expression, but develop normally and later die as 1st instar larvae. These observations are consistent with the fact that maternally supplied Dcas protein persists throughout embryogenesis (Tekotte, 2002).

Surprisingly, hypomorphic dcas mutations lead to very specific cell fate transformations in the peripheral nervous system (PNS). The wild-type mechano-sensory organs of the PNS consist of two outer cells, the shaft (bristle) and socket, and three inner cells: the neuron, sheath and a less prominent glial cell, that migrates away from the other cells. The organ is derived from a single mechano-sensory organ precursor (SOP) that is selected from a sheet of epithelial cells by lateral inhibition of neuronal cell fate. Each SOP divides asymmetrically in a fixed lineage, initially to generate two secondary precursor cells, pIIa and pIIb. Subsequently, pIIa divides to produce the shaft and socket and pIIb divides to generate the neuron and the sheath. Each SOP gives rise to a stereotypic complement of cells through the segregation of determinants into one of each of the two daughter cells. Such determinants include the membrane-associated Numb protein that binds and antagonizes Notch signaling (Tekotte, 2002).

In hypomorphic dcas mutants almost all shafts are transformed into sockets leading to a double socket phenotype in pupae and adults. Furthermore, in stronger alleles inner cells are often transformed into sockets, resulting in triple and quadruple sockets. Such phenotypes are characteristic of mutations that cause an increase in Notch signaling. The Notch receptor functions in restricting neural-fate specification during lateral inhibition. Once cleaved, the Notch intracellular domain is imported into the nucleus, where it activates the transcription of downstream genes together with its co-activator, Suppressor of Hairless {Su(H)}. Hairless (H) is a Notch antagonist, thought to act by inhibiting Su(H) function (Tekotte, 2002).

This study shows that imp-α3 accumulates in the nucleoplasm of all cells of the developing mechano-sensory organ and the surrounding epidermal cells, suggesting that Dcas is the export receptor for imp-α3 and presumably the other imp-α family members in Drosophila. Taken together, these results suggest that Dcas is a ubiquitous export receptor required in all tissues throughout development. The possible reasons why dcas mutations show defects characteristic of developmental control genes is discussed, and it is proposed that tissue-specific requirements for imp-α function in nuclear protein import could provide the answer (Tekotte, 2002).

Drosophila importin α1 performs paralog-specific functions essential for gametogenesis

Importin αs mediate nuclear transport by linking nuclear localization signal (NLS)-containing proteins to importin beta1. Animal genomes encode three conserved groups of importin α's, α1's, α2's, and α3's, each of which are competent to bind classical NLS sequences. Using Drosophila melanogaster, the isolation and phenotypic characterization of the first animal importin α1 mutant (Drosophila karyopherin α1) is described. Animal α1's are more similar to ancestral plant and fungal α1-like genes than to animal α2 and α3 genes. Male and female importin α1 (Dα1) null flies developed normally to adulthood (with a minor wing defect) but were sterile with defects in gametogenesis. The Dα1 mutant phenotypes were rescued by Dα1 transgenes, but not by Dα2 or Dα3 transgenes. Genetic interactions between the ectopic expression of Dα1 and the karyopherins CAS and importin β1 suggest that high nuclear levels of Dα1 are deleterious. It is concluded that Dα1 performs paralog-specific activities that are essential for gametogenesis and that regulation of subcellular Dα1 localization may affect cell fate decisions. The initial expansion and specialization of the animal importin α-gene family may have been driven by the specialized needs of gametogenesis. These results provide a framework for studies of the more complex mammalian importin α-gene family (Ratan, 2008).

The genetic interactions between coectopic expression of Dα1 and Dcas and Ketel are consistent with the idea that the tergite defects and lethality are the result of increases in the levels of importin α in nuclei. Genetic manipulations that would be expected to decrease nuclear levels of Dα1 (overexpression of Dcas or loss-of-function Ketel mutants) mitigated the effects of overexpressing Dα1. Likewise, manipulations that would be expected to increase nuclear levels of Dα1 (overexpression of Ketel or loss-of-function Dcas mutants) enhanced Dα1 overexpression phenotypes. Interestingly, an increase in cNLS cargo levels also enhanced the Dα1 overexpression defects. Here, higher cNLS cargo levels could be expected to recruit more Dα1 into targeting complexes with importin β1 (Ketel), resulting in higher steady state nuclear levels of Dα1. Taken together, these results argue that higher than normal nuclear levels of Dα1 are deleterious, and that the nucleocytoplasmic trafficking of nuclear transport factors must be carefully balanced during development (Ratan, 2008).

Search PubMed for articles about Drosophila Cas

Brinkmann, U., Brinkmann, E., Gallo, M., and Pastan, I. (1995). Cloning and characterization of a cellular apoptosis susceptibility gene, the human homolog to the yeast chromosome segregation gene Cse1. Proc. Natl. Acad. Sci. 92: 10427-10431. PubMed ID: 7479798

Bouton, A. H., Riggins, R. B. and Bruce-Staskal, P. J. (2001). Functions of the adapter protein Cas: signal convergence and the determination of cellular responses. Oncogene 20: 6448-6458. PubMed ID: 11607844

Carter, N., Nakamoto, T., Hirai, H. and Hunter, T. (2002). EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas). Nat. Cell Biol. 4: 565-573. PubMed ID: 1213415

Cho, S. Y. and Klemke, R. L. (2000). Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix. J. Cell Biol. 149: 223-236. PubMed ID: 10747099

Chodniewicz, D. and Klemke, R. L. (2004). Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold. Biochim. Biophys. Acta 1692: 63-76. PubMed ID: 15246680

Defilippi, P., Di Stefano, P. and Cabodi, S. (2006). p130Cas: a versatile scaffold in signaling networks. Trends Cell Biol. 16: 257-263. PubMed ID: 16581250

Fonseca, P. M., Shin, N. Y., Brabek, J., Ryzhova, L., Wu, J. and Hanks, S. K. (2004). Regulation and localization of CAS substrate domain tyrosine phosphorylation. Cell. Signal 16: 621-629. PubMed ID: 14751547

Honda, H., et al. (1998). Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas. Nat. Genet. 19: 361-365. PubMed ID: 9697697

Honda, H., Nakamoto, T., Sakai, R. and Hirai, H. (1999). p130(Cas), an assembling molecule of actin filaments, promotes cell movement, cell migration, and cell spreading in fibroblasts. Biochem. Biophys. Res. Commun. 262: 25-30. PubMed ID: 10448062

Huang, J., Sakai, R. and Furuichi, T. (2006). The docking protein Cas links tyrosine phosphorylation signaling to elongation of cerebellar granule cell axons. Mol. Biol. Cell 17(7): 3187-96. PubMed ID: 16687575

Huang, Z., et al. (2007). Crk-associated substrate (Cas) signaling protein functions with integrins to specify axon guidance during development. Development 134: 2337-2347. PubMed ID: 17537798

Kutay, U., Bischoff, F. R., Kostka, S., Kraft, R. and Gorlich, D. (1997). Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell 90: 1061-1071. PubMed ID: 9323134

Li, W., et al. (2004). Activation of FAK and Src are receptor-proximal events required for netrin signaling. Nat. Neurosci. 7: 1213-1221. PubMed ID: 15494734

Liu, G., Beggs, H., Jurgensen, C., Park, H. T., Tang, H., Gorski, J., Jones, K. R., Reichardt, L. F., Wu, J. and Rao, Y. (2004). Netrin requires focal adhesion kinase and Src family kinases for axon outgrowth and attraction. Nat. Neurosci. 7: 1222-1232. PubMed ID: 15494732

Liu, G., Li, W., Gao, X., Li, X., Jurgensen, C., Park, H. T., Shin, N. Y., Yu, J., He, M. L., Hanks, S. K. et al. (2007). p130(CAS) is required for netrin signaling and commissural axon guidance. J. Neurosci. 27: 957-968. PubMed ID: 17251438

O'Neill, G. M., Fashena, S. J. and Golemis, E. A. (2000). Integrin signalling: a new Cas(t) of characters enters the stage. Trends Cell Biol. 10: 111-119. PubMed ID: 10675905

Ratan, R., Mason, D. A., Sinnot, B., Goldfarb, D. S. and Fleming, R. J. (2008). Drosophila importin α1 performs paralog-specific functions essential for gametogenesis. Genetics 178(2): 839-50. PubMed ID: 18245351

Ren, X. R., Ming, G. L., Xie, Y., Hong, Y., Sun, D. M., Zhao, Z. Q., Feng, Z., Wang, Q., Shim, S., Chen, Z. F. et al. (2004). Focal adhesion kinase in netrin-1 signaling. Nat. Neurosci. 7: 1204-1212. PubMed ID: 15494733

Sawada, Y., Tamada, M., Dubin-Thaler, B. J., Cherniavskaya, O., Sakai, R., Tanaka, S. and Sheetz, M. P. (2006). Force sensing by mechanical extension of the Src family kinase substrate p130Cas. Cell 127: 1015-1026. PubMed ID: 17129785

Suzuki, T., Nakamoto, T., Ogawa, S., Seo, S., Matsumura, T., Tachibana, K., Morimoto, C. and Hirai, H. (2002). MICAL, a novel CasL interacting molecule, associates with vimentin. J. Biol. Chem. 277: 14933-14941. PubMed ID: 11827972

Tekotte, H., et al. (2002). Dcas is required for importin-α3 nuclear export and mechano-sensory organ cell fate specification in Drosophila. Dev. Biol. 244: 396-406. PubMed ID: 11944946

Terman, J. R., Mao, T., Pasterkamp, R. J., Yu, H. H. and Kolodkin, A. L. (2002). MICALs, a family of conserved flavoprotein oxidoreductases, function in plexin-mediated axonal repulsion. Cell 109: 887-900. PubMed ID: 12110185

Tikhmyanova, N., et al. (2010). Dcas supports cell polarization and cell-cell adhesion complexes in development. PLoS One 5(8): e12369. PubMed ID: 20808771

Wiesner, S., Legate, K. R. and Fassler, R. (2005). Integrin-actin interactions. Cell. Mol. Life Sci. 62: 1081-1099. PubMed ID: 15761669

Yang, L. T., Alexandropoulos, K. and Sap, J. (2002). c-SRC mediates neurite outgrowth through recruitment of Crk to the scaffolding protein Sin/Efs without altering the kinetics of ERK activation. J. Biol. Chem. 277: 17406-17414. PubMed ID: 11867627

date revised: 20 June 2012

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