misshapen

Transcriptional Regulation

In the course of a screen designed to identify genes regulated by the photoreceptor transcription factor Glass, a set of lethal non-complementing P-element insertions mapping to 62E6-7 were isolated. Expression of lacZ from these insertions is completely Glass-dependent in photoreceptors. This gene has been named misshapen (Treisman, 1997).

Targets of Activity

To determine whether Msn activates Basket/JNK, cultured cells were transfected with msn together with an epitope-tagged JNK, and kinase activity assays were performed on JNK precipitates. Overexpression of either msn or mammalian NIK leads to about a four- to fivefold increase in JNK kinase activity as assessed by in vitro kinase reaction. In agreement with previous studies using NIK, a mutation abolishing Msn's kinase activity markedly reduces Msn's ability to activate JNK. The ability of Msn to activate JNK was confirmed by examining its effect on an activated transcription factor 2 (ATF2)-stimulated luciferase reporter gene; JNK has been shown to phosphorylate and activate ATF2. Overexpression of Msn in cultured cells leads to an approximately 10-fold increase in the transcriptional activity of ATF2. These findings indicate that msn and NIK are both structurally and functionally similar and suggests that msn may function to activate JNK in Drosophila (Su, 1998).

The role of DJNK in dorsal closure is to phosphorylate and activate Djun, resulting in transcriptional activation of the dpp gene at the leading edge of the dorsal epidermis. In turn, the binding of Dpp to its receptors Thick veins (Tkv) and Punt (Put) on the ventrally adjacent epithelial cells induces reorganization of the cytoskeleton, leading to epithelial cell elongation and subsequent closure over the amnioserosa. This is supported by the findings that dpp expression is decreased in the dorsal-most epithelial cells in embryos lacking bsk and hep, and that expression of activated forms of Djun or Tkv rescues embryos zygotically mutant for bsk or Jra. Therefore, if msn functions to initiate dorsal closure by activating Basket/JNK, dpp expression in the leading edge epithelial cells should be decreased in msn mutant embryos. In agreement with the idea that msn functions upstream of bsk, dpp expression in leading edge cells surrounding the amnioserosa is observed to be decreased in embryos lacking msn to a degree comparable to bsk¹ embryos. About 20% of embryos derived from the msn¹⁰²/+; msn¹⁰²/+ cross display a decrease in dpp expression in the dorsal leading edge. The decrease in dpp staining in msn mutant embryos is limited to the dorsal rim cells (Su, 1998 and references)

Protein Interactions

Two families of protein kinases that are closely related to Ste20 in their kinase domain have been identified: the p21-activated protein kinase (Pak) and SPS1 families. In contrast to Pak family members, SPS1 family members do not bind and are not activated by GTP-bound p21Rac and Cdc42. A member of the SPS1 family, called Misshapen (Msn), has been placed genetically upstream of the c-Jun amino-terminal (JNK) mitogen-activated protein (MAP) kinase module in Drosophila. The failure to activate JNK in Drosophila leads to embryonic lethality due to the failure of these embryos to stimulate dorsal closure. Msn probably functions as a MAP kinase kinase kinase kinase in Drosophila, activating the JNK pathway via an as yet undefined MAP kinase kinase kinase. A Drosophila TNF-receptor-associated factor, DTRAF1, has been identified by screening for Msn-interacting proteins using the yeast two-hybrid system. In contrast to the mammalian TRAFs that have been shown to activate JNK, DTRAF1 lacks an amino-terminal 'Ring-finger' domain, and overexpression of a truncated DTRAF1, consisting of only its TRAF domain, activates JNK. Another DTRAF, DTRAF2, has been identified that contains an amino-terminal Ring-finger domain. Msn specifically binds the TRAF domain of DTRAF1 but not that of DTRAF2. The region between the kinase and C-terminal regulatory domains of Msn is sufficient to bind DTRAF1, whereas neither the kinase domain nor the C-terminal domain alone can bind the protein. A stretch of about 250 amono acids that lies in the N-terminal portion of the interdomain is sufficient for Msn to bind DTRAF1. The C-terminal portion of this region does not interact with DTRAF1 but does interact with the SH3 domains of Dreadlocks, supporting the idea that the central region couples Msn and related Ste20 kinases to multiple upstream targets. DTRAF1 and DTRAF2 can dimerize with themselves but cannot form heterodimers. In Drosophila, DTRAF1 is thus a good candidate for an upstream molecule that regulates the JNK pathway by interacting with, and activating, Msn. Consistent with this idea, expression of a dominant-negative Msn mutant protein blocks the activation of JNK by DTRAF1. Furthermore, coexpression of Msn with DTRAF1 leads to the synergistic activation of JNK. A truncated Msn lacking the kinase domain functions as a dominant-negative inhibitor by blocking activation of JNK by DTRAF1. Some of these observations are extended to the mammalian homolog of Msn, Nck-interacting kinase (NIK), suggesting that TRAFs also play a critical role in regulating Ste20 kinases in mammals (Liu, 1999).

Recent studies suggest that the SH2/SH3 adaptor Dock/Nck transduces tyrosine phosphorylation signals to the actin cytoskeleton in regulating growth cone motility. The signaling cascade linking the action of Dock/Nck to the reorganization of cytoskeleton is poorly understood. Dock is shown to interact with the Ste20-like kinase Misshapen (Msn) in the Drosophila photoreceptor (R cell) growth cones. Loss of msn causes a failure of growth cones to stop at the target, a phenotype similar to loss of dock, whereas overexpression of msn induces pretarget growth cone termination. Physical and genetic interactions between Msn and Dock indicate a role for Msn in the Dock signaling pathway. It is proposed that Msn functions as a key controller of growth cone cytoskeleton in response to Dock-mediated signals (Ruan, 1999).

To investigate the potential role of msn in R cell growth cones, the effect of msn mutations on R cell projections was assessed. As strong loss-of-function alleles of msn are embryonic lethal, R cell projections were examined in third-instar larvae homozygous for a hypomorphic allele of msn [msn^l(3)03349]. While in msn mutants R cell growth cones are able to extend into the developing optic lobe, their innervation patterns within the lamina and the medulla are altered. The msn phenotype exhibits a certain similarity to that of dock loss-of-function mutants. In dock mutants, many R1-R6 growth cones pass over their normal target (i.e., lamina) and extend further into the medulla layer, generating gaps in the lamina R1-R6 termination site (a smooth continuous line of immunoreactivity in wild type). In addition, dock affects R cell fasciculation and growth cone morphology. Similarly, it has been found that loss of msn function causes defects in R cell targeting and fasciculation; gaps are observed in the R1-R6 termination site, coincident with projections of abnormal, large bundles into the medulla. R cell growth cone morphology is also altered in msn mutants. Unlike in dock, however, in msn, R cell growth cones are able to expand upon reaching the target. While all msn mutants examined exhibited defects in R cell innervation pattern, the severity of the phenotype varies from individual to individual (Ruan, 1999).

To determine whether msn is required in the developing eye for R cell projections, genetic mosaic analysis was carried out. Mutant eye clones homozygous for msn¹⁰², a strong loss of function allele, were generated in an otherwise wild-type fly by eye-specific mitotic recombination. R cell projections in mosaic larvae were visualized with mAb 24B10. Similar defects in R cell innervation pattern are observed. The percentage (~46%) of larvae showing obvious defects was close to the percentage (~50%) of individuals with relatively large mutant eye patches (identified as white eye tissue in adult). To specifically assess the role of Msn in R1-R6 growth cones, msn mutant eye patches were generated in msn heterozygous flies carrying the adult R1-R6-specific marker Rh1-lacZ. In wild-type adult flies, all R1-R6 axons terminate in the lamina, as assessed by lacZ staining. In contrast, in all mosaic adults examined, R1-R6 axons from msn mutant patches pass over the lamina and terminate abnormally in the medulla. These results indicate that msn, like dock, is genetically required in the eye for R1-R6 growth cone targeting. Similarly, no obvious defects are detected in the differentiation of the R1-R6 targeting region (i.e., lamina) in msn mutants, as assessed by anti-Dachshund staining. Moreover, eye-specific expression of a msn transgene rescues R cell projection defects in homozygous msn mutants (Ruan, 1999).

Dock protein is enriched in R cell axons and growth cones. If Msn has a functional relationship with Dock in R cell growth cones, it would be expected that Msn protein is expressed in R cells and is localized to growth cones. The expression pattern of Msn in third-instar larval eye-brain complexes was determined with a rabbit anti-Msn serum. Msn staining is seen in R cell axons along the path of projections (from the developing eye disc to the lamina) in wild-type whole-mount preparations. The lamina plexus is strongly stained as a continuous layer of immunoreactivity, a pattern that is indistinguishable from that stained with anti-Dock antibody. Since at this stage the vast majority of axonal processes in the lamina neuropil are expanded R1-R6 growth cones, the uniform staining of Msn and Dock in the lamina neuropil suggests strongly that Msn and Dock colocalize to R1-R6 growth cones.The localization of Msn in R1-R6 growth cones is consistent with a role for Msn in coordinating the response to target-derived signals (Ruan, 1999).

The fact that loss of msn caused the failure of R1-R6 growth cones to stop at their target lamina suggests a role for msn in the shutdown of growth cone motility when axons reach their target. Target-derived stop signals may activate Msn, which in turn coordinates cytoskeletal reorganization in decelerating growth cone motility. If this model is correct, one may predict that ectopic activation of Msn should induce abnormal termination of R cell growth cones. To test this, the endogenous msn gene was overexpressed in differentiating R cells using the eye-specific promoter GMR. Overexpression of Msn in R cell axons was confirmed by immunohistochemical staining. Compared with wild-type, overexpression of msn causes a large number of R1-R6 growth cones to stop before they reached their normal target lamina. Overexpression of msn also causes defects in the medulla terminal field. In contrast, neither the shape of R cells nor their localization on the developing eye disc is affected. Overexpression of msn from a transgene containing msn cDNA under control of the GMR promoter causes a similar early stop phenotype. The severity of the phenotype is dose dependent, because the increase in the copies of msn transgene enhanced the phenotype (Ruan, 1999).

One possible explanation for the gain-of-function phenotype is that overexpression of Msn activates the Msn pathway prematurely: this sends a terminating signal to growth cone cytoskeleton to induce pretarget termination. Alternatively, the hyperactivation of the Msn pathway may cause some general defects in the reorganization of growth cone cytoskeleton, leading to the arrest of growth cones before they reach the target. The former interpretation, that Msn plays an instructive role in terminating R1-R6 growth cones, is favored for the following reasons: (1) in msn gain-of-function mutants, the early stop growth cones expand, similar to growth cones that terminate correctly in the lamina. In wild type, R cell growth cones expand only when they terminate in the target; (2) the fact that the early stop R cell growth cones are still able to expand in the lamina argues against a general defect in the reorganization of growth cone cytoskeleton (Ruan, 1999).

To examine whether Msn interacts with Dock physically, a glutathione S-transferase (GST) fusion protein was generated containing a fragment of Msn that encompasses multiple consensus PXXP motifs for SH3 domain-binding. The immobilized GST-Msn fusion protein precipitates Dock from adult fly lysates in a dose-dependent manner, indicating the direct association of Msn with Dock. To test whether Msn associates with Dock in intact flies, coimmunoprecipitation experiments were carried out. Fly lysates were prepared from third-instar larval eye-brain complexes or adult heads. Anti-Dock antibody was used to precipitate Dock and its interacting proteins from the lysates. Msn protein is detected in anti-Dock precipitates but not in control serum precipitates, indicating an in vivo association of Msn with Dock in flies at both developmental and adult stages (Ruan, 1999).

To define the domains of Dock and Msn that mediate the binding, the yeast two-hybrid system was used to analyze their interactions. Consistent with binding experiments using GST-Msn fusion protein, the PXXP fragment of Msn binds to Dock in yeast. The binding of Dock to Msn is mediated mainly by its SH3-1 and SH3-2 domains. Mutations in either SH3-1 or SH3-2 inhibit the association of Dock with Msn, indicating that a stable association requires the simultaneous binding of SH3-1 and SH3-2 to the PXXP sequence in the polypeptide of Msn, whereas SH3-3 is less necessary for the binding (Ruan, 1999).

To determine the biological relevance of the physical association of Msn with Dock, a test was performed to see whether dock and msn interact genetically. The dosage of dock gene was reduced in larvae homozygous for the hypomorphic msn allele [msn^l(3)03349]. The reduction by half of dock gene dosage dramatically enhances the msn phenotype. The R1-R6 termination site at the lamina becomes more disorganized. R cell growth cones are much less expanded and appear more similar to those of dock mutants. This enhanced phenotype is completely penetrant. It is estimated that in each hemisphere, ~70%-100% of growth cones are less expanded, as compared with those in controls. In dock and msn double mutants, R cell projections are indistinguishable from those in dock mutants. These results, together with the physical association of Msn with Dock, strongly suggest that Msn and Dock function in the same signaling pathway controlling R cell projections (Ruan, 1999).

That Dock/Nck is capable of binding activated receptor tyrosine kinases via its SH2 domain, together with the above phenotypic analysis of dock and msn mutants, suggests that Msn is activated by Dock-mediated stop signals in terminating R1-R6 growth cones in the lamina. This model makes the simple prediction that gain of function in msn should suppress the R1-R6 nonstop phenotype in dock mutants. To assess this possibility, the endogenous msn gene was overexpressed in homozygous dock mutants. In dock mutants, the medulla layer is hyperinnervated, as many R1-R6 axons fail to stop at the lamina termination site. Overexpression of Msn in dock mutants largely suppresses the R1-R6 nonstop phenotype ; R cell axons in the medulla are dramatically reduced in all larvae examined. The fact that gain of function in msn is capable of terminating R1-R6 growth cones in dock null mutants is consistent with the prediction that Dock functions upstream of Msn activation in decelerating R1-R6 growth cone motility. Surprisingly, overexpression of msn in the absence of dock also causes the premature termination of many R cell growth cones within the optic stalk, a phenotype that is not observed in wild-type flies overexpressing msn. This result raises the intriguing possibility that Dock is also able to negatively regulate the function of Msn at certain stages of axonal projections (Ruan, 1999).

To further investigate the relationship between msn and dock in the control of growth cone motility, the effect of overexpressing Dock on the msn gain-of-function phenotype was examined. Dock was overproduced in R cells under control of the GMR promoter. In wild type, overexpression of Dock has no effect on R cell projections, suggesting that Dock is not rate limiting in the termination of growth cones. Overexpression of Dock in msn gain-of-function mutants largely suppresses the pretarget termination phenotype, confirming that Dock also negatively regulates the function of msn. SH3 mutants incapable of binding Msn in yeast either completely fail to suppress the phenotype or only weakly suppress the phenotype. In contrast, the SH3-3 mutant, displaying Msn-binding activity, suppresses the phenotype as efficiently as wild-type Dock. These results argue that the physical association of Dock with Msn is essential for the regulation of Msn by Dock. Interestingly, although the R336Q mutation (eliminating phosphotyrosine-binding activity of the SH2 domain) does not affect the binding of Dock to Msn, it completely abolishes the ability of Dock to suppress the msn gain-of-function phenotype. These data suggest that the negative regulation of Msn function by Dock involves an SH2-dependent tyrosine phosphorylation signal (Ruan, 1999).

It is proposed that Dock couples different signals to Msn at different stages of axonal projection. At an early stage, signals promoting growth cone extension may induce tyrosine phosphorylation on specific proteins (e.g., docking protein), which then recruit Msn through Dock (via the SH2 domain) to specific regions within the growth cone. Consequently, this may segregate Msn from its substrates, thus preventing the premature activation of the Msn pathway. In growth cones overexpressing Msn, however, excessive Msn that cannot be recruited by a limited amount of endogenous Dock may diffuse freely into certain regions to activate its (Msn's) substrates, which then induce pretarget growth cone termination. Similarly, the pretarget termination phenotype is enhanced by loss of dock and is suppressed by overexpression of dock. Once the growth cone reaches the target, upregulation of Msn may be accomplished in two steps through the combination of reducing the extension signal and increasing the stop signal. (1) The Dock-Msn complex needs to be released from those docking sites, which would be achieved by dephosphorylation through the activation of some protein tyrosine phosphatases. One such candidate is the receptor tyrosine phosphatase PTP69D, which has recently been shown to be required for the proper targeting of R1-R6 growth cones. (2) The stop signal activates the function of Msn through Dock by either positioning Msn close to its substrate or directly stimulating its activity, leading to the termination of the growth cone in the target. In the absence of Dock, endogenous Msn may not reach a threshold local concentration or activity required for growth cone termination. The observation that reduction of dock gene dosage enhances the hypomorphic msn loss-of-function phenotype is consistent with this view. While the above model fits with the results, understanding of the exact biochemical mechanism underlying the regulation of Msn by Dock awaits identification of upstream regulators of Dock in R cell growth cones (Ruan, 1999).

Recent studies suggest that Dock/Nck plays a highly conserved role in growth cone signaling. Nck can be recruited into signaling complexes in response to the activation of the vertebrate guidance receptors EphB1 and EphB2, two Eph receptor tyrosine kinase family members (see Drosophila Eph receptor tyrosine kinase). Moreover, Nck can functionally replace Dock in R cell growth cones. Furthermore, Dock, like Nck, is capable of binding ligand-activated EphB1. Given the extraordinary sequence conservation between Msn and NIK, it is highly likely that in vertebrate growth cones, NIK plays a similar role in response to Nck-mediated signals. Hence, the interaction between Dock/Nck and Msn/NIK may represent an evolutionarily conserved mechanism linking tyrosine phosphorylation to changes in growth cone behavior (Ruan, 1999 and references therein).

While extensive studies in several systems have made considerable progress in defining the general mechanisms that direct growth cone extension, much less is known of the mechanism that makes growth cones stop at a specific target layer underlying the formation of layer-specific connections. Misshapen (Msn) has been proposed to shut down Drosophila photoreceptor (R cell) growth cone motility in response to targeting signals linked to Msn by the SH2/SH3 adaptor protein Dock. To identify downstream targets of Msn in R cell growth cones, a genetic dissection was undertaken to search for second-site mutations that modify a Msn hyperactivation phenotype. bifocal (bif), a gene encoding a putative cytoskeletal regulator, shows strong interaction with msn. Bif binds to F-actin in vitro and colocalizes with F-actin during development. Bifocal is a component of the Msn pathway for regulating R cell growth cone targeting. Phenotypic analysis indicates a specific role for Bif to terminate R1-R6 growth cones. Biochemical studies show that Msn associates directly with Bif and phosphorylates Bif in vitro. Cell culture studies demonstrate that Msn interacts with Bif to regulate F-actin structure and filopodium formation. It is proposed that Bif functions downstream of Msn to reorganize actin cytoskeleton in decelerating R cell growth cone motility at the target region (Ruan, 2002).

A genetic approach was undertaken to search for genes encoding other components of the Dreadlocks-Msn signaling pathway. A msn gain-of-function phenotype was generated by overexpressing Msn in R cell growth cones. In wild-type, after exiting the optic stalk, R1–R6 growth cones migrate over a distance of ~20 µm within the lamina, then stop extension and expand significantly in size to form the lamina plexus, while R7 and R8 growth cones migrate through the lamina into the medulla. In larvae overexpressing msn, however, many R1–R6 growth cones terminate before reaching the lamina plexus, a phenotype in marked contrast to that in msn loss-of-function mutants in which many R1–R6 growth cones failed to stop at the lamina layer. Overexpression of msn also disrupts the regular array of R7 and R8 growth cones in the medulla. It was reasoned that if a gene functions downstream of msn, then reducing the dosage of this gene by half would decrease the level of signaling through the Msn pathway, thereby suppressing the Msn hyperactivation phenotype. Thus, a screen for modifiers of this msn hyperactivation phenotype might lead to the identification of other components of the Dock-Msn signaling pathway (Ruan, 2002).

To determine the feasibility of this approach, an examination was carried out to see if reducing the dosage of other genes in the genome would dominantly modify the pretarget termination phenotype in flies overexpressing msn. Analysis of deficiency lines shows that reducing the dosage of a number of cytological regions could dominantly modify the msn overexpression phenotype, indicating that this msn gain-of-function genetic background is indeed sensitive to the dosage of other genes. This approach was undertaken to examine the potential interaction between msn and a set of genes that had been previously implicated in regulating cytoskeletal changes in Drosophila. Interestingly, it was found that reducing the dosage of bif largely suppresses the pretarget msn hyperactivation phenotype. Suppression was observed using two different bif alleles R38 (~56%, n = 34) and R47 (~75%, n = 20). In contrast, reducing the dosage of bsk, a gene that encodes the fly homolog of C-Jun N-terminal kinase and has been shown previously to function downstream of Msn to regulate dorsal closure in early embryos, showed no effect. This result argues against the idea that msn and bsk interact similarly in R cell growth cones (Ruan, 2002).

Among other genes examined, reducing the dosage of cdc42 (~10%, n = 34 hemispheres) or disabled (dab) (~30%, n = 12 hemispheres) enhances the msn overexpression phenotype. In eye-brain complexes showing enhanced phenotype, R cell axons terminate within the optic stalk or the eye disc, which is never observed in wild-type larvae overexpressing msn. No modification of the msn overexpression phenotype is observed by reducing the dosage of either dpak, Rho1, all three Rac genes Rac1-Rac2-Mtl, or chickadee (chic), which encodes the fly homolog of profilin (n = 26 hemispheres) (Ruan, 2002).

The observation that Msn hyperactivation phenotype is sensitive to the dosage of bif, together with previous reports showing the link between Bif and the actin cytoskeleton, raises the interesting possibility that Bif functions downstream of Msn to regulate cytoskeletal changes in R cell growth cones (Ruan, 2002).

How could upstream stop signals be relayed through Dock and Msn to Bif? While the molecular nature of upstream regulators of Dock in R cell growth cones remains unknown, it has been shown that Dock functions downstream of the guidance receptor Dscam, a member of immunoglobulin superfamily, in larval photoreceptor growth cones. Dock mediates growth cone signaling through recruiting Pak to activated Dscam. It is speculated that Dock relays stop signals to Msn similarly in the adult visual system to regulate R1–R6 growth cone targeting. Dock and Msn form an in vivo complex at both larval and adult stages. It is speculated that when R1–R6 growth cones reach the lamina, stop signals produced by the intermediate target (i.e., lamina marginal glia) activate their growth cone receptors, which subsequently recruit the Dock-Msn complex. Consequently, this might bring Msn in proximity to Bif or directly stimulate the activity of Msn through a conformational change, leading to increased phosphorylation on Bif (Ruan, 2002).

While Msn associates directly with Bif and phosphorylates Bif in vitro, it is not known if Msn and Bif constitutively associate in R cell growth cones or if the association between them is transient and dependent on stop signals from the target region. Given that Bif is predominantly associated with the plasma membrane, it is speculated that the recruitment of Dock-Msn complex by activated growth cone receptors might relocate Msn from cytoplasm to plasma membrane, thus bringing Msn and Bif together. The formation of such a signaling complex could allow Msn to regulate the function of Bif through phosphorylation, or relocate Bif into a specific region within the growth cone to initiate downstream events. Testing these speculations awaits the identification of upstream stop signals and R cell growth cone receptors (Ruan, 2002).

How does the interaction between Msn and Bif regulate the changes in growth cone cytoskeleton? One possible scenario is that Bif, activated by Msn, induces the redistribution of F-actin within the growth cone, leading to the withdrawal of the growth cone leading edge. Consistently, cell culture studies show that Msn can reorganize Bif-induced actin fibers and reduce the number and the length of filopodia-like structures. Several studies have also demonstrated that the arrest of growth cone extension in vitro could be achieved through growth cone collapse, which is due at least in part to the loss of actin bundles at the leading edge of the growth cone. While the fact that R cell growth cones expand significantly in size upon reaching the target region argues against a mechanism involving the collapse of the whole growth cone, it remains possible that the initial termination involves partial growth cone collapse at the leading edge after exposure to stop signals. Since R cell growth cone morphology remains normal in msn and bif mutants, the view is favored that the interaction between Msn and Bif regulates the reorganization of actin filaments in spatially restricted domains within the growth cone without affecting the general structure of growth cone cytoskeleton (Ruan, 2002).

That Bif colocalizes with F-actin and can promote actin polymerization in cultured cells, together with a report that immobilized F-actin could pull down Bif from fly lysates, suggest strongly that Bif associates either directly or indirectly with F-actin filaments. Such association may stabilize actin filaments, thus contributing to the dramatic increase in the level of F-actin observed in cultured cells. Whether Bif also plays a similar role in promoting actin polymerization in R cells remains unclear since loss of bif affects neither growth cone outgrowth nor the amount of F-actin in R cell bodies and growth cones. One possible explanation is that other functionally redundant proteins maintain the level of F-actin in the absence of Bif. It is speculated that Bif may have at least two activities in R cell growth cones. Bif may be functionally redundant with other proteins to promote actin polymerization. Additionally, it may also play a role in restructuring F-actin in terminating R cell growth cones. The latter activity of Bif may resemble that of the Dictyostelium actin binding protein Severin. Severin and its mammalian homolog Gelsolin can bind to F-actin and fragment actin filaments. Interestingly, both Severin and Gelsolin are also phosphorylated by members of the GCK family of Ste20-like kinases in vitro. It is speculated that phosphorylation of Bif by Msn might directly increase such activity of Bif, thus inducing the shortening and aggregation of F-actin filaments leading to growth cone termination (Ruan, 2002).

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