Expression of DLIMK gene in Drosophila embryos was analyzed by in situ hybridization with the digoxigenin-labeled antisense RNA probe. DLIMK mRNA was detected uniformly at embryonic stages 2 to 5, with the signal intensity gradually decreased during embryogenesis, and was no longer detectable at and after the stage These results suggest that DLIMK transcripts in Drosophila embryos are supplied maternally and are likely to be degraded after stage 5. Zygotic transcription of DLIMK gene may occur around after stages 4-5, but if so the level seems to be weak (Ohashi, 2000).
Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Transforming growth factor β (TGFβ) signals regulate axon growth. In most contexts, TGFβ signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFβ responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFβ receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFβ receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo (Ng, 2008).
Once growing axons reach the correct postsynaptic target, axon outgrowth terminates and synaptogenesis begins. These studies suggest that TGFβ signals play a role. When Babo is inactivated, MB axon growth does not terminate properly and overextends across the midline. Consistent with this, CA Babo expression results in precocious termination, forming axon truncations. How Babo is spatially and temporally regulated remains to be determined. Analogous to the Drosophila NMJ, MB axon growth might be terminated through retrograde signalling. Target-derived TGFβ ligands could signal to Babo (on MB axon growth cones) and stop axons growing further. In an alternative scenario, TGFβ ligands might act as a positional cue that prevents MB axons from crossing the midline. Recent data have shown that Babo acting through Smad2 restricts individual R7 photoreceptor axons to single termini. Loss of Babo, Smad2, or the nuclear import regulator Importin α3 (Karyopherin α3 - FlyBase), results in R7 mutant axons invading neighbouring R7 terminal zones. With the phenotype described in this study, Babo could similarly be restricting MB axons to appropriate termination zones, its loss resulting in inappropriate terminations on the contralateral side (Ng, 2008).
In contrast to MB neurons, Babo inactivation in AL and OL neurons resulted in axon extension and targeting defects. This might reflect cell-intrinsic differences in the response in different neurons to a common Babo signalling program. This may be the case for MB axon pruning and DC axon extension, which require Babo/Smad2 signals. Whether these differences derive from cell-intrinsic properties, or from Babo signal transduction, they underline the importance of Smad-independent signals in many aspects of axonal development (Ng, 2008).
The results suggest that Smad-independent signals involve Rho GTPases. One caveat in genetic interaction experiments is that the loss of any given gene might not be dosage-sensitive with a particular assay. Nevertheless, all the manipulations together suggest that Babo-regulated axon growth requires Rho1, Rac and LIMK1. How Babo signals involve Rho GTPases remains to be fully determined. In addition to LIMK1, which binds to Wit, one possibility, as demonstrated for many axon guidance receptors, is that the RhoGEFs, RhoGAPs and Rho proteins might be linked to the Babo receptor complex. Thus, ligand-mediated changes in receptor properties would lead to spatiotemporal changes in Rho GTPase and LIMK1 activities (Ng, 2008).
The data suggest that a RhoGEF2/Rho1/Rok/LIMK1 pathway mediates Babo responses. Whether Rac activators are required is unclear, as tested RacGEFs do not genetically interact with babo. In this respect, rather than through GEFs, Babo might regulate Rac through GAPs, by inhibiting Tumbleweed (Tum) activity (Ng, 2008).
Do mutations in Rho1 and Rac components phenocopy babo phenotypes? β lobe overextensions are observed in Rok, Rho1 and Rac mutant neurons. In MB neurons, Rac GTPases also control axon outgrowth, guidance and branching. Rho1 also has additional roles in MB neurons. Although Rho1 mutant neuroblasts have cell proliferation defects, single-cell αβ clones do show β lobe extensions. RhoGEF2 strong loss-of-function clones do not exhibit axon overextension. As there are 23 RhoGEFs in the Drosophila genome, there might well be redundancy in the way Rho1 is activated. LIMK1 inactivation in MB neurons was reported previously. Axon overextensions were not observed as LIMK1 loss results in axon outgrowth and misguidance phenotypes. This suggests that LIMK1 mediates multiple axon guidance signals, of which TGFβ is a subset in MB morphogenesis (Ng, 2008).
Although their phenotypes are similar, several lines of evidence indicate that CA Babo does not simply reflect LIMK1 misregulation in MB neurons. First, whereas LIMK1 genetically interacts with most Rho family members and many Rho regulators, CA babo is dosage-sensitive only to Rho1 and Rac and specific Rho regulators, suggesting that Babo regulates LIMK1 only through a subset of Rho signals (Ng, 2008).
Second, the LIMK1 misexpression phenotype is suppressed by expression of wild-type cofilin (Twinstar Tsr), S3A Tsr, or the cofilin phosphatase Slingshot (Ssh). By contrast, only wild-type Tsr, but not S3A Tsr or Ssh, suppresses CA Babo. The suppression by wild-type Tsr might reflect a restoration of the endogenous balance or spatial distribution of cofilin-on (unphosphorylated) and -off (phosphorylated) states within neurons. Indeed, optimal axon outgrowth requires cofilin to undergo cycles of phosphorylation and dephosphorylation. Since S3A forms of cofilin cannot be inactivated and recycled from actin-bound complexes, wild-type cofilin is more potent in actin cytoskeletal regulation (Ng, 2008).
CA Babo might not simply misregulate LIMK1 but also additional cofilin regulators. Recent data suggest that extracellular cues (including mammalian BMPs) can regulate cofilin through Ssh phosphatase and phospholipase Cγ activities. In different cell types, cofilin phosphorylation and phospholipid binding (which also inhibits cofilin activity) states vary and potently affect cell motility and cytoskeletal regulation. Whether a combination of LIMK1, Ssh and phospholipid regulation affects cofilin-dependent axon growth remains to be determined (Ng, 2008).
Third, by phalloidin staining, LIMK1, but not CA Babo, misexpression results in a dramatic increase in F-actin in MB neurons. Thus, CA Babo does not in itself lead to actin misregulation. Fourth, Babo also regulates axon growth independently of LIMK1 (Ng, 2008).
This study differs significantly from the canonical model of Smad signalling, in which type 1 receptors function downstream of the ligand-type 2 receptor complex. In this study, the gain- and loss-of-function results suggest that type 2 receptors act downstream of type 1 signals. Since ectopic only Wit and Put suppress the babo axon overextension phenotype, this implies that Smad-dependent and -independent signals have distinct type 1/type 2 receptor interactions. How these interactions propagate Smad-independent signals remains to be fully determined. Babo could act as a ligand-binding co-receptor with Wit and Put. In addition, Babo kinase activity could regulate type 2 receptor or Rho functions. The results suggest, however, that provided that Wit or Put signals are sufficiently high, Babo is not required. Whatever the mechanism(s), it is likely that Babo requires the Wit C-terminus-LIMK1 interaction to relay cofilin phosphoregulatory signals. How Put functions is unclear. Since the put135 allele (used in this study) carries a missense mutation within the kinase domain, this suggests that kinase activity is essential. put does not genetically interact with LIMK1. Since Put lacks the C-terminal extension of Wit that is necessary for LIMK1 binding, this suggests that Put acts independently of LIMK1. One potential effector is Rac, which, in the context of Babo signalling, also appears to be Pak1- and thus LIMK1-independent (Ng, 2008).
In MB neurons, Wit and Put can function interchangeably. In other in vivo paradigms, type 2 receptors are not interchangeable. However, since the Wit C-terminal tail is required to substitute for Put, this suggests that Wit axon growth signals are independent of its kinase activity. Together, this suggests that Smad-independent signals involve LIMK1-dependent and -independent mechanisms (Ng, 2008).
This study shows that Babo mediates two distinct responses in related MB populations. How do MB neurons choose between axon pruning and axon growth? The babo rescue studies suggest that whereas Baboa or Babob elicits Smad-independent responses, only Baboa mediates Smad-dependent responses. Since Babo isoforms differ only in the extracellular domain, differences in ligand binding could determine Smad2 or Rho GTPase activation. However, it is worth noting that in DC neurons, either isoform mediates axon extension through Smad2 and Medea. In addition, although expressed in all MB neurons, CA babo misexpression (which confers ligand-independent signals) perturbs only αβ axons. Thus, cell-intrinsic properties might also be essential in determining Babo responses (Ng, 2008).
Many TGFβ ligands signal through Babo. For example, Dawdle, an Activin-related ligand, patterns Drosophila motor axons, whereas Activin (Activin-β, FlyBase) is required for MB axon pruning. Whether these ligands regulate Babo MB, AL and OL axonal morphogenesis is unclear. Taken together, the evidence suggests that Babo signalling is varied in vivo and is involved in many aspects of neuronal development (Ng, 2008).
TGFβ signals are responsible for many aspects of development and disease and, throughout different models, Smad pathways are closely involved. Although Smad-independent pathways are known, their mechanisms and roles in vivo are unclear. TGFβ signals often drive cell shape changes in vivo. During epithelial-to-mesenchymal transition (EMT), cells lose their epithelial structure and adopt a fibroblast-like structure that is essential for cell migration during development and tumour invasion. TGFβ-mediated changes in the actin cytoskeleton and adherens junctions are necessary for EMT. Although Smads are crucial, TGFβ signals also involve the Cdc42-Par6 complex, resulting in cell de-adhesion and F-actin breakdown through Rho1 degradation. In other studies, however, TGFβ-mediated EMT has been shown to require Rho1, which can be regulated by Smad activity (Ng, 2008).
Many TGFβ-driven events in Drosophila are Smad-dependent. Whether Smad-independent roles exist beyond those identified in this study remains to be tested. This study therefore provides a framework to understand how non-Smad signals regulate cell morphogenesis during development (Ng, 2008).
The steroid hormone 20-hydroxyecdysone (ecdysone) is the key regulator of postembryonic developmental transitions in insects and controls metamorphosis by triggering the morphogenesis of adult tissues from larvae. The Rho GTPase, which mediates cell shape change and migration, is also an essential regulator of tissue morphogenesis during development. Rho activity can modulate gene expression, in part, by activating LIM kinase (LIMK) and consequently affecting actin-induced SRF transcriptional activity. A link has been established between Rho-LIMK-SRF signaling and the ecdysone-induced transcriptional response during Drosophila development. Specifically, it has been determined that the Rho GTPase, via LIMK, regulates the expression of several ecdysone-responsive genes, including those encoding the ecdysone receptor itself, a downstream transcription factor (Br-C), and Stubble, a transmembrane protease required for proper leg formation. Stubble and Br-C mutants exhibit strong genetic interactions with several Rho pathway components in the formation of adult structures, but not with Rac or Cdc42. In cultured SL2 cells, inhibition of Rho, F-actin assembly, or SRF blocks the transcriptional response to ecdysone. Together, these findings indicate a link between Rho-LIMK signaling and steroid hormone-induced gene expression in the context of metamorphosis and thereby establish a novel role for the Rho GTPase in development (Chen, 2004).
Metamorphosis in Drosophila is stringently controlled by pulses of the steroid hormone ecdysone at discrete developmental stages. During larval-pupal transition, ecdysone triggers coordinated changes in tissue morphology that involve histolysis of larval tissues and the initiation of adult structures. Rho GTPase-mediated signaling pathways have been implicated in several aspects of morphogenesis during Drosophila embryo formation. However, a role for Rho signaling in metamorphosis has not yet been reported. Among the downstream mediators of Rho signaling are the LIM kinases, and a closely related Drosophila ortholog of mammalian LIM kinases (designated Dlimk) is specifically expressed at relatively high levels in late larval and pupal stages, suggesting a potential role in Rho-LIMK signaling during this transition. In adult flies, Dlimk is expressed at substantially higher levels in males than in females, consistent with a potential evolutionarily conserved role in spermatogenesis, a process in which mammalian LIMK2 has been implicated. Dlimk mRNA is uniformly expressed throughout eye, wing, and leg imaginal discs (Chen, 2004).
The GAL4/UAS transgene system was used to examine Dlimk function in vivo. Overexpression of Dlimk in the imaginal wing disc via several different wing-specific GAL4 drivers causes notched wings, missing wing veins, vein fusion, and blistered wings. The notched wing phenotype appears to reflect an increase in apoptosis and is rescued by the p35 viral caspase inhibitor. In addition, wings exhibit enlarged cells (indicated by low wing hair density) and alterations in the number and polarity of wing hairs. Notably, similar defects in wing hair number and polarity are also seen in rho1 and Drok (the Rho effector kinase that activates LIMK) mutants, suggesting that Dlimk functions in the same pathway. Mammalian LIMKs promote actin assembly in cultured cells, and prominent F-actin accumulation and aberrant actin organization is observed in the wing discs of transgenic flies specifically overexpressing Dlimk. Thus, Dlimk can regulate actin assembly in developing tissues (Chen, 2004).
To verify that Dlimk normally regulates morphogenesis during the larval-pupal transition, a kinase-deficient form of Dlimk (DlimkD522A) was used as a dominant-negative protein. An analogous mutation in mammalian LIMK1 gives rise to a protein that specifically interferes with LIMK function in cultured cells. Use of a T80-GAL4/UAS-DlimkD522A transgenic line to express DlimkD522A during development results in viable and fertile animals, with approximately 85% of adults exhibiting malformed wings and legs, consistent with a normal requirement for Dlimk in proper disc morphogenesis. In wild-type adult legs, the femur and tibia are elongated and slender structures; however, in DlimkD522A mutant flies, the femur is bent and twisted, and the tibia is often shorter and twisted. In addition, wings are malformed and are approximately 40% smaller than those of wild-type flies. Coexpression of DlimkD522A and wild-type Dlimk results in flies whose wings appear normal, indicating that the effects of dominant-negative Dlimk result from specific inhibition of the endogenous wild-type Dlimk as opposed to nonspecific interference with an unrelated signaling pathway (Chen, 2004).
The malformed legs in DlimkD522A flies closely resemble leg defects in flies in which Rho signaling is perturbed through genetic disruption of Rho1, DrhoGEF2 (a guanine nucleotide exchange factor for Rho1), sqh (myosin light chain), and zipper (nonmuscle myosin heavy chain). Sqh and zipper are downstream targets of Drok and regulate actomyosin contractility. Loss-of-function mutants of Rho1 or DrhoGEF2 strongly suppress the severity of wing defects associated with Dlimk expression. Reducing Rho activity by overexpressing the potent Rho inhibitor, p190 RhoGAP, also efficiently suppresses Dlimk-induced wing defects. Moreover, reducing levels of Diaphanous or Drok, two Rho targets that promote actin assembly, also substantially reduces the severity of Dlimk-induced wing defects. A loss-of-function allele of blistered, the Drosophila SRF ortholog, also suppresses the Dlimk-induced wing defects, suggesting that regulation of SRF-dependent transcription by Rho-LIMK signaling plays a role in wing morphogenesis. Significantly, in mammalian cells, LIMK and Diaphanous cooperate to regulate SRF activity. Reducing levels of the Rho-related GTPases, Rac1, Rac2, and Cdc42, or the Rac activator, Myoblast City (Mbc), or the Rac/Cdc42 effector target, PAK, has very little effect on the Dlimk-induced wing phenotype. Thus, it appears that in the developing leg and wing, Dlimk specifically mediates a Rho-actin signaling pathway required for imaginal-disc morphogenesis (Chen, 2004).
Defects in leg morphogenesis resembling those in DlimkD522A flies are seen in mutants of several ecdysone-inducible genes, including those encoding the transcription factor, Broad-complex (BR-C or br), and Stubble (Sb), a transmembrane serine protease. Both genes are required for disc morphogenesis during larval-pupal transition. Significantly, br mutants interact genetically with mutants of Sb, zipper, and blistered during imaginal-disc morphogenesis, suggesting that the observed role for a Rho-Dlimk pathway in leg morphogenesis could reflect a requirement for this pathway in the response to ecdysone (Chen, 2004).
To determine if the Rho-Dlimk pathway interacts genetically with br or Sb, a heat-shock-inducible DlimkD522A transgene that exhibits a low-penetrance malformed leg phenotype was crossed with with br and Sb mutants. DlimkD522A and mutants of several Rho signaling components strongly interact with Sb63b and Sb70, two dominant-negative alleles of Stubble, to produce malformed legs at a high frequency. However, mutants of the Rho-related GTPases, Rac1, Rac2, and Cdc42, do not enhance the frequency of leg defects. Similarly, several components of the Rho-Dlimk pathway, but not Rac and Cdc42, strongly interact with br1 in an analogous genetic interaction test (Chen, 2004).
The observed interactions among Rho1, Dlimk, br, and Sb support a role for Rho signaling in ecdysone-regulated metamorphosis. However, it was determined that neither Rho1 expression nor activation is ecdysone inducible. In light of previous studies linking Rho-LIMK signaling to effects on gene expression, BR-C and Sb expression was examined in flies overexpressing Rho1, Dlimk, or p190 RhoGAP during early puparium stages, when disc morphogenesis is underway. Expression of BR-C and Sb mRNA normally peaks approximately 2–4 hr after puparium formation. However, in flies overexpressing Rho1 or Dlimk, expression of these genes persists well beyond the normal peak of expression seen in "driver-only" control flies (approximately 8–10 hr after puparium formation). Moreover, expression of these genes is greatly reduced at all stages of pupation in flies expressing p190 RhoGAP. Significantly, although most of the transgenic flies that overexpress p190 RhoGAP die at a late pupal stage, the few "escapers" that eclose exhibit malformed wings and twisted and bent leg phenotypes that are very similar to those seen in flies expressing DlimkD522A. In addition, the pupal lethality that is frequently observed with overexpression of p190 RhoGAP is efficiently rescued by coexpressing Dlimk, indicating that the late developmental defects that arise as a consequence of Rho inactivation largely reflect defects in Rho-LIMK signaling (Chen, 2004).
Expression of the ecdysone receptor (EcR) itself is similarly regulated by Rho1 and Dlimk. However, loss-of-function alleles of the EcR or Sb fail to rescue the effects of overexpressing Dlimk, suggesting that Rho-LIMK signaling controls additional aspects of metamorphosis independently of its effects on the ecdysone response. Thus, Rho-LIMK signaling may play a role in coordinating Rho-directed cell shape changes and movements with ecdysone-induced gene expression during tissue morphogenesis (Chen, 2004).
Many of the identified transcriptional targets of the ligand-activated ecdysone receptor are, themselves, transcription factors, which are not the actual effectors of tissue morphogenesis. However, the Stubble gene, which is highly sensitive to Rho-LIMK signaling, encodes a protein that participates directly in morphogenesis through its ability to promote remodeling of the extracellular matrix. The ability of Rho to direct both actin-mediated cell shape changes and the expression of a cell surface protease provides a potential mechanism for coordinately regulating these two major components of tissue morphogenesis during development (Chen, 2004).
To examine more directly a requirement for a Rho-actin-SRF pathway in the transcriptional response to ecdysone, Drosophila SL2 cells were used. In SL2 cells, as in developing discs, ecdysone induces the expression of EcR mRNA. Transfection of cells with the Rho-inhibitory C3 toxin or pretreatment with the actin polymerization inhibitor, latrunculin B, substantially reduces the ecdysone-induced increase in EcR mRNA but does not affect transcription of the ecdysone-insensitive gene rp49 or the Rho1 gene. As expected, latrunculin B completely inhibits morphogenesis of leg appendages, indicating a requirement for F-actin assembly. To examine the role of SRF in ecdysone-induced EcR expression, SL2 cells were treated with RNAi corresponding to the blistered gene. RNAi-treated cells exhibit reduced SRF expression and an absence of ecdysone-induced EcR mRNA expression. Together, these results suggest that the ability of Rho and Dlimk to promote F-actin assembly and SRF activation is responsible for their effects on ecdysone-responsive gene expression and tissue morphogenesis. In addition, the findings in SL2 cells indicate that the observed effects of Rho-SRF signaling on the ecdysone response are cell-autonomous effects. Interestingly, genetic interactions were observed between zipper and sb and between zipper and br, suggesting that Rho-regulated actomyosin contractility, in addition to F-actin assembly, may also influence the ecdysone response. In this regard, it is interesting to note that mechanical stretching of cells reportedly promotes SRF activity. Alternatively, actomyosin contractility may play a parallel role in disc morphogenesis that is independent of any direct regulation of the ecdysone response (Chen, 2004).
A DNA element that matches the reported SRF binding consensus site within the 5′ and 3′ regulatory sequences (2 kb each) of the EcR gene has not been identified. Hence, it remains possible that an SRF-regulated coactivator of ecdysone receptor gene expression is a primary target of Rho-Dlimk signaling. It is interesting to note that the Drosophila transcription factor, Crooked legs, regulates expression of ecdysone receptor mRNA and is encoded by an ecdysone-inducible gene that is also required for wing and leg morphogenesis. Such findings highlight the complexity of the gene expression hierarchy involved in the morphogenetic response to ecdysone and indicate a likely role for transcriptional feedback mechanisms (Chen, 2004).
Thus, Rho GTPase-mediated signal transduction to the actin cytoskeleton and ecdysone-induced gene expression are both critical regulatory components of tissue morphogenesis during Drosophila development. In these studies, a direct relationship between these two pathways was identified in the context of metamorphosis. Specifically, these findings indicate that Rho, through its ability to activate LIMK and promote actin polymerization, regulates the expression of several ecdysone-responsive genes, including the ecdysone receptor itself. By modulating the expression of ecdysone-responsive genes, including a cell surface protease, the Rho-LIMK signaling pathway appears to play a critical role in regulating the proper morphogenesis of adult structures from the imaginal discs of larvae. This connection represents a previously unrecognized link between Rho GTPase signaling and nuclear hormone signaling that potentially plays a broader role in additional developmental contexts (Chen, 2004).
Lim Kinase (Limk) belongs to a phylogenetically conserved family of serine/threonine kinases, which have been shown to be potent regulators of the actin cytoskeleton. Despite accumulating evidence of its biochemical actions, its in vivo function has remained poorly understood. The association of the Limk1 gene with Williams Syndrome indicates that proteins of this family play a role in the nervous system. To unravel the cellular and molecular functions of Limk, the Limk gene in Drosophila has been either knocked out or activated. At the neuromuscular junction, loss of Limk leads to enlarged terminals, while increasing the activity of Limk leads to stunted terminals with fewer synaptic boutons. In the antennal lobe, loss of Limk abolishes the ability of p21-activated kinase (Pak) to alter glomerular development. In contrast, increase in Limk function leads to ectopic glomeruli, a phenotype suppressible by the coexpression of a hyperactive Cofilin gene. These results establish Limk as a critical regulator of Cofilin function and synapse development, and a downstream effector of Pak in vivo (Ang, 2006).
Limk regulates the development of the neuromuscular junction: The Drosophila Limk gene was cloned by screening the fly EST database with the rat Limk1 sequence (Ohashi, 2000). A search of the genome database showed that Limk is the only fly ortholog and maps to the cytological region 11B2 on the X chromosome. Using a P element, EP(X)1313, located 2.8 kb downstream of Limk, 5 deletion alleles of the gene were generated and named Limk1 to Limk5. Three of the alleles, Limk2, Limk3, and Limk5, remove all or most of the gene. Limk5 was used in most of these analyses. Limk hemizygotes are viable, fertile, and morphologically normal externally (Ang, 2006).
Mutation in the mouse Limk-1 gene resulted in the aberrant development of synapses (Meng, 2002), which raises the possibility that synapse development may be impaired in the fly Limk mutant. To address this possibility, the neuromuscular structures of the third instar larva were examined. At this stage, each abdominal hemisegment contains a stereotyped pattern of 30 muscles, which are innervated by ~35 motoneurons. Each motoneuron makes connections with specific muscles through NMJs that are highly stereotyped in terms of the number of synaptic boutons and the complexity of arborization. Third instar larval fillet of wild type and the mutant were stained with an antibody against Synaptotagmin (Syt) to visualize the morphology of the NMJ. Limk mutants have slightly smaller muscle fibers than wild type, and their NMJs exhibit increased bouton number. The NMJ overgrowth was quantified in the Limk mutant by counting the number of Type Ib and Is boutons per μm2 of muscle area on muscles 6/7, 12, and 13. In the Limk mutants, there are increases in bouton numbers to 73%-112% greater than those of wild type, with the largest increase shown by muscle 12. To determine whether Limk functions pre- or post-synaptically to regulate NMJ development, attempts were made to rescue the mutant phenotype by cell type-specific cDNA expression. When wild-type Limk cDNA was expressed using the motoneuron-specific OK6-Gal4 driver, the bouton numbers are reduced to 18%-41% greater than those of wild type. In contrast, expression of the cDNA using the muscle-specific 24B-Gal4 driver did not significantly alter the mutant NMJ phenotype. It is concluded from these results that Limk functions in the presynaptic cell to regulate the structural development of the synapse. To assess axonal projections in the Limk mutant, mutant embryos were stained with antibodies (anti-HRP, BP102, and 1D4) that label both central and peripheral trajectories. The patterns of axon trajectories in the CNS and the periphery of the Limk mutant are normal compared with those in wild type. Thus, Limk is not required for axonal pathfinding in the motoneurons (Ang, 2006).
The overgrowth of the Limk NMJ suggests that the normal function of Limk is to suppress synaptic sprouting. To test this idea, Limk activity was increased in larval motoneurons during synapse development. The constitutively active UAS-Limkkd gene was specifically expressed in motoneurons using the OK6-Gal4 driver. In animals expressing Limkkd, the synaptic terminals are strongly reduced in size. Although the NMJs retain an overall normal shape, the bouton numbers are reduced to 53%-76% of those of the wild type. In summary, loss of Limk function leads to synapse overgrowth while gain of Limk function leads to synapse undergrowth. The size of the NMJ synapse is thus sensitive to, and inversely correlated with Limk activity. The correlation of synapse size to the dose of Limk function indicates that Limk plays a key role in governing synapse sprouting (Ang, 2006).
Limk does not regulate the function of the neuromuscular junction: To ascertain if Limk plays a role in synaptic function, the synaptic physiology of the Limkmutant neuromuscular synapses was examined. Intracellular recordings were performed from muscle 6/7, 12, and 13 from segments A3 or A4 of the third instar larva to measure the postsynaptic responses to spontaneous and evoked transmitter release. The amplitude of evoked postsynaptic potentials (EPSPs) at the various muscles in the Limk mutant is not significantly different from that of the wild type. Both the size and amplitude distribution of the unitary potentials are also similar between Limk and the control. These results indicate that the synaptic function of the NMJ is not altered in the Limk mutant, despite the expansion in NMJ size. The similarity in synaptic functions implies that the extra boutons are either less active or each has the same number of actives zones as normal, but the active zones have a decreased probability of release. To distinguish between these possibilities, the NMJs were stained with the nc82 antibody, and the number of active zones at the Limk NMJs was determined. Limk mutant has slightly less active zones per bouton compared with the control. It is possible that the slight decrease in active zone number helps to compensate for the increase in bouton number, resulting in normal synaptic function in the Limk mutant (Ang, 2006).
Limk regulates the development of the antennal lobe glomeruli: The observation that the antennal lobe (AL) structure is aberrant in the Pak mutant (Ang, 2003), together with current findings, led to experiments to see whether Limk also regulates the development of AL synapses. The AL is the first relay station of the olfactory pathway, where olfactory receptor neuron (ORN) axons synapse on dendrites of the projection neurons (PNs). The synapses are not evenly distributed through the AL neuropil, but are concentrated in anatomically distinct structures, the glomeruli, each of which is unique based on its characteristic size, shape, and position in the AL neuropil. Axons from ORNs expressing a given odorant receptor terminate in a specific glomerulus (Ang, 2006).
The morphology of the ALs of the Limk mutants was examined by staining with the nc82 mAb, which stains the AL neuropil revealing the glomeruli. Glomerular development is assessed by several criteria: (1) whether each glomerulus is surrounded by a distinct boundary, a margin devoid of nc82 staining and (2) the size, shape, and location of the glomeruli are taken into account. Glomerular development is quantified by determining the frequencies with which four representative glomeruli (VA1d, DA4, VA6, DM6) can be unambiguously identified based on the above criteria. In the wild type, the AL is 72.7 ± 1.17 μm in diameter and is partitioned into distinct glomeruli. The indicator glomeruli can be identified in 100% of the ALs. In the Limk5 mutant, the ALs are 70.68 ± 1.10 μm in diameter, a value similar to that of the wild type. The indicator glomeruli can be also discerned in all the ALs (Ang, 2006).
Synaptic development was assessed by examining Synaptotagmin::GFP in the DM2, DM3, and VA1v glomeruli using the Or22a-Gal4, Or47a-Gal4, and Or47b-Gal4 drivers, respectively. In the wild type, these glomeruli have relatively smooth boundaries and accumulate Syt::GFP. When examined under constant laser power, the level of Syt::GFP accumulation in the Limk5 mutant is not significantly different from that of the wild type. To assess projections of the ORN axons, a membrane-anchored GFP (encoded by UAS-mCD8::GFP) was expressed under the control of the Or22a-Gal4, Or47a-Gal4, and Or47b-Gal4 drivers. In the mutant antennae, the cell bodies of Or22a, Or47a, and Or47b neurons are found in numbers and distribution similar to those of wild type. The Or22a and Or47a axons also converge precisely to their cognate glomeruli. It is concluded from these observations that Limk is not required for the differentiation of the ORNs or the targeting of their axons (Ang, 2006).
The lack of obvious synaptic defects in the ALs of the loss-of-function Limk mutant suggests that a redundant gene, acting in parallel with Limk may partially compensate for its role in AL development. To further examine Limk's function in the ORNs, Limk activity was increased in the ORNs during glomerular development. The N-terminally truncated, hyperactive Limkkd molecule (encoded by the UAS-Limkkd transgene) is expressed under the control of the SG18.1-Gal4 driver, which is preferentially expressed in a large subset of ORNs. In the Limkkd overexpressing animal, the AL is elongated dorsoventrally. The structures of many glomeruli are disrupted. Inspection of the marker glomeruli shows that while those located at the lateral position (VA1d and DA4) are easily identifiable, those located more medially (VA6, DM6) can only be identified from 0% to 64% of the time. In addition to the disruption in glomerular structures, nc82-stained structures appear at the midline, a situation not seen in the wild type. These midline structures accumulate synaptobrevin::GFP (Nsyb::GFP), indicating that they contain synapses. Labeling of the ORN axons with a membrane-anchored GFP indicates the midline structures reside within the antennal commissure, which normally contains only axons. Globose structures also appear at the midline when the full-length Limk gene is expressed in ORNs, indicating that this effect is not due to a neomorphic function caused by truncation of the Limk protein. These results show that Limk has a powerful effect on the glomeruli, supporting a role for Limk in AL development (Ang, 2006).
Presynaptic Limkkd expression induces ectopic glomeruli: To probe the nature of the midline structures in the Limkkd-expressing animal, the DM2 glomerulus were labeled using the Or22a-Nsyb::GFP transgene. In the wild type, DM2 is located at the dorsomedial corner in all ALs. No nc82-stained structures are seen at the midline. In the Limkkd-expressing animal, DM2 is located entirely at the midline in 60% of the ALs. In the remaining ALs, DM2 shows only minor medial displacement. Thus, the ectopic structures at the midline of the Limkkd-expressing animals are innervated by ORN axons. It was of interest to know if the midline structures are also innervated by PN dendrites. The GH-mGFP transgene was created, that is expressed in a subset of the PNs. In the GH-mGFP transgenic animal, a stereotyped subset of PN dendrites is labeled creating a characteristic patchwork pattern in the AL neuropil. Careful examination of the confocal sections showed that a pair of dorsomedial arbors extend posteriorly, but were never found at the level of the ellipsoid body. In the Limkkd-expressing animal, GH-mGFP labels the same stereotyped set of dendritic arbors. However, the pair of dorsomedial arbors extend to the level of the ellipsoid body where they converge on the midline. The PN dendrite arbors overlap with nc82-stained midline structures. These results indicate that the midline structures are innervated by ORN axons and PN dendrites. Together, the observations suggest that the midline structures of the Limkkd-expressing animals are displaced endogenous glomeruli (Ang, 2006).
Limk is necessary for Pak's ability to direct glomerular development: In vitro experiments with the human proteins show that Pak1 phosphorylates and activates Limk1 (Edwards, 1999). It was hypothesized that, in the fly, Pak acts upstream of Limk to regulate glomerular development in the ALs. In the Pak4/Pak6 null mutant, the ALs are smaller compared with those of wild type. Staining with the nc82 mAb shows that the boundaries surrounding individual glomeruli are frequently missing, resulting in loss in distinctiveness of most of the marker glomeruli. Thus, Pak activity is necessary for glomerular development. To determine if elevated Pak is sufficient to alter AL development, the hyperactive Pakmyr molecule was expressed under the control of SG18.1-Gal4. Expression of Pakmyr in the ORNs results in strong disruption in the anatomy of the ALs. Although the neuropil is partitioned, the compartments correspond poorly to known glomeruli. Consequently, the marker glomeruli are identifiable in only 0% to 79% of ALs. The abnormal structures of the glomeruli are reflected in the DM2 glomerulus (labeled with the Or22a-Nsyb::GFP transgene). Unlike the wild type where it is distinct and symmetrically positioned, in the Pakmyr animal, it is poorly demarcated from the surrounding neuropil and asymmetrically positioned. It is possible that adjacent glomeruli are fused to form larger structures in the Pakmyr-expressing animal. Thus, increased presynaptic Pak activity is sufficient to disrupt glomerular morphogenesis. To determine if Limk mediates Pak function, UAS-Pakmyr was expressed in the Limk5 null mutant background. In the Limk5/Y; SG18.1-Gal4/UAS-Pakmyr animal, the AL neuropil is partitioned into compartments that correspond well to known glomeruli. Marker glomeruli can now be identified in 79% to 100% of the ALs. The DM2 glomeruli are morphologically distinct and symmetrically positioned. Thus, mutation in Limk blocks the ability of Pakmyr to interfere with glomerular development. This result supports the idea that Limk functions downstream of Pak to regulate glomerular development (Ang, 2006).
Cofilin functions downstream of Limk to regulate AL innervation: Biochemical experiments have shown that the Drosophila Limk specifically phosphorylates the serine 3 amino acid of Cofilin, a key regulator of actin turnover (Ohashi, 2000). In human, phosphorylation of serine 3 of Cofilin has been shown to shut down its actin depolymerizing activity. Indeed, mutation of the serine 3 amino acid of Cofilin to an alanine leads to a constitutively activated Cofilin protein. It was therefore hypothesized that Limk down-regulates Cofilin function during glomerular development. To test this hypothesis, Limkkd was expressed either alone, with wild-type Cofilin, or with CofilinS3A in ORNs. Expression of Limkkd results in disruption of glomerular structures and ectopic glomeruli at the midline in 100% of the ALs. Coexpression with wild-type Cofilin did not modify the Limkkd phenotype. In contrast, coexpression with CofilinS3A leads to the reappearance of distinct glomerular structures and the loss of ectopic midline structures in 83% of the ALs. These results support the idea that Limk negatively regulates Cofilin function by acting through the serine-3 amino acid during glomerular development (Ang, 2006).
By either knocking out or activating Limk, this study has shown that Limk regulates synapse structures at the NMJ and in the AL of Drosophila. At the NMJ, the loss of Limk leads to overgrowth of the synaptic terminal. Conversely, activation of Limk leads to stunted terminals. In the AL, the loss of Limk does not induce dramatic structural changes. However, overexpression of Limk results in ORN axons establishing glomeruli at ectopic locations. These results support the idea that Limk regulates structural development of synapses in vivo. These findings are in accord with the study by Meng (2002), which showed that the loss of mouse Limk1 gene results in abnormal dendritic spines morphology in the hippocampus. Interestingly, hippocampal slices from the Limk1 knockout mouse also exhibit changes in synaptic functions, an effect not seen at the fly Limk mutant. Despite the structural defects, Limk mutant NMJs display normal synaptic physiology. Collectively, these observations across different systems indicate that proteins of the Limk family play a conserved role in synapse development. Disruption in synapse development may underlie the behavioral and developmental symptoms of WS patients (Ang, 2006).
How does Limk regulate synaptic growth? The inverse correlation of NMJ synapse size with the dose of Limk activity indicates that the function of Limk at the NMJ is to inhibit synapse expansion. It is proposed that, by phosphorylating Cofilin and stabilizing the actin cytoskeleton, Limk prevents synapse remodeling and sprouting. Since the motoneuron synapse grows 10-fold in size between the first and the third larval instar stages, expression of Limkkd early during larval development (with the OK6-Gal4 driver) would arrest the NMJ in a premature state. Although the role of Limk in the olfactory system is not precisely understood, the model of Limk function that is proposed here could also account for the ability of Limkkd to cause ORNs to form glomeruli in aberrant positions. During pupal development, terminal arbors from adjacent glomeruli initially overlap but subsequently draw apart to form discrete glomeruli. Expression of Limkkd early in development (with the SG18.1-Gal4 driver) would be expected to inhibit the retraction of neighboring arbors from one another. Subsequent maturation of the overlapping terminal arbors may then result in the formation of aberrant glomeruli. Another possibility is that Limk directly induces glomerular expansion, a role opposite that postulated above for NMJ development. How Limkkd instructs the formation of ectopic glomeruli is currently being investigated (Ang, 2006).
Eaton and Davis have reported that Limk functions downstream of the BMP receptor, wit, to increase NMJ stability and stimulate NMJ growth (Eaton, 2005). That Limk promotes synapse stability is compatible with the findings that Limk restricts synaptic remodeling. However, Eaton and Davis also reported that NMJs have normal sizes in the LimkP1 allele; this contrasts with finds of enlarged NMJs in the Limk5 allele. Furthermore, they found that expression of a wild-type Limk transgene restored NMJ growth in the wit mutant, leading to the proposal that Limk stimulates NMJ growth. In contrast, this study found that expression of a gain-of-function Limk transgene produced stunted NMJs, leading to the conclusion that Limk represses NMJ growth. These divergent results can be reconciled by the idea that Limk can both restrict and promote NMJ growth, and that the dose of Limk determines its effects. It is likely that the synaptic stability induced by Limk is critical not only to prevent unregulated growth, but also for growth elicited by stimulatory signals by inhibiting retraction of newly formed boutons. In the wild type, Limk would be present at a “threshold” level where dynamic remodeling and synaptic stability coexist. In this scenario, the complete loss of Limk, as in the Limk5 null mutant, would cause excessive NMJ sprouting. A residual level of activity, as in the hypomorphic LimkP1 mutant, would be sufficient for a normal NMJ size. Finally, a high level of activity, as in the Limkkd-expressing animal, would prevent NMJ growth. This model suggests that Limk may play a pivotal and dosage-sensitive role in specifying NMJ development (Ang, 2006).
In vitro studies have implicated molecules functioning both up- and downstream of Limk. For example in human, Pak1 phosphorylates and activates Limk1 (Edwards, 1999). In the fly, Limk phosphorylates Cofilin in vitro (Ohashi, 2000). It is not known if these proteins function in a signaling pathway in vivo. This study presents evidence that Pak, Limk, and Cofilin are components of a signaling cascade that govern glomerular development in the olfactory system. (1) Loss of Pak results in an aglomerular phenotype whereas its activation disrupts glomerular structure, indicating that Pak plays a critical role in glomerular development. (2) A functional Limk is needed for Pak to disrupt glomerular structure, consistent with the idea that Pak activates Limk. (3) The glomerular-inducing activity of Limkkd is suppressed by coexpression with CofilinS3A, showing that Limk represses Cofilin. These genetic results thus show that Pak activates Limk, which in turn down-regulates Cofilin during glomerular development. The results also hint at the presence of a gene that acts in parallel with Limk: whereas elevation of Limk in the ORNs leads to profound glomerular assembly, loss of Limk only slightly impairs olfactory synapses. It is hypothesized that a redundant gene masks the requirement for Limk in glomerular development (Ang, 2006).
Paxillin is a prominent focal adhesion docking protein that regulates cell adhesion and migration. Although numerous paxillin-binding proteins have been identified and paxillin is required for normal embryogenesis, the precise mechanism by which paxillin functions in vivo has not yet been determined. An ortholog of mammalian paxillin in Drosophila (DPax) has been identified and a genetic analysis of paxillin function during development was undertaken. Overexpression of Dpax disrupts leg and wing development, suggesting a role for paxillin in imaginal disc morphogenesis. These defects may reflect a function for paxillin in regulation of Rho family GTPase signaling since paxillin interacts genetically with Rac and Rho in the developing eye. Moreover, a gain-of-function suppressor screen identified a genetic interaction between Dpax and center divider cdi in wing development. Cdi belongs to the cofilin kinase family, which includes the downstream Rho target, LIM kinase (LIMK). Significantly, strong genetic interactions were detected between Dpax and Dlimk, as well as downstream effectors of Dlimk. Supporting these genetic data, biochemical studies indicate that paxillin regulates Rac and Rho activity, positively regulating Rac and negatively regulating Rho. Taken together, these data indicate the importance of paxillin modulation of Rho family GTPases during development and identify the LIMK pathway as a critical target of paxillin-mediated Rho regulation (Chen, 2005).
Paxillin is a scaffolding protein found in focal adhesions. Targeted disruption of paxillin in mice results in an early embryonic lethal phenotype with defects in multiple mesodermally derived structures. The recent completion of the Drosophila genome revealed the evolutionary conservation of many of the key molecules found in focal adhesions, including integrins, paxillin, vinculin, FAK, p130CAS (see CAS/CSE1 segregation protein), and ILK. The Drosophila paxillin is predominantly expressed in embryos, pupae, and male adults. In situ analysis of staged embryos reveals a restricted expression pattern of Dpax. In particular, Dpax is highly expressed in tissues undergoing cell shape changes or cell migration. Overexpression of Dpax in late larval stages results in a pupal lethal phenotype with few escapers bearing malformed phenotypes, suggesting that Dpax also plays an important role during later stages of development (Chen, 2005).
A loss-of-function mutant of Drosophila paxillin has not yet been reported. Therefore, the UAS/GAL4 system was employed to investigate the function of Dpax in the later stages of development. As has been reported for Drosophila FAK, overexpressing Dpax results in a blistered-wing phenotype. In mammals, paxillin is a substrate of FAK in transducing signals from integrins. FAK regulates focal adhesion disassembly and has been shown to be involved in Drosophila Wnt4-mediated cell movement during ovarian morphogenesis and is also required for border cell migration during oogenesis. The function of Dpax in oogenesis is not clear; however, Dpax is also highly expressed in the border cells (Chen, 2005).
The blistered-wing phenotype is also found in integrin mutant flies. In the prepupal stage, the wing is a single epithelial sheet, and integrins have been suggested to play a regulatory role. As development progresses this sheet folds into a dorsal and ventral side, and the integrins play an adhesive role at these later stages. Using drivers that are expressed at different stages of development, the studies suggest that paxillin could be important for both the regulatory and adhesive functions of the integrins. Such functions would be consistent with studies of mammalian systems in which paxillin functions downstream of multiple integrins and can regulate both inside out and outside in signaling. In addition, both paxillin and FAK are important for focal adhesion turnover. Thus, too much paxillin or FAK may increase the turnover of focal complexes and perturb the stable adhesion between two epithelia, thereby resulting in the blistering phenotype (Chen, 2005).
Using a gain-of-function screen for modifiers that can rescue the Dpax-induced wing blistering, Cdi/TESK was identified. Like LIMK, Cdi/TESK phosphorylates the actin-depolymerizing factor cofilin and stabilizes F-actin. Cdi/TESK is highly homologous to LIMK in the kinase domain; however, a recent study has demonstrated that Cdi/TESK functions downstream of Rac1 during spermatogenesis. Drosophila LIMK functions downstream of Rho1 in regulating disk morphogenesis (Chen, 2005).
Dlimk and components in the Rho-LIMK pathway, including ssh, tsr, and bs/DSRF, also rescue the blistering phenotype. In addition, another regulator of SRF and actin, diaphanous, also shows genetic interactions with Dpax. Diaphanous is a direct effector of Rho which cooperates with LIMK to regulate SRF activation. All of these components play important roles in regulating F-actin synthesis. Taken together, these data indicate that it is possible that an increase in actin levels can prevent the increase in focal adhesion turnover caused by the excess level of paxillin, therefore suppressing the blistering phenotype. It is possible that simply overexpressing actin might be sufficient to rescue the blistering phenotype, although the results suggest that paxillin itself does not affect F-actin synthesis or actin organization. The ability of paxillin, however, to coimmunoprecipitate with LIMK and the increased cofilin phosphorylation in Pxl–/– MEFs suggests that paxillin can modulate LIMK function. These data, combined with the genetic and biochemical evidence that paxillin can regulate Rho, suggest that paxillin could act at multiple points to regulate the Rho pathway (Chen, 2005).
Interestingly, while modulation of some components downstream of Rho is able to suppress the blistering phenotype, overexpression of other components such as ROK does not alter this phenotype. While this could reflect insufficient expression levels or more complex regulation of ROK, the data suggest that paxillin's regulation of the Rho pathway may involve either modulation of only certain downstream components or a lack of function for these components in the paxillin-induced phenotypes (Chen, 2005).
Rho GTPases play an important role in regulating actin cytoskeleton organization. Genetic and biochemical analysis reveal that paxillin activates Rac signaling but inactivates Rho signaling. Previous binding and localization studies suggest that mammalinan paxillin may regulate Rac through its indirect association with at least two Rac exchange factors. Pix/Cool is linked to paxillin via PKL/Git2, the ARF-GAP, and overexpression studies with mutants of paxillin and other members of this complex have led to the suggestion that paxillin may be important for recruiting this complex to focal contacts. A second binding partner, Crk, can also link paxillin to Rac activation via a nontraditional exchange factor, Dock180. Mislocalization of one or both complexes in Pxl–/– mouse embryo fibroblasts (MEF)s could therefore lead to defects in Rac activation and subsequent defects in lamellipodium dynamics and migration. Both Pix/Cool and Crk localization were examined in rescued and Pxl–/– MEFs and only a minor decrease was detected in Cool and Crk positive peripheral adhesions in Pxl–/– cells. In MEFs, therefore, paxillin is not required for localization of these proteins to peripheral adhesions. This may be due to functional redundancy, as the paxillin family member Hic-5 can also bind the PKL-Pix complex and Crk can bind to other focal adhesion proteins, including p130Cas. In any case, mislocalization of these complexes is unlikely to account for the differences in Rac activation. In contrast, genetic studies of Drosophila have shown that deletion of a region encompassing the Drosophila homolog of Cool was able to suppress the Dpax-induced blistering. Thus, one potential mechanism by which paxillin may control Rac activation in Drosophila is through regulation of Pix/Cool. Since Rac and Rho have been shown to antagonize each other, it remains possible that in higher eukaryotes, paxillin could indirectly regulate Rac via regulation of Rho (Chen, 2005).
It is not clear how paxillin down-regulates Rho activity. Paxillin might be important for spatial regulation of Rho activity and/or controlling the activity or localization of a Rho GAP or GEF. Two Rho GAPs have been linked to mammalian paxillin. Graf is a Rho GAP that was originally identified as a Fak-binding partner, and a homolog of this protein has been identified in Drosophila studies. Since paxillin can interact with Fak, it is possible that loss of paxillin may somehow affect Graf localization or activation. While Fak localization to focal adhesions is less efficient in Pxl–/– MEFs, the effects are minimal and thus this is unlikely to account for the enhanced Rho activity. It is worth noting that it has recently been reported that mammalian paxillin binds to the p120 RasGAP and competes with p120 RasGAP for binding to p190 RhoGAP. It has been suggested that paxillin inhibits Rho by promoting the formation of free p190 RhoGAP. The Drosophila ortholog of p190 RhoGAP does not bind to the Drosophila p120 RasGAP. In addition, only minor changes in p190 localization to the leading edge were detected in Pxl–/– MEFs. Thus, paxillin may antagonize Rho function through multiple distinct regulatory mechanisms (Chen, 2005).
Taken together, these data suggest that while paxillin has the ability to interact with multiple proteins involved in diverse signaling pathways, a major function of this scaffolding protein in vivo is to regulate Rho family GTPases. Thus, misregulation of these GTPases is likely to account for the adhesion defects observed during development in mouse and Drosophila studies (Chen, 2005).
Aizawa, H., Wakatsuki, S., Ishii, A., Moriyama, K., Sasaki, Y., Ohashi, K., Sekine-Aizawa, Y., Sehara-Fujisawa, A., Mizuno, K., Goshima, Y. and Yahara, I. (2001). Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse. Nat. Neurosci. 4: 367-373. 11276226
Ang, L. H., Kim, J., Stepensky, V. and Hing, H. (2003). Dock and Pak regulate olfactory axon pathfinding in Drosophila. Development 130: 1307-1316. 12588847
Ang, L. H., Chen, W., Yao, Y., Ozawa, R., Tao, E., Yonekura, J., Uemura, T., Keshishian, H. and Hing, H. (2006). Lim kinase regulates the development of olfactory and neuromuscular synapses. Dev. Biol. 293(1): 178-90. 16529736
Arber, S., Barbayannis, F.A., Hanser, H., Schneider, C., Stanyon, C. A., Bernard, O. and Caroni, P. (1998). Regulation of actin dynamics through phosphorylation of cofilin by LIM-kinase. Nature 393: 805-809. 9655397
Ball, R. W., Warren-Paquin, M., Tsurudome, K., Liao, E. H., Elazzouzi, F., Cavanagh, C., An, B. S., Wang, T. T., White, J. H., Haghighi, A. P. (2010). Retrograde BMP signaling controls synaptic growth at the NMJ by regulating trio expression in motor neurons. Neuron 66: 536-549. PubMed ID: 20510858
Barber, C. F., Jorquera, R. A., Melom, J. E., Littleton, J. T. (2009). Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains. J Cell Biol 187: 295-310. PubMed ID: 19822673
Bernard, O., Ganiatsas, S., Kannourakis, G. and Dringen, R. (1994). Kiz-1, a protein with LIM zinc finger and kinase domains, is expressed mainly in neurons, Cell Growth Differ. 5: 1159-1171. 7848918
Chen, G. C., Gajowniczek, P. and Settleman, J. (2004). Rho-LIM kinase signaling regulates ecdysone-induced gene expression and morphogenesis during Drosophila metamorphosis. Curr. Biol. 14: 309-313. 14972681
Chen, G. C., Turano, B., Ruest, P. J., Hagel, M., Settleman, J., Thomas, S. M. (2005). Regulation of Rho and Rac signaling to the actin cytoskeleton by paxillin during Drosophila development. Mol. Cell. Biol. 25(3): 979-87. 15657426
Chen, X. and Macara, I. G. (2006). Par-3 mediates the inhibition of LIM kinase 2 to regulate cofilin phosphorylation and tight junction assembly. J. Cell. Biol. 172(5): 671-8. 16505165
Cheng, A. K. and Robertson, E. J. (1995). The murine LIM-kinase gene (limk) encodes a novel serine threonine kinase expressed predominantly in trophoblast giant cells and the developing nervous system. Mech. Dev. 52: 187-197. 8541208
Cziko, A. M., McCann, C. T., Howlett, I. C., Barbee, S. A., Duncan, R. P., Luedemann, R., Zarnescu, D., Zinsmaier, K. E., Parker, R. R. and Ramaswami, M. (2009). Genetic modifiers of dFMR1 encode RNA granule components in Drosophila. Genetics 182: 1051-1060. PubMed ID: 19487564
Delorme, V., et al. (2007). Cofilin activity downstream of Pak1 regulates cell protrusion efficiency by organizing lamellipodium and lamella actin networks. Dev. Cell 13(5): 646-62. PubMed citation: 17981134
Eaton, B. A. and Davis, G. W. (2005). LIM Kinase1 controls synaptic stability downstream of the type II BMP receptor. Neuron 47(5): 695-708. 16129399
Edwards, D. C., et al. (1999). Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics. Nat. Cell Biol. 1: 253-259. 10559936
Endo, M., Ohashi, K., Sasaki, Y., Goshima, Y., Niwa, R., Uemura, T. and Mizuno, K. (2003). Control of growth cone motility and morphology by LIM kinase and Slingshot via phosphorylation and dephosphorylation of cofilin. J. Neurosci. 23: 2527-2537. 12684437
Foletta, V. C., et al. (2003). Direct signaling by the BMP type II receptor via the cytoskeletal regulator LIMK1. J. Cell. Biol. 162: 1089-1098. 12963706
Frangiskakis, J. M., et al. (1996). LIM-kinase1 hemizygosity implicated in impaired visuospatial constructive cognition. Cell 86: 59-69. 8689688
Gorovoy, M., et al. (2005). LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells. J. Biol. Chem. 280(28): 26533-42. 15897190
Goyal, P., Pandey, D., Behring, A. and Siess, W. (2005). Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283. J. Biol. Chem. 280(30): 27569-77. 15923181
Heredia, L., et al. (2006). Phosphorylation of actin-depolymerizing factor/cofilin by LIM-kinase mediates amyloid beta-induced degeneration: a potential mechanism of neuronal dystrophy in Alzheimer's disease. J. Neurosci. 26(24): 6533-42. 16775141
Honma, M., Benitah, S. A. and Watt, F. M. (2006). Role of LIM kinases in normal and psoriatic human epidermis. Mol. Biol. Cell 17(4): 1888-96. 16467374
Hsieh, S. H., Ferraro, G. B. and Fournier, A. E. (2006). Myelin-associated inhibitors regulate cofilin phosphorylation and neuronal inhibition through LIM kinase and Slingshot phosphatase. J. Neurosci. 26(3): 1006-15. 16421320
Kobayashi, M., Nishita, M., Mishima, T., Ohashi, K. and Mizuno, K. (2006). MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. EMBO J. 25(4): 713-26. 16456544
Korkut, C., Li, Y., Koles, K., Brewer, C., Ashley, J., Yoshihara, M., Budnik, V. (2013). Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron 77: 1039-1046. PubMed ID: 23522040
Lawler, S. (1999). Regulation of actin dynamics: The LIM kinase connection. Curr. Biol. 9: R800-R802. 10556082
Lee-Hoeflich, S. T., et al. (2004). Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis. EMBO J. 23: 4792-4801. 15538389
Manser, E., et al. (1994). A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature 367: 40-46. 8107774
Meng, Y., et al. (2002). Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice, Neuron 35: 121-133. 12123613
Mizuno, K., et al. (1994). Identification of a human cDNA encoding a novel protein kinase with two repeats of the LIM/double zinc finger motif. Oncogene 9: 1605-1612. 8183554
Mori, T. et al. (1997). Comparison of tissue distribution of two novel serine/threonine kinase genes containing the LIM motif (LIMK-1 and LIMK-2) in the developing rat. Brain Res. Mol. Brain Res. 45: 247-254. 9149099
Nagata, K., et al. (1999). The N-terminal LIM domain negatively regulates the kinase activity of LIM-kinase 1. Biochem. J. 343: 99-105. 10493917
Ng, J. and Luo, L. (2004). Rho GTPases regulate axon growth through convergent and divergent signaling pathways. Neuron 44: 779-793. 15572110
Ng, J. (2008). TGF-β signals regulate axonal development through distinct Smad-independent mechanisms. Development 135(24): 4025-35. PubMed Citation: 19004854
Nishita, M., Aizawa, H. and Mizuno, K. (2002). Stromal cell-derived factor 1alpha activates LIM kinase 1 and induces cofilin phosphorylation for T-cell chemotaxis. Mol. Cell Biol. 22(3): 774-83. 11784854
Nishita, M., et al. (2005). Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration. J. Cell. Biol. 171(2): 349-59. 16230460
Niwa, R., Nagata-Ohashi, K., Takeichi, M., Mizuno, K. and Uemura T. (2002). Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108(2): 233-46. 11832213
Ohashi, K., Hosoya, T., Takahashi, K., Hing, H. and Mizuno, K. (2000). A Drosophila homolog of LIM-kinase phosphorylates cofilin and induces actin cytoskeletal reorganization. Biochem. Biophys. Res. Commun. 276(3): 1178-85. 11027607
Piccioli, Z. D., Littleton, J. T. (2014). Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin. J Neurosci 34: 4371-4381. PubMed ID: 24647957
Proschel, C., et al. (1995). Limk1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro, Oncogene 11: 1271-1281. 7478547
Schratt, G. M., et al. (2006). A brain-specific microRNA regulates dendritic spine development. Nature 439(7074): 283-9. 16421561
Soosairajah, J., Maiti, S., Wiggan, O., Sarmiere, P., Moussi, N., Sarcevic, B., Sampath, R., Bamburg, J. R. and Bernard O. (2005). Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin. EMBO J. 24(3): 473-86. 15660133
Sumi, T., et al. (1999). Cofilin phosphorylation and actin cytoskeletal dynamics regulated by rho- and Cdc42-activated LIM-kinase 2. J. Cell. Biol. 147: 1519-1532. 10613909
Sumi, T., Hashigasako, A., Matsumoto, K. and Nakamura, T. (2006). Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition. Exp. Cell Res. 312(7): 1021-30. 16455074
Tolias, K. F., Duman, J. G., Um, K. (2011). Control of synapse development and plasticity by Rho GTPase regulatory proteins. Prog Neurobiol 94: 133-148. PubMed ID: 21530608
Tursun, B., et al. (2005). The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones. Genes Dev. 19(19): 2307-19. 16204183
Vardouli, L., Moustakas, A. and Stournaras, C. (2005). Lim-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta. J. Biol. Chem. 280(12): 11448-57. 15647284
Verdier, V., Guang-Chao-Chena and Settleman, J. (2006b). Rho-kinase regulates tissue morphogenesis via non-muscle myosin and LIM-kinase during Drosophila development. BMC Dev. Biol. 6: 38. Medline abstract: 16882341
Wang, J. Y., et al. (2000). LIM kinase 1 accumulates in presynaptic terminals during synapse maturation, J. Comp. Neurol. 416: 319-334. 10602091
Wang, W., et al. (2006). The activity status of cofilin is directly related to invasion, intravasation, and metastasis of mammary tumors. J. Cell Biol. 173(3): 395-404. 16651380
Wen, Z., Han, L., Bamburg, J. R., Shim, S., Ming, G. L. and Zheng, J. Q. (2007). BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin. J. Cell Biol. 178: 107-119. PubMed Citation: 17606869
White-Grindley, E., Li, L., Mohammad Khan, R., Ren, F., Saraf, A., Florens, L. and Si, K. (2014). Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2. PLoS Biol 12: e1001786. PubMed ID: 24523662
Yang, E. J., Yoon, J. H., Min do, S. and Chung, K. C. (2004). LIM kinase 1 activates cAMP-responsive element-binding protein during the neuronal differentiation of immortalized hippocampal progenitor cells. J. Biol. Chem. 279(10): 8903-10. 14684741
Yang, N., Higuchi, O., Ohashi, K., Nagata, K., Wada, A., Kangawa, K., Nishida, E. and Mizuno, K. (1998). Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature 393: 809-812. 9655398
Yang, X., et al. (2004). LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1. Nat. Cell Biol. 6: 609-617. 15220930
Yoshihara, M., Adolfsen, B., Galle, K. T., Littleton, J. T. (2005). Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth. Science 310: 858-863. PubMed ID: 16272123
Zebda, N., Bernard, O., Bailly, M., Welti, S., Lawrence, D. S. and Condeelis J. S. (2000). Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension. J. Cell. Biol. 151(5): 1119-28. 11086013
date revised: 10 July 2014
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.