InteractiveFly: GeneBrief

mushroom bodies tiny: Biological Overview | References

BIOLOGICAL OVERVIEW

The p21 activated kinase (Pak) family of protein kinases are involved in many cellular functions like re-organisation of the cytoskeleton, transcriptional control, cell division, and survival. These pleiotropic actions are reflected in a plethora of known interacting proteins and phosphorylation substrates. Yet, the integration of a single Pak protein into signalling pathways controlling a particular developmental process are less well studied. For two of the three known Pak proteins in Drosophila melanogaster, D-Pak and Mushroom bodies tiny (Mbt), distinct functions during eye development have been established. This study undertook a genetic approach to identify proteins acting in the Mbt signalling pathway during photoreceptor cell morphogenesis. The genetic screen identified the actin depolymerisation factor Twinstar/Cofilin as one target of Mbt signalling. Twinstar/Cofilin becomes phosphorylated upon activation of Mbt. However, biochemical and genetic experiments question the role of the LIM domain protein kinase (Limk) as a major link between Mbt and Twinstar/Cofilin as it has been suggested for other PAK proteins. Constitutive activation of Mbt not only disturbs the actin cytoskeleton but also affects adherins junctions organisation indicating a requirement of the protein in cell adhesion dependent processes during photoreceptor cell differentiation (Menzel, 2007).

Morphogenesis is a fundamental process during the development of a multi-cellular organism, which not only requires the specification of the various cell types, but also their assembly into complex tissues by means of cell shape changes, cell movement, sorting processes, and elimination of surplus cells. Many of these events depend on precisely controlled modulation of cell-cell or cell-extracellular matrix contacts (Menzel, 2007).

Adherens junctions are specialized membrane structures that mediate adhesion between epithelial cells and provide a link to the actin cytoskeleton. The central component of adherens junctions is the cadherin-catenin complex. Cadherins constitute a family of transmembrane proteins that mediate Ca²⁺-dependent cell-cell adhesion. The intracellular domain of cadherin binds to β-Catenin (Drosophila: Armadillo), which in turn can associate with the actin binding protein α-Catenin. However, the prevailing dogma that cadherins at adherens junctions are linked to the actin cytoskeleton through β-Catenin and α-Catenin in a stable quaternary complex has recently been questioned by the finding that α-Catenin is associated in a mutually exclusive manner with either cadherin-β-Catenin or with actin (Menzel, 2007).

A role of adherens junctions as a focal point for intracellular signalling has emerged. Notably, the formation of cadherin-mediated cell contacts influences the activity of the RhoGTPases Cdc42, Rac and Rho. These proteins function as molecular switches, cycling between an active GTP-bound conformation and an inactive, GDP-bound state. In its active form, RhoGTPases bind to a vast number of downstream effector proteins and one of the major effects is the reorganisation of the actin cytoskeleton (Menzel, 2007).

A family of proteins, which are influenced by RhoGTPases in its activity and localisation, are the p21-activated kinases (Pak). Pak proteins are characterised by a C-terminal serine/threonine kinase domain and a N-terminal p21-binding domain (PBD) required for binding of Rac- or Cdc42-like RhoGTPases. Based on additional structural features, which determine the regulation of the kinase activity and the binding to other proteins, Pak proteins can be classified into two subgroups. From the six Pak proteins known in Homo sapiens (Hs), three (HsPak1-3) have been assigned to group 1, whereas HsPak4-6 belong to the group 2 (Bokoch, 2003 and Hofmann, 2004). Similar, the three Pak proteins found in Drosophila melanogaster can be classified as group 1 (D-Pak, D-Pak3) and group 2 (Mbt/D-Pak2) members, respectively (Mentzel, 2005). The role of Pak proteins as regulators of the cytoskeleton, cell morphology and cell motility is well established. Different Pak proteins can induce the formation of lamellipodia, filopodia, and membrane ruffles as well as the dissolution of stress fibers and the disassembly of focal adhesions (Bokoch, 2003; Hofmann et al., 2004). A function of Pak proteins at the centrosomes of dividing cells has been described (Zhao, 2005). Deregulation of Pak protein activity can result in oncogenic transformation (Kumar, 2006). The pleiotropic functions of Pak proteins are reflected in the plethora of known interacting proteins and phosphorylation substrates (Kumar, 2006). This obviously raises the question of differential activation of effector pathways by different Pak proteins in a cell-type specific manner (Menzel, 2007).

The developing Drosophila eye is used as a model system to study the role of the group 2 Pak protein Mbt in tissue morphogenesis. The adult eye with its 800 single eye units (ommatidia), each containing eight photoreceptor cells as well as non-neuronal cone, pigment, and bristle cells, arises from a monolayer epithelium, the eye imaginal disc. Differentiation of the different cell types of the eye is initiated in third instar larvae, when an indentation known as the morphogenetic furrow starts to move from posterior to anterior across the eye disc. Anterior to the furrow, cells continue to proliferate, posterior to it, the photoreceptor cells and lens-secreting cone cells become specified in a sequential manner through a series of inductive cell-cell interactions. At pupal stage, pigment and bristle cells are added to complete the ommatidium structure and surplus cells are eliminated by programmed cell death. After recruitment and determination, photoreceptor cells undergo massive morphological changes to form the rhabdomeres, the light-sensitive structures. Rhabdomeres are apical membrane specializations. Alignment of rhabdomeres to the ommatidial optical axis is achieved by involution of the apical domains and adherens junctions into the epithelial layer followed by their massive expansion in distal-proximal direction (Menzel, 2007).

Loss of function studies of the Pak proteins Mbt and D-Pak provided evidence for distinct localisation and function of these proteins during photoreceptor cell development. D-Pak is required in growth cones to control guidance of photoreceptor cell axons, whereas recruitment of Mbt to adherens junctions is dependent on a functional RhoGTPase binding site (Schneeberger, 2003). In mbt mutant animals, recruitment of the photoreceptor cells appear largely normal, but their final differentiation is disturbed. The adherens junctions become highly disorganized and rhabdomeres fail to elongate properly leading to the suggestion that Mbt acts as a downstream effector of activated RhoGTPases at adherens junctions to regulate photoreceptor cell morphogenesis (Schneeberger, 2003). This study shows that activation of Mbt disturbs the co-ordinated assembly of the ommatidium structure by interfering with the actin cytoskeleton and adherens junctions. Given the multitude of potential candidate targets of Mbt, a genetic screen was establised to identify components that act downstream of Mbt to control photoreceptor cell morphogenesis. This screen identified the actin depolymerisation factor Twinstar as one target of Mbt signalling. Yet, biochemical and genetic experiments question the role of LIM domain protein kinase (Limk) as a major link between Mbt and Twinstar (Menzel, 2007).

The view of adherens junctions as static structures that maintain epithelial integrity appears to be at odds with the requirement of dynamic cell shape changes during development. The assembly of the ommaditia of the Drosophila eye from an initially unpatterned epithelium provides an example. Photoreceptor cells become specified during third larval instar, but their final morphology is established at pupal stage. Simultaneously, the non-neuronal cone, pigment, and bristle cells must form a precise cell lattice that requires cell shape changes, movements and elimination of surplus cells. In spite of these dynamic processes, the integrity of adherens junctions as a basic feature of epithelia cells has to be maintained throughout eye development. The prevailing view that E-Cadherin is stably linked via β-Catenin and α-Catenin to the actin cytoskeleton has recently been questioned. It was shown that monomeric α-Catenin binds to E-Cadherin/β-Catenin, whereas dimeric α-Catenin does not and instead binds with high affinity to actin to influence Arp2/3-mediated actin polymerisation. If it is not α-Catenin that provides a physical link between adherens junctions and the actin cytoskeleton, what other proteins could mediate such a link (Menzel, 2007)?

Genetic and biochemical evidence is presented that the Pak protein Mbt, which localizes in a RhoGTPase dependent manner to adherens junctions, could provide a link to the actin cytoskeleton during photoreceptor cell morphogenesis. Constitutive activation of Mbt in photoreceptor cells causes clustering of F-actin, which finally leads to complete disruption of the adult eye morphology. In addition, the positioning of nuclei at apical levels is disturbed. The finding that mutations in twinstar and the phosphatase-encoding slingshot act as modifiers of the eye phenotype of a hypomorphic mbt allele suggest that these proteins work together to control photoreceptor cell morphogenesis. Additional evidence comes from the phenotypic analysis of the corresponding mutants. In twinstar mutants, elevated levels of F-actin, disruption of the ommatidial architecture and misshapen rhabdomeres are observed. Loss of Slingshot function results in increased F-actin levels and defects in the apical movement of photoreceptor cell nuclei. So far, no evidence is available for a direct interaction between Slingshot and Mbt. In vertebrates, Pak4 inactivates Slingshot and activates Limk by phosphorylation. Slingshot plays a dual role. It dephosphorylates and thereby down-regulates the activity of Limk to phosphorylate Cofilin at serine 3 and it directly dephosphorylates Cofilin at serine 3 (Soosairajah, 2005). Thus Pak4 activation results in a decrease of Cofilin activity and actin filament turnover. A major role of D-Limk in Mbt signalling is questioned by biochemical and genetic experiments. Activated Mbt is able to induce Twinstar phosphorylation. However, despite binding and phosphorylation of D-Limk by activated Mbt, no strong increase of D-Limk activity was seen. Furthermore, genetic experiments with a recently identified null mutation in D-Limk show that Mbt signalling is not completely blocked in the absence of D-Limk function. Yet, the strong synergistic effect upon complete removal of Mbt and D-Limk function indicates the requirement of both proteins in eye development. If it is not Mbt, what other proteins could be involved in regulation of D-Limk function during eye morphogenesis? Recent experiments implicate a role of the Par3 protein as a regulator of vertebrate Limk-2 activity towards Cofilin. Interestingly, the Drosophila Par3 homologue Bazooka localizes to and is required for maintenance of adherens junctions during photoreceptor cell morphogenesis (Menzel, 2007).

It is also evident that regulation of Twinstar activity by Pak proteins is not restricted to eye development. Mutations in twinstar lead to proliferation defects of central brain neuroblasts and the neurons derived from these neuroblasts show axon growth defects. In this study, genetic evidence was presented that Twinstar function in axon growth is activated by Slingshot and inhibited by D-Limk, which in turn is regulated by D-Pak and Rock-kinases. In vertebrates, Limk and Slingshot control growth cone motility and morphology via Cofilin phosphorylation. Knock-out of Limk in mice leads to changes in Cofilin phosphorylation and the actin cytoskeleton resulting in abnormalities in spine morphology and in synaptic function. A dominant-negative version of mouse Pak1 causes changes in spine number and synaptic morphology. Thus, different Pak proteins might not only act as general regulators of Cofilin activity, but they could also provide spatial and temporal control even in a single cell (Menzel, 2007).

In addition to the role in re-organisation of the actin cytoskeleton, overexpression experiments indicate that Mbt is also able to influence the adhesive properties of cells. Differential adhesion is crucial to establish the final retinal pattern. It has been demonstrated that an apical protein complex consisting of Crumbs, Stardust, and DPATJ controls assembly of adherens junctions and is essential for rhabdomere maintenance. Differential adhesion, by expression of DE-Cadherin alone or together with DN-Cadherin has been suggested as a mechanism that controls cell shape changes. Expression of DN-Cadherin in cone cells (in addition to DE-Cadherin, which is expressed in all cells in an ommatidium) confers on them specific adhesion properties in order to adopt a cell shape that minimizes surface contacts with surrounding cells. Integration of other proteins into the adhesive complex could be another mechanism to modify adhesive contacts. Hibris and IrreC/rst, transmembrane proteins of the immunoglobulin superfamily, become integrated into the adhesion complex and form an intercellular link only at the interface between the presumptive primary pigment cells and secondary/tertiary pigment cells to control proper cell sorting during pupal eye development. Interference with DE-Cadherin function blocks proper localisation of IrreC/rst and results in cell sorting and survival defects. It is therefore conceivable that neuronal expression of activated Mbt influences DE-Cadherin-mediated cell adhesion of photoreceptor and bristle cells with the surrounding pigment and cone cells. In this way, motility, sorting or survival of all retinal cells can be affected. Evidence for a function of other group 2 PAK proteins in regulation of cell adhesion comes from studies with human PAK4 and Xenopus X-Pak5. Expression of an activated version of human Pak4 leads to anchorage-independent growth of fibroblasts (Qu, 2001). X-Pak5 is expressed in regions of the embryo that undergo extensive cell movements during gastrulation. It was shown that X-Pak5 localises to cell-cell junctions and regulates in a calcium-dependent manner cell-cell adhesion. An activated version of X-Pak5 decreased cell adhesiveness, while kinase-dead X-Pak5 increased adhesion (Faure, 2005). However, in all cases the molecular targets of the PAK proteins to exert these effects remain to be identified (Menzel, 2007).

Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor

In Drosophila epithelial cells, apical exclusion of Bazooka/Par3 defines the position of the Zonula Adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. This study shows that the small GTPase Rap1, its effector AF6/Canoe (Cno) and the Cdc42-effector Pak4/Mushroom bodies tiny (Mbt), converge in regulating epithelial E-Cadherin, and Bazooka retention at the ZA. Furthermore, the results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating their functions during ZA morphogenesis are interlinked. In this context, the Rap1-GEF Dizzy was found to be enriched at the ZA and the results suggest it promotes Rap1 activity during ZA morphogenesis. Altogether, it is proposed the Dizzy, Rap1/Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis (Walther, 2018).

In the pupal photoreceptor, ZA morphogenesis is orchestrated by a conserved protein network that includes Cdc42, Par6, aPKC, Baz, Crb and its binding partner Sdt, and Par1. In turn, AJ material is an essential part of the regulatory network that orchestrates polarity. Previous work has shown that Mbt regulates pupal photoreceptor development by promoting ZA morphogenesis. During this process Mbt contributes in preventing Baz from spreading to the lateral membrane, a regulation that this study found to depend in part on the phosphorylation of Arm by Mbt at S561 and S688. It is proposed that Mbt regulates photoreceptor polarity by promoting the retention of Baz at the developing ZA. Failure in ZA retention leads to Baz spreading to the lateral membrane where it is eliminated through Par1-mediated displacement. In these cells, failure to retain AJ material, including Baz, at the ZA leads to its shortening along the apical basal axis and can impact on the polarization program of the photoreceptor (Walther, 2018).

This study shows that Mbt function is linked to that of Dzy, Rap1 and Cno. First, Cno and Mbt accumulation at the ZA is interdependent, reflecting a tight coupling between the Rap1 and Cno pathway and Mbt. Second, it was found that Cno promotes Baz retention at the ZA, as cnoIR leads to shorter ZAs that can be depleted of Arm and Baz. This phenotype resembles that of mbt mutant cells and is also seen when overexpressing a version of Arm that cannot be phosphorylated by Mbt. These observations prompted a test or the hypothesis that Rap1, Cno and Mbt might function as part of a linear pathway promoting Baz retention at the ZA. In this pathway, it was reasoned that Mbt could mediate Rap1 function through Arm phosphorylation. In testing this hypothesis, it was found that this is not the case. Instead, the observation that expressing a version of Arm that mimics its constitutive phosphorylation by Mbt does not ameliorate the cnoIR phenotype suggests that Rap1, Cno, and Mbt converge in promoting Baz retention at the ZA, and cannot compensate for each other during this process. This conclusion is well supported by the finding that overexpressing cno in mbt mutant cells does not lead to an amelioration of the mbt phenotype. Third, it was found that Mbt influences the distribution of Rap1 along the apical-basal axis of the cell in that Rap1::GFP no longer accumulates preferentially at the ZA. This correlates with a loss of Dzy::GFP at the plasma membrane, raising the possibility that Mbt might regulate Rap1 through Dzy. However, the dzy phenotype is milder than that seen with Rap1 or cno, in that loss of dzy does not lead to cell delamination from the retina. This suggests that, as has been reported in the cellularizing embryo, other GEFs regulate Rap1 during epithelial morphogenesis (Walther, 2018).

An interesting aspect of the cnoIR phenotype is the defects in apical accumulation of aPKC and Crb. These defects are not observed in the dzy mutant or Rap1IR cells, indicating that Cno might function independently of Rap1 during this process. However, it is noted that while Cno was not detected at the ZA of cnoIR cells, it can still be detected in Rap1IR cells. It is therefore hypothesized that residual Cno in Rap1IR cells supports optimum aPKC and Crb accumulation at the apical membrane. In this model, Dzy, Rap1 and Cno function as part of the same pathway, which includes a function in promoting optimum apical accumulation of Crb and aPKC. Baz is required for Par complex assembly and associated aPKC and Crb recruitment at photoreceptor apical membrane. It is hypothesized that the defects in Crb and aPKC that were detect in cnoIR cells are linked to the failure in retaining Baz at the ZA, which leads to its elimination from the lateral membrane by Par1. More work will be required to understand how exactly AJ material and ZA retention of Baz influences apical membrane specification (Walther, 2018).

Rap1 and cno have been shown to regulate apical-basal polarity in the cellularizing embryo. In this model system, Rap1 and Cno regulate the apical localization of Baz and Arm, which precedes the apical recruitment of Crb. In turn, Baz influences the localization of Cno. This work indicates that similar complex regulations are at play in the pupal photoreceptor. However, unlike in the early embryo, AJ material (Arm) is absolutely required for Baz (and Par6-aPKC) accumulation or retention at the cell cortex in the developing pupal photoreceptor. A model is therefore favored whereby Mbt, Rap1 and Cno influence ZA morphogenesis primarily through regulating the interface between E-Cad or Arm, Baz and the F-actin cytoskeleton. In this model, Mbt regulates this interface both through Arm phosphorylation and cofilin-dependent regulation of F-actin, and Cno contributes to this process, at least in part, through its ability to bind to F-actin (Walther, 2018).

To probe Rap1 and Cno function during photoreceptor ZA morphogenesis, the effect of decreasing Rap1 expression on E-Cad stability was assessed. Consistent with the notion that the function of mbt and Rap1 are linked during ZA morphogenesis, it as found that, as it is the case for Mbt , Rap1 is required to stabilize E-Cad::GFP at the photoreceptor ZA. However, the mobile fraction estimated for E-Cad is much higher in Rap1IR cells than in mbt^P1 null cells (evaluated at ~70% for Rap1IR and 45% for mbt^P1). Together with the finding that Mbt accumulation at the ZA is decreased in Rap1IR cells, FRAP data are therefore compatible with Mbt mediating part of the function of Rap1 in promoting E-Cad stability. However, the much larger mobile fraction were estimated in the Rap1IR genotype when compared to mbt^P1 photoreceptors indicates that Rap1 must also regulate E-Cad stability independently of Mbt. The longer time scale for E-Cad::GFP to recover in Rap1IR cells when compared to mbt^P1 mutant cells is compatible with Rap1 functioning, in part, through promoting E-Cad delivery (Walther, 2018).

Mbt, a Drosophila PAK protein, combines with Cdc42 to regulate photoreceptor cell morphogenesis

The gene mushroom bodies tiny (mbt) encodes a putative p21-activated kinase (PAK), a family of proteins that has been implicated in a multitude of cellular processes including regulation of the cytoskeleton, cell polarization, control of MAPK signalling cascades and apoptosis. The mutant phenotype of mbt is characterized by fewer neurones in the brain and the eye, indicating a role for the protein in cell proliferation, differentiation or survival. Mutations in mbt interfere with photoreceptor cell morphogenesis. Mbt specifically localizes at adherens junctions (AJs) of the developing photoreceptor cells, and Cdc42, an Mbt interacting protein, is responsible for localization of Mbt to AJs. A structure-function analysis of the Mbt protein in vitro and in vivo revealed that the Mbt kinase domain and the GTPase binding domain, which specifically interacts with GTP-loaded Cdc42, are important for Mbt function. A role for Mbt is proposed as a downstream effector of Cdc42 in photoreceptor cell morphogenesis (Schneeberger, 2003).

Based on their distinct molecular structure, mammalian PAK 1-3 and Drosophila PAK are classed together as the group I PAKs, whereas mammalian PAK4-6 and Mbt constitute the group II PAKs. All PAK proteins share a C-terminal kinase domain and a N-terminal binding domain for proteins of the Rho family of small GTPases (p21-binding domain, PBD). The group I PAKs show some additional structural features that are missing in group II PAKs. Most importantly, the PBD of group I PAKs is C-terminally flanked by the kinase inhibitory domain (KID), which negatively regulates kinase activity through interaction with the kinase domain. Binding of GTP-bound forms of Cdc42 or Rac releases this intramolecular association, resulting in autophosphorylation and full activation of the kinase. Group I PAKs also possess several proline-rich sequences that bind to SH3 domain-containing proteins. Interaction with the SH2/SH3 domain adaptor proteins Nck and the corresponding Drosophila homolog Dock provides a link to cell-surface receptors. SH3 domain-mediated binding to Cool/PIX proteins can positively or negatively regulate PAK kinase activity (Schneeberger, 2003 and references therein).

Mbt mutant flies display a rough eye phenotype (Melzig, 1998). Tangential sections through adult eyes reveal the absence of a variable number of photoreceptor cells in many ommatidia. The rhabdomeres of the remaining photoreceptor cells show morphological defects. The cross-section profiles of the rhabdomeres are enlarged and neighbouring rhabdomeres often contact each other, a condition not seen in the wild type. Longitudinal sections reveal that the rhabdomeres are twisted, fragmented and do not extend from the pseudocone to the floor of the retina. These phenotypes suggested that Mbt is required for recruitment and/or proper differentiation of photoreceptor cells (Schneeberger, 2003).

To analyse the function of Mbt during eye development, a polyclonal antiserum was generated and eye-antennal imaginal discs from third instar larvae and pupae were stained. Differentiation of the cells that comprise the single eye units (ommatidia) occurs in a step-wise fashion and is initiated in the morphogenetic furrow, which moves across the eye disc from posterior to anterior. Staining of third-instar eye-imaginal discs with the Mbt antiserum revealed an accumulation of the Mbt protein at apical membrane sites of the photoreceptor cells as soon as they become recruited to the ommatidial clusters and initiate differentiation. Low levels of Mbt protein were detected at the membranes of undifferentiated cells. Staining was completely absent in eye discs derived from mbt^P1 mutant larvae, demonstrating the specificity of the antiserum and confirming the notion that mbt^P1 is a complete loss-of-function allele (Melzig, 1998). To determine the subcellular localization of Mbt more precisely, eye discs were co-stained with anti-Armadillo (Arm, Drosophila ß-Catenin) antibodies, a marker for adherens junctions (AJs). Staining for both largely overlaps in the photoreceptor cells. From apical to basal cross sections it became evident that Mbt is less abundant in the most apical domain of Arm expression in the photoreceptor cells (Schneeberger, 2003).

The final architecture of the ommatidia is established during pupal development and is accompanied by major morphological changes. At 37% of pupal development (p.d.), the apical domains of the photoreceptor cells have involuted. Thus, the apical domains of the photoreceptors point toward the center of the ommatidial cluster. After involution, the apical membranes of the photoreceptor cells start to expand to form the rhabdomeres. Each rhabdomere is surrounded by the stalk membrane, which connects it to the zonula adherens. As shown by anti-Arm staining, the AJs span the whole proximal-to-distal length of the photoreceptors at 50% p.d. Mbt remains colocalized with Arm at AJs of pupal photoreceptor cells at different stages of their development. Higher levels of Mbt expression can also be seen in the future bristle cells, whereas cone and pigment cells express low levels of Mbt. A 3D reconstruction of a wild-type ommatidial cluster stained with anti-Arm and anti-Mbt antibodies shows the colocalization of both proteins at AJs of the photoreceptor cells along the whole proximodistal length. In summary, these data provide evidence that Mbt is localized at AJs of photoreceptor cells from the initial recruitment to their final differentiation (Schneeberger, 2003).

The observed phenotypes in mbt^P1 eyes could result from a defect in cell proliferation, photoreceptor cell recruitment or differentiation. To determine whether mbt mutations affect recruitment or early neuronal differentiation of photoreceptor cells, wild-type and mbt^P1 third instar larval eye discs were stained with an antibody against the neuronal differentiation marker Elav. Only rarely did mbt^P1 ommatidia contain fewer Elav-positive cells than wild-type clusters. This result was confirmed by using HRP as an independent differentiation marker. This suggests that a failure in recruitment of photoreceptor cells is not the major cause of the mbt phenotype (Schneeberger, 2003).

The specific localization of Mbt at AJs prompted a look for AJ defects in mbt^P1 third instar and pupal eye imaginal discs with an anti-Arm antibody. In third instar eye discs, the AJs of the developing photoreceptor cells appear disorganized. Frequently, the AJs extend laterally. The AJ defects become much more pronounced at pupal stages. At 37% p.d., AJs fail to extend in proximodistal direction. At 50% p.d., AJs are fragmented and form patchy and disorganized structures. To verify these results and to exclude the possibility that mbt^P1 disturbs only Arm localization without affecting AJs, mbt^P1 eye imaginal discs were co-stained with anti-Canoe antibodies as an independent AJ marker. Canoe and Arm remain colocalized in mbt^P1 eye discs. In addition, wild-type and mbt^P1 pupal eye discs were stained with antibodies against the apical determinant Crumbs (Crb) and the Discs large (Dlg) protein, which is a marker for septate junctions in epithelial cells. Crumbs is essential to maintain AJ integrity during photoreceptor cell morphogenesis and is localized at the stalk membrane between AJs and the rhabdomeres. Compared with wild-type ommatidia, Crb and Dlg are de-localized in mbt^P1 mutant cells. In summary these data suggest that Mbt function is required in the developing photoreceptor cells to undergo their morphological changes (Schneeberger, 2003).

To gain insight into the molecular mechanisms that control Mbt function, the binding of Mbt to Rho-type GTPases was tested. Group I PAKs have been shown to interact via the p21-binding domain (PBD) with GTP-loaded Rac and Cdc42 but not with Rho, whereas the group II PAK proteins PAK4 and PAK5 preferentially bind to GTP-bound Cdc42. Myc-tagged versions of the Drosophila homologs of Cdc42, Rac1 and Rho1 were co-expressed with HA-tagged Mbt in HEK293 cells. Co-immunoprecipitation experiments revealed a nearly exclusive binding of Cdc42 to Mbt. Rac1 showed only a very weak interaction whereas no binding of Rho1 to Mbt was detected. The specificity of the interaction between Cdc42 and Mbt was tested by mutation of two conserved histidine residues in the PBD to leucine (Mbt^H19,22L). The mutant Mbt protein was unable to bind to Cdc42. Thus, the interaction between Cdc42 and Mbt is indeed mediated by the PBD. To determine whether activation of Rho-type GTPases influences binding to Mbt in vivo, the constitutively activated variants Cdc42^G12V, Rac1^G12V and Rho^G14V were co-expressed with Mbt in HEK293 cells. Cdc42^G12V showed an enhanced interaction with Mbt when compared to wild-type Cdc42. This result indicates that only the active, GTP-bound form of Cdc42 binds to Mbt. To verify this result, Cdc42 was expressed as a GST-fusion protein in bacteria and used in pull-down experiments upon loading with GDP or GTP. Mbt selectively binds to GTP-loaded Cdc42 but not to unloaded or GDP-loaded Cdc42. Thus, the preference for binding GTP-bound Cdc42 appears to be a common feature among group II PAKs (Schneeberger, 2003).

The PBD of group I PAKs is C-terminally flanked by the kinase inhibitory domain (KID). Binding of activated Cdc42 and Rac relieves the inhibitory influence of the KID on PAK kinase activity. In addition, group II PAKs share significant sequence homology C-terminal to the PBD, but the sequences differ significantly from the group I PAK KID (Schneeberger, 2003).

To analyse the influence of Cdc42 binding on Mbt kinase activity the Cdc42 binding-deficient Mbt^H19,22L construct was used. A second Mbt construct used in this study bears a mutation in the kinase domain. This mutation (T525A), located in the linker region between subdomains VII and VIII, corresponds to the T777A mutation in the Saccharomyces cerevisiae PAK protein Ste20p and has been found to disrupt autophosphorylation and catalytic activity of Ste20p. HEK293 cells were transfected with HA-tagged wild-type or the presumptive kinase-dead version of Mbt and the immunopurified protein complexes were incubated with kinase buffer and gamma-³²P]ATP together with myelin basic protein (MBP) as a substrate. Compared with wild type Mbt, the T525A mutation strongly reduced autophosphorylation and substrate phosphorylation. Co-expression of Cdc42 with Mbt did not increase autophosphorylation or MBP phosphorylation when compared with cells transfected with Mbt alone. Importantly, co-expression of Mbt and the constitutively activated Cdc42^G12V construct slightly reduced rather than enhanced the ability of wild-type Mbt to phosphorylate MBP. Conversely, the Cdc42-binding defective

Mbt^H19,22L protein showed a moderate increase of MBP phosphorylation independent of co-expression with Cdc42 or Cdc42^G12V. Autophosphorylation was not affected by removal of the Cdc42-binding site. These results fit with previous observations that kinase activity of PAK4, 5 and 6 is not upregulated upon Cdc42 binding, whereas deletion of the PBD can lead to enhanced kinase activity. Thus, group II PAKs appear to differ from group I PAKs in their mechanism to regulate kinase activity (Schneeberger, 2003).

In order to test the requirement of the Cdc42-binding domain and the kinase domain for Mbt function in vivo, wild-type or mutated Mbt proteins were expressed during eye development in the absence of the endogenous Mbt protein. Northern blot analysis and antibody staining indicates that mbt^P1, which carries a P-element insertion in the protein encoding sequence, is a complete loss-of function allele. Gal4:238Y-driven expression of a mbt cDNA in the brain is sufficient to rescue the mbt^P1 brain phenotype (Tettamanti, 1997; Melzig, 1998). Gal4:238Y is also expressed in the eye-antennal imaginal disc in a manner that closely resembles the expression pattern of the endogenous Mbt protein. Consistent with this observation, the eye phenotype of mbt^P1 flies was completely rescued by Gal4:238Y-driven expression of the wild-type mbt cDNA. By contrast, the Cdc42-binding deficient Mbt^H19,22L protein was unable to rescue the mbt^P1 eye phenotype, whereas the kinase-defective Mbt^T525A construct partially rescues the mbt^P1 eye phenotype (Schneeberger, 2003).

In summary, these experiments have verified the importance of the Cdc42-binding domain and the kinase domain for the in vivo function of Mbt during eye development. The partial rescue ability of the Mbt^T525A construct indicates that some functions of Mbt are independent of kinase activity. The differences observed in the rescue ability of the kinase defective Mbt^T525A and the Cdc42 binding-deficient Mbt^H19,22L proteins also suggests that Cdc42 binding to Mbt influences Mbt function in a kinase-independent manner. One possibility that was investigated was the proper localization of the Mbt protein to AJs (Schneeberger, 2003).

Group II PAKs lack the N-terminal binding site for the Nck/Dock adaptor protein, which could provide a link to membrane-bound proteins. To investigate whether the Cdc42-binding domain is responsible for the observed localization of Mbt to AJs, wild-type and mutated mbt cDNAs were expressed with the Gal4:238Y driver line in a mbt^P1 mutant background and the subcellular localization of the corresponding Mbt proteins were analysed in pupal eye discs. The expression pattern of the transgenic, non-mutated Mbt protein in the eye imaginal disc closely resembles the expression pattern of the endogenous Mbt protein. High levels of transgenic Mbt accumulate at the AJs of the developing photoreceptor cells. Consistent with the complete rescue of the adult mbt^P1 eye phenotype, no morphological abnormalities were observed when pupal eye discs were stained with anti-Mbt or anti-Arm antibodies. An identical localization pattern was observed when the kinase-defective Mbt^T525A protein was expressed in the mbt^P1 mutant background, indicating that eliminating kinase activity does not influence the subcellular distribution of the Mbt protein. However, as revealed by co-staining with an anti-Arm antibody, the transgenic Mbt^T525A protein only partially rescued the AJ defects in mbt^P1 animals. The AJs extend to some degree in proximal-to-distal direction but still do not have a regular architecture. This result correlates with the partial rescue observed in adult eyes. By contrast, the Cdc42 binding-deficient Mbt^H19,22L protein did not accumulate at AJs but instead was distributed within the cytoplasm. Anti-Arm staining revealed that the Mbt^H19,22L protein is unable to rescue the AJs defects seen in mbt^P1 mutant eye discs. Thus, there is an absolute requirement of the Cdc42 binding domain for localization and function of the Mbt protein during eye development. To exclude the possibility that the failure of the Mbt^H19,22L protein to localize at AJs is not a secondary effect of the mbt^P1 phenotype itself, the Mbt^H19,22L protein was also expressed in a wild-type background. Although endogenous and transgenic Mbt protein cannot be distinguished in this case, two observations were made: (1) the Mbt^H19,22L protein did not cause any obvious AJs defects when expressed in a wild-type background; (2) Mbt protein was found at AJs and in the cytoplasm. Because no cytoplasmic Mbt protein was detected upon expression of the non-mutated Mbt protein in a wild-type background, it is concluded that the Mbt^H19,22L protein localizes in the cytoplasm (Schneeberger, 2003).

To show that Cdc42 and not another protein bound to the PBD of Mbt is responsible for localization of Mbt to AJs, animals that either lacked Cdc42 function or ectopically expressed mutated versions of the Cdc42 protein were examined. Because removal of Cdc42 function causes lethality (Genova, 2000), homozygous mutant Cdc42³ or Cdc42⁴ cell clones were generated using the MARCM system. Only those cells that are homozygous for the Cdc42 mutation express the membrane localized mCD8 marker. Most of the Cdc42³ or Cdc42⁴ clones obtained in the eye disc contain only a few mCD8-positive cells. Consistent with findings that Cdc42 mutant cells can initiate their differentiation into photoreceptor cells (Genova, 2000), the majority of mCD8-positive (Cdc42 mutant) photoreceptor cells analysed extend an axon. From single apical sections and apical-to-basal cross-sections it is evident that Mbt is localized at the apical side of photoreceptors, whereas the mCD8 marker labels the whole cell surface of the Cdc42 mutant photoreceptor cell, including the axonal projection. Loss of Cdc42 function is accompanied by the loss of apical Mbt protein (Schneeberger, 2003).

The influence of the constitutively activated (GTP-loaded) Cdc42^G12V and of the dominant-negative (GDP-loaded) Cdc42^T17N protein on Mbt localization was examined. Because expression of these constructs with the Gal4:238Y driver line results in embryonic lethality, the eye-specific GMR-Gal4 driver line was used. Consistent with the finding that Mbt only binds to GTP-loaded Cdc42, expression of Cdc42^T17N has only minor effects on Mbt localization and the AJs morphology. By contrast, Cdc42^G12V causes a dramatic change in the Mbt and Arm expression pattern. Mbt accumulates at membrane sites of all cells. Arm expression can only be seen at early stages of photoreceptor cell recruitment, indicating that Cdc42^G12V completely disrupts the integrity of AJs in the developing photoreceptor cells (Schneeberger, 2003).

One major difference between group I and group II PAKs is the regulation of kinase activity. For group I PAK proteins, binding of GTP-bound Cdc42 or Rac releases the inhibitory effect of the KID on catalytic activity. The lack of an obvious KID in group II PAKs is reflected by their distinct biochemical properties. In contrast to group I PAKs, a slightly reduced rather than enhanced kinase activity is observed upon co-expression of Mbt and a constitutively active variant of Cdc42 in serum starved cells. A Cdc42 binding-deficient Mbt protein showed enhanced kinase activity in vitro. Similar results have been reported for other group II PAKs. Kinase activity of PAK4 was not further enhanced upon co-transfection of activated Cdc42 but deletion or mutation of the PBD of PAK4 and PAK6 results in enhanced kinase activity (Schneeberger, 2003).

From these data, the question remains of what role activated Cdc42 plays in regulating the functions of group II PAKs. Genetic studies have verified the importance of the kinase domain and the PBD for the in vivo function of Mbt. Despite increased kinase activity in vitro, a construct lacking the PBD is unable to rescue the mbt^P1 mutant phenotype in the eye. However, a kinase-dead Mbt protein partially rescued the mbt^P1 phenotype. This indicates that Cdc42 binding to Mbt fulfils some additional essential functions that are independent of kinase activity. Localization studies show that one major function of the PBD is to recruit Mbt specifically to adherens junctions. These data are also supported by the observation that localization of a PBD-deficient Mbt protein to the cellular membrane, by fusing it to a general membrane targeting sequence, is not sufficient to restore the wild-type function of the protein. It is therefore proposed that Cdc42 has a dual function: specific recruitment of Mbt to AJs and regulation of the catalytic activity of Mbt. The importance of proper targeting of PAK proteins to distinct subcellular compartments for their in vivo function is also evident from other studies. PAK4 recruitment to Golgi membranes by activated Cdc42 is dependent on an intact PBD. Activated Rac and Cdc42 also promote the relocalization of a recently described group II PAK protein in Xenopus laevis, X-PAK5, from microtubule networks to actin-rich regions. In the case of group I PAKs, autophosphorylation of the Nck and PIX SH3 domain binding sites has been suggested as a mechanism to control cycling between different cellular compartments. Also, D-PAK function in the photoreceptor axons and growth cones is dependent on the interaction with the Drosophila Nck homolog dreadlocks (Dock), which binds to the tyrosine phosphorylated axon guidance receptor DSCAM through its SH2 domain (Schneeberger, 2003).

Rho GTPases are important regulators of the actin cytoskeleton and are involved in many developmental processes that require morphological changes of epithelial and neuronal cells. Each GTPase regulates a diverse range of effector molecules and thus induces multiple defects when misregulated. For this reason, it is difficult to reconcile all the data obtained with the loss-of-function Cdc42 alleles and the various ectopically expressed Cdc42 variants, but a number of conclusions can be drawn with respect to the function of Mbt and D-PAK. (1) Activated Cdc42 has an effect on the levels of D-PAK accumulating at the dorsal most ends (leading edge) of epidermal cells flanking the amnioserosa. This is consistent with the result that overexpression of activated Cdc42 in the eye disc leads to accumulation of Mbt at the membrane. (2) The mbt and the Cdc42 mutant phenotypes in the eye display some similarities. In the eye disc, cells devoid of endogenous Cdc42 or Mbt function can initiate their differentiation into photoreceptor cells (Genova, 2000). In the adult eye, loss of Cdc42 function also causes the loss of photoreceptor cells and defects in rhabdomere morphology of the remaining photoreceptor cells. At first sight the similarities in the loss-of-function phenotypes contradict the biochemical data, implying a negative role for Cdc42 in kinase activation. (3) The Cdc42 binding-deficient Mbt protein, despite enhanced kinase activity in vitro, does not cause visible phenotypes in the eye even when expressed in a wild-type background. There are several possibilities to reconcile the data. In all cases, Mbt is either not present or not localized to AJs. Some functions of Mbt might be independent of kinase activity. Alternatively, the moderate increase in kinase activity of the Mbt^H19,22L protein might not be sufficient to induce dominant phenotypes. A more detailed analysis of Mbt kinase activity in vivo requires the identification of physiological substrates (Schneeberger, 2003).

The described roles of PAK proteins and RhoGTPases in regulating the actin cytoskeleton and the results presented in this study imply that Mbt localized at AJs mediates signals to the cytoskeleton to ensure proper photoreceptor cell morphogenesis. Because Mbt interacts only with GTP-loaded Cdc42, recruitment of Mbt to AJs would require localized Cdc42 activation. Although there is no direct evidence so far for a selective Cdc42 activation at AJs in photoreceptor cells, studies in mammalian epithelial cell lines have demonstrated Cdc42 activation by the AJ protein E-cadherin. In addition, the molecular link between Mbt and the actin cytoskeleton remains to be defined. As has been reported for PAK4, the Drosophila homolog of LIMK could provide a link between Mbt and the actin cytoskeleton. Based on the similar mutant phenotype, the PDZ domain protein Canoe could be another interaction partner of Mbt. Canoe is localized at AJs and the mammalian ortholog Afidin has been shown to bind to actin filaments. Unravelling the precise molecular functions of Mbt in cell morphogenesis awaits the identification of interaction partners and physiological substrates (Schneeberger, 2003).

A protein related to p21-activated kinase (PAK) that is involved in neurogenesis in the Drosophila adult central nervous system

Brains are organized by the developmental processes generating them. The embryonic neurogenic phase of Drosophila has been studied in detail at the genetic, cellular and molecular level. In contrast, much of what is known of postembryonic brain development has been gathered by neuroanatomical and gene expression studies. The molecular mechanisms underlying cellular diversity and structural organisation in the adult brain, such as the establishment of the correct neuroblast number, the spatial and temporal control of neuroblast proliferation, cell fate determination, and the generation of the precise pattern of neuronal connectivity, are largely unknown. In a screen for viable mutations affecting adult central brain structures, the mushroom bodies tiny (mbt) gene, which encodes a protein related to p21-activated kinase (PAK), was isolated. Mutations in mbt primarily interfere with the generation or survival of the intrinsic cells (Kenyon cells) of the mushroom body, a paired neuropil structure in the adult brain involved in learning and memory (Melzig, 1998; full text of article).

The MBT protein sequence revealed significant homology with the PAK family of serine/threonine kinases. Several distinct PAK proteins were identified in different vertebrate species including human, mouse and rat. PAK proteins contain an amino-terminal binding domain for the Rho subfamily of Ras-related small GTPases (p21 proteins), a carboxy-terminal kinase domain, and a heterotrimeric G-protein β-subunit binding site at the carboxyl terminus. There are some differences between MBT and other PAK family members, however. The p21-binding domain of PAK is limited to 57 amino acids, with an amino-terminal 16 amino acid core critical for binding to the Rho-type GTPases Cdc42 and Racin vitro. The core sequence of the MBT p21-binding domain is well conserved with other PAK proteins, but the homology is less significant in the carboxy-terminal half of the complete putative MBT p21-binding domain. Interestingly, a Caenorhabditis elegans PAK-related protein (referred to here as CePAK2), predicted from an open-reading frame analysis of cosmid C45B11, shares extensive homology with the complete MBT p21-binding domain. A second difference between the PAK proteins so far described and MBT or CePAK2 is that MBT and CePAK2 lack an amino-terminal proline-rich Src homology 3 (SH3) domain binding motif. In hPAK1, this sequence mediates binding to the SH3 domain of the NCK adaptor protein. The structural features of MBT and CePAK2 indicate that these proteins may form a new subclass of the PAK family (Melzig, 1998).

Although PAK proteins were discovered by their ability to bind Rac and Cdc42, the biological function of PAK proteins in mediating GTPase functions such as cytoskeleton reorganization, neurite outgrowth, cell cycle progression and activation of the Jun N-terminal kinase (JNK) signaling cascade is not well understood. The isolation of mutations in the mbt gene allowed an investigation of the putative role of this PAK-related protein in vivo. The dramatic reduction in mushroom body neuropil volume in mbt correlates with a reduction in the Kenyon cell body layer volume. Compared to wild-type flies, neither Kenyon cell body size nor packaging density are altered in mbtflies (8–10 Kenyon cell bodies per 100μm²). It is concluded that the Kenyon cell number is reduced in mbtflies. Thus, the mbt phenotype could be caused by a loss of mushroom body neuroblasts, a defect in mushroom body neuroblast or ganglion mother cell proliferation, or increased cell death among Kenyon cells (Melzig, 1998).

Labeling mitotically active mushroom body neuroblasts at early larval stages with bromodeoxyuridine (BrdU) showed that most mbt larvae contained the wild-type number of four mushroom body neuroblasts in each brain hemisphere. Very few mbt larvae had a reduced number of labeled mushroom body neuroblasts. In wild-type animals, the four mushroom body neuroblasts in each brain hemisphere proliferate throughout development and contribute equally to the entire adult mushroom body structure. The resulting four-fold clustering within the cell body layer and the fiber projections can be visualized in three-dimensional reconstructions of Gal4 enhancer trap lines labeling subsets of Kenyon cells in each of the four cell clusters. In most adultmbt brains, the four-fold clustering of the mushroom bodies was preserved whereas a dramatic decrease in the number of labeled cells in each cluster and in the fiber tract diameter was observed. Occasionally, mbt mushroom bodies showed only two or three clusters indicating a loss of mushroom body neuroblasts or a complete failure of some neuroblasts to proliferate. No Kenyon cell fiber misrouting was observed inmbt mutant flies, however (Melzig, 1998).

In addition to the defects in the central brain, mbt flies have a rough eye phenotype. Tangential sections revealed that a variable number of photoreceptor cells in many ommatidia were missing. At least in the case of the R7 and the R8 photoreceptor axons, the innervation pattern in the medulla appeared to be normal. In summary, these experiments indicate that the MBT protein could be part of a more general mechanism regulating cell number in a variety of neuronal tissues. Utilizing both genetic approaches and in vitro assays will allow identification of components of the MBT signaling pathway and to analyse their function during neuronal development (Melzig, 1998).

Search PubMed for articles about Drosophila Mbt

Abo, A., Qu, J., Cammarano, M. S., Dan, C., Fritsch, A., Baud, V., Belisle, B. and Minden, A. (1998). PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia. EMBO J. 17: 6527-6540. 9822598

Bokoch, G. M. (2003) Biology of the p21-activated kinases. Annu. Rev. Biochem. 27: 743-781. PubMed ID: 12676796

Cau, J., Faure, S., Comps, M., Delsert, C. and Morin, N. (2001). A novel p21-activated kinase binds the actin and microtubule networks and induces microtubule stabilization. J. Cell Biol. 155: 1029-1042. 11733543

Dan, C., Kelly, A., Bernard, O. and Minden, A. (2001). Cytoskeletal changes regulated by the PAK4 serine/threonine kinase are mediated by LIM kinase 1 and cofilin. J. Biol. Chem. 276: 32115-32121. 11413130

Eaton, S., Auvinen, P., Luo, L., Jan, Y. N. and Simons, K. (1995). CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium. J. Cell Biol. 131: 151-164. 7559772

Faure, S., et al. (2005). Xenopus p21-activated kinase 5 regulates blastomeres’ adhesive properties during convergent extension movements. Dev. Biol. 277: 472-492. PubMed ID: 15617688

Genova, J. L., Jong, S., Camp, J. T. and Fehon, R. G. (2000). Functional analysis of Cdc42 in actin filament assembly, epithelial morphogenesis, and cell signaling during Drosophila development. Dev. Biol. 221: 181-194. 10772800

Harden, N., Ricos, M., Ong, Y. M., Chia, W. and Lim, L. (1999). Participation of small GTPases in dorsal closure of the Drosophila embryo: Distinct roles for Rho subfamily proteins in epithelial morphogenesis. J. Cell Sci. 112: 273-284. 9885281

Hofmann, C., Shepelev, M and Chernoff, J. (2004). The genetics of Pak. J. Cell Sci. 117: 4343-4354. PubMed ID: 15331659

Kim, S. H., Li, Z. and Sacks, D. B. (2000). E-cadherin-mediated cell-cell attachment activates Cdc42. J. Biol. Chem. 275: 36999-37005. 10950951

Kumar, R., Gururaj, A. E. and Barnes, C. J. (2006). p21-activated kinases in cancer. Nat. Rev. Cancer 6: 459-471. PubMed ID: 16723992

Luo, L., Liao, Y. J., Jan, L. Y. and Jan, Y. N. (1994). Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8: 1787-1802. 7958857

Melzig, J., Rein, K. H., Schaefer, U., Pfister, H., Jaeckle, H., Heisenberg, M. and Raabe, T. (1998). A protein related to p21-activated kinase (PAK) that is involved in neurogenesis in the Drosophila adult central nervous system. Curr. Biol. 8: 1223-1226. PubMed ID: 9811608

Mentzel, B. and Raabe, T. (2005). Phylogenetic and structural analysis of the Drosophila melanogaster p21-activated kinase DmPAK3. Gene 349: 25-33. PubMed ID: 15777717

Menzel, N., Schneeberger, D. and Raabe, T. (2007). The Drosophila p21 activated kinase Mbt regulates the actin cytoskeleton and adherens junctions to control photoreceptor cell morphogenesis. Mech. Dev. 124: 78-90. PubMed ID: 17097274

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: 1178-1185. 11027607

Qu, J., et al. (2001). Activated PAK4 regulates cell adhesion and anchorage-independent growth, Mol. Cell. Biol. 21: 3523-3533. PubMed ID: 11313478

Riesgo-Escovar, J. R., Jenni, M., Fritz, A., and Hafen, E. (1996). The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye. Genes Dev. 10: 2759-2768. 8946916

Schneeberger, D. and Raabe, T. (2003). Mbt, a Drosophila PAK protein, combines with Cdc42 to regulate photoreceptor cell morphogenesis. Development 130: 427-437. PubMed ID: 12490550

Soosairajah, J., et al. (2005). Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin. EMBO J. 24(3): 473-86. PubMed ID: 15660133

Tettamanti, M., Armstrong, J. D., Endo, K., Yang, M. Y., Furukubo-Tokunaga, K., Kaiser, K. and Reichert, H. (1997). Early development of the Drosophila mushroom bodies, brain centres for associative learning and memory. Dev. Genes Evol. 207: 242-252

Walther, R. F., Burki, M., Pinal, N., Rogerson, C. and Pichaud, F. (2018). Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor. J Cell Sci 131(6). PubMed ID: 29507112

Zhao, Z. S., et al. (2005). The GIT-associated kinase PAK targets to the centrosome and regulates Aurora-A. Mol. Cell 20: 237-249. PubMed ID: 16246726

date revised: 10 August 2018

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