InteractiveFly: GeneBrief

tricornered: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The Drosophila Ndr serine/threonine kinase Tricornered (Trc) is required for the normal morphogenesis of epidermal hairs, bristles, laterals, and dendrites. Mutations in trc and its interacting factor furry (fry) cause cells to form ectopic wing hairs, implying that these genes function temporally before hair initiation (Geng, 2000; Cong, 2001). In vivo evidence has been obtained that Trc function is regulated by phosphorylation and that mutations in key regulatory sites result in dominant negative alleles. Wild-type, but not mutant Trc, is found in growing hairs, and no Trc has been detected in pupal wing nuclei, implying that in this developmental context Trc functions in the cytoplasm. The furry gene and its homologues in yeast and Caenorhabditis elegans have been implicated as being essential for the function of the Ndr kinase family. Drosophila furry is also found in growing hairs, its subcellular localization is dependent on Trc function, and it can be coimmunoprecipitated with Trc. These data suggest a feedback mechanism involving Trc activity that regulates the accumulation of Fry in developing hairs (He, 2005).

Tricornered is involved in a repulsion mechanism regulating the distribution of dendrites in dendritic fields. To cover neuronal receptive fields completely but without redundancy, neurons of certain functional groups exhibit tiling of their dendrites via dendritic repulsion. Two evolutionarily conserved proteins, the Tricornered and Furry, are essential for tiling and branching control of Drosophila sensory neuron dendrites. Dendrites of fry and trc mutants display excessive terminal branching and fail to avoid homologous dendritic branches, resulting in significant overlap of the dendritic fields. Trc control of dendritic branching involves regulation of RacGTPase, a pathway distinct from the action of Trc in tiling. Time-lapse analysis further reveals a specific loss of the ability of growing dendrites to turn away from nearby dendritic branches in fry mutants, suggestive of a defect in like-repels-like avoidance. Thus, the Trc/Fry signaling pathway plays a key role in patterning dendritic fields by promoting avoidance between homologous dendrites as well as by limiting dendritic branching (Emoto, 2004).

Dendritic tiling refers to the complete but nonredundant coverage of a receptive field by dendrites of functionally homologous neurons, like tiles covering a floor. Tiling has been well characterized in the mammalian retina. Retinal ganglion cells (RGCs) in the rabbit retina, for example, can be grouped into at least 11 distinct physiological classes. Dendrites of some RGCs of the same subtype typically cover the whole retina with minimal overlap, whereas dendrites of different subtypes overlap extensively. Similarly, amacrine cells in the rabbit retina are classified into at least 22 subclasses based on the branching pattern of their dendrites, and several subtypes appear to tile the retina. Tiling thus ensures efficient and unambiguous representation of the entire visual field and is likely to be of general importance. Indeed, dendritic tiling among sensory neurons of moths and flies suggest that tiling is an evolutionarily conserved mechanism for dendritic field organization (Emoto, 2004 and references therein).

Drosophila dendrite arborization (da) sensory neurons provide a suitable system to study cellular and molecular mechanisms underlying dendritic development. The 15 da neurons in each abdominal hemisegment are classified into four subtypes based on their unique dendritic arborization profiles (Grueber, 2002). In addition, class III and class IV da neurons exhibit tiling in a subtype-specific manner (Grueber, 2002; Sugimura, 2003). Several lines of evidence suggest that the dendritic tiling of class IV neurons arises from class-specific competition between dendrites of neighboring neurons. (1) Dendrites of class IV neurons often appear to stop growing, make a turn, or retract as they come within a short distance of each other, whereas there is extensive overlap between dendrites of neurons belonging to different classes (Grueber, 2002, 2003a; Sugimura, 2003). (2) Laser ablation of certain class IV neurons during late embryonic stages causes dendrites of the surrounding class IV neurons to grow into the territories of the ablated neurons (Grueber, 2003a; Sugimura, 2003). Conversely, duplication of a class IV neuron results in a reduction rather than any overlap of their respective fields (Grueber, 2003a). (3) Despite the dendritic overgrowth and/or overbranching in mutants, such as flamingo and sequoia, there is still tiling between the extra branches within the same neuron as well as between different neurons (Grueber, 2002). These observations suggest that a like-repels-like mechanism is responsible for the dendritic tiling of class IV neurons; however, the underlying molecular mechanisms remain unknown (Emoto, 2004 and references therein).

tricornered (trc) and furry (fry) are evolutionally conserved genes implicated in regulating cell morphology (Adler, 2002). The trc and fry genes encode a serine/threonine kinase of the ACG family (Tamaskovic, 2003b) and a large (380 kDa) protein with no known functional domain (Cong, 2001), respectively. In budding yeast, the Trc homolog Cbk1p promotes nuclear translocation of the Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes (Colman-Lerner, 2001; Weiss, 2002). Cbk1p also controls polarized cell growth through an Ace2p-independent mechanism (Colman-Lerner, 2001; Weiss, 2002). The Fry homolog Tao3p (also named Pag1p) is required for both Cbk1p functions (Du, 2002; Nelson, 2003). Mutation of a fungal homolog of trc, cot1, causes a drastic increase of the hyphal branching in Neurospora (Yarden, 1992). Interestingly, mutations of the trc homolog sax-1 in C. elegans cause sensory neurons to have ectopic neurites (Zallen, 2000). In Drosophila, mutations of either trc or fry result in branched bristles and multiple wing hair phenotypes (Geng, 2000; Cong, 2001). However, the roles of Trc and Fry in neurons are obscure (Emoto, 2004 and references therein).

Trc and Fry are shown to function cell autonomously in Drosophila da neurons to regulate dendritic tiling and branching. Trc-kinase activity is required for the dendritic branching and tiling in vivo and is positively regulated by Fry. The control of dendritic branching but not tiling involves negative regulation of the RacGTPase signaling pathway by Trc. These findings suggest that Trc/Fry utilizes two distinct signaling pathways to shape the dendritic fields: one pathway to limit dendritic branching and a separate pathway to promote like-repels-like response of dendritic processes (Emoto, 2004).

Thus, Trc/Fry signaling pathway plays an essential role in regulation of dendritic branching. trc and fry mutants typically displayed excessive terminal branches, whereas the major dendrite architecture appeared normal in all four classes of da neurons. For example, the primary, secondary, and tertiary branches of class IV neurons did not show overbranching in trc and fry mutants whereas the terminal branches were increased by a factor of two. It is thus likely that Trc and Fry specifically regulate the branching of fine structures, but not overall architecture, of dendrites. Consistent with this idea, no obvious defect was observed in neurons with simple dendrites, such as bipolar neurons, external sensory neurons, and chordotonal neurons (Emoto, 2004).

Trc and Fry homologs have been identified in a variety of species including S. cerevisiae (Colman-Lerner, 2001; Weiss, 2002; Du, 2002), S. pombe (Verde, 1998; Hirata, 2002), N. Crassa (Yarden, 1992), C. elegans (Zallen, 2000 ; Gallegos, 2004), Drosophila (Geng, 2000 and Cong, 2001), and mammals (Tamaskovic, 2003b). Moreover, Fry homologs show a strong genetic interaction with Trc homologs. In S. cerevisiae and S. pombe, mutations in either of these genes cause severe cell growth defect as well as cell morphology defect. In Drosophila, however, other than the dendritic branching phenotype in neurons, no obvious cell growth defects were found in any tissues in the trc and fry mutant third instar larvae. Zallen reported that mutations in the trc homolog sax-1 gene cause extra neurite formation in sensory neurons of C. elegans while the overall structure of the neuron appears normal. Thus, the Trc/Fry signaling appears to mediate similar functions in sensory neurons of flies and worms. In addition, mutations of trc or fry result in branched bristles and multiple wing hair phenotypes in Drosophila (Geng, 2000; Cong, 2001), and mutation of a fungal homolog of trc, cot1, causes a drastic increase of the hyphal branching in Neurospora. Thus, the Trc/Fry signaling is likely to be a general mechanism to regulate branching of cellular processes (Emoto, 2004).

Dendritic tiling has been proposed to play a key role in patterning the dendritic fields of particular neurons, including Drosophila class IV sensory neurons (Jan, 2003; Grueber, 2004). Previous studies suggest that tiling in class IV neurons arises from repulsion between homologous dendrites (Grueber, 2003a; Sugimura, 2003). Consistent with this idea, time-lapse study reveals no overlap of the dendrites in late embryonic, first, second, or third instar larval stages, suggesting that tiling in class IV neurons is established when dendrites first meet and is maintained throughout the larval stage (Emoto, 2004).

Genetic and molecular evidence has been provided that Trc and Fry play a crucial role in establishing and maintaining the dendritic tiling of class IV neurons. In trc or fry mutants, terminal branches of class IV dendrites fail to avoid crossing each other, not only within the same neuron, but also between different neurons, leading to a significant overlap of dendritic fields. Since trc and fry null mutants display both overbranching and tiling phenotypes, one obvious possibility is that the tiling phenotype simply results from the overbranching phenotype. The following lines of evidence support the notion that the dendritic tiling defect in trc and fry mutants is not secondary to the overbranching phenotype. (1) When normalized by the total branch number or the total branch length, the number of dendritic crossings in trc and fry null mutants remains significantly greater than that in wild-type controls. (2) In the absence of dendritic overbranching, a robust tiling phenotype is still observed in the fry⁶ hypomorphic allele, as well as larvae transheterozygous for fry¹and fry⁶ or for fry¹ and trc¹. (3) trc and fry mutants display not only iso-neuronal tiling defects but also hetero-neuronal tiling defects. Such a significant overlap of the dendritic fields between neighboring class IV neurons is unlikely to result from a simple increase of terminal dendritic branches if each dendritic branch retains a like-repels-like activity. Indeed, a series of mutants, such as flamingo and sequoia, with dendritic overgrowth and/or overbranching have been isolated in previous studies; however, they appear not to have tiling defects in class IV dendrites (Grueber, 2002). The independence of tiling and branching phenotypes is further supported by the observation that the dominant-negative RacN17 suppresses the dendritic branching but not the tiling phenotype due to expression of the dominant-negative TrcK112A mutant (Emoto, 2004).

Time-lapse observations show that in wild-type larvae, ~70% of terminal branches appear to make a dramatic turn before they cross nearby branches, again supporting the idea that the like-repels-like mechanism plays a central role in class IV dendritic tiling. Compared to wild-type, fry mutant dendrites fail to turn away from nearby branches, but they showed normal net growth and retraction. Taken together, these data strongly suggest a role for Trc and Fry in the like-repels-like behavior of the class IV dendrites. It remains possible, however, that other mechanisms function in parallel with the Trc/Fry signaling pathway to establish tiling, since some dendritic branches of class IV neurons still appear to tile in trc and fry mutants (Emoto, 2004).

Sax-1 and Sax-2 (the worm homologs of Trc and Fry) also have an essential role in mechanosensory neurite tiling (Gallegos, 2004). This finding, together with the current study, strongly suggest an evolutionarily conserved role for the Trc/Fry signaling pathway in dendritic tiling. Indeed, of the two mammalian trc homologs ndr1 and ndr2, ndr2 is highly expressed in various tissues including the brain, while ndr1 displays a relatively specific localization in muscles (Devroe, 2004). Although it has not been established whether a tiling mechanism contributes to dendritic field specification in the central nervous system outside of retina, cerebral cortical neurons are known to exhibit contact-mediated growth inhibition of neurites (Sestan, 1999). It will be of interest to examine potential roles of Trc and Fry homologs in regulation of dendritic tiling as well as branching in vertebrate nervous systems. Additionally, considering a close correlation between abnormal dendrite patterning and mental retardation, it might be intriguing to examine the relationship between ndr1/2 genes and mental retardation diseases (Emoto, 2004).

The MARCM analyses and the rescue studies, together with the expression of Trc and Fry in da neurons, indicate that Trc and Fry function cell autonomously in neurons. In addition, Trc kinase activity is indispensable for the control of dendritic branching and tiling in vivo, and Fry is required for Trc kinase activity, indicative of an important role of intracellular kinase signaling. The kinase domain of Trc is closely related to Rho-kinase (Rok), with 45% amino acid identity and 71% similarity to Drosophila Rok (Drok), but Trc lacks a Rho binding domain and other regulatory domains (Tamaskovic, 2003b). Indeed, C. elegans Sax-1 has a partial genetic interaction with Rho in neuronal cell shape regulation (Zallen, 2000). Whereas no obvious interaction between Trc/Fry and Rho/Drok was observed in Drosophila da neurons, Rac signaling likely plays an important role in Trc/Fry regulation of dendritic branching of class IV neurons. Trc partially suppresses the overbranching phenotype induced by RacWT. Moreover, the dominant-negative RacN17 suppresses the overbranching but not tiling phenotypes in neurons expressing dominant-negative Trc mutant. One possible scenario is that Trc/Fry negatively regulates Rac to control dendritic branching. This notion is further supported by analyses of the active RacGTP protein level in cells expressing wild-type or mutant Trc (Emoto, 2004).

In contrast to the involvement of Rac in dendritic branching, there is no indication that tiling depends on Rac signaling. Rather, two distinct pathways seem to be employed by Trc/Fry to control dendritic branching and tiling. This idea is consistent with genetic data indicating that tiling defects can be separated from branching defects in trc and fry mutants. It is of interest to note that in budding yeast, Cbk1p (Trc homolog) and Tao3p (Fry homolog) play multiple roles involving different downstream pathways (Colman-Lerner, 2001; Weiss, 2002; Nelson, 2003; Emoto, 2004 and references therein).

The repulsion between dendrites likely involves either contact-mediated dendritic interactions or signaling via a short-range diffusible substance -- a signal that is likely to have class-specific components (Lohmann, 2001; Jan, 2003). It is unlikely that Trc and Fry determine the class specificity since Trc and Fry are expressed in all da neurons. Conceivably, Trc may transmit subtype-specific repulsion signals, generated by class-specific factors to downstream components including the cytoskeleton, and induce a like-repels-like behavior of dendrites. The finding that Fry and Trc are involved in the like-repels-like response in class IV neurons has provided an entry point for studying the molecular mechanisms that control dendritic tiling. As shown in this study, phosphorylation of the conserved Ser/Thr of Trc appears critical for the Trc/Fry signaling in vivo, yet little is known about the upstream kinase(s) and the downstream substrate(s) of Trc in any species. A molecular dissection of the Trc kinase signaling pathway in neurons will help elucidate how the Trc/Fry signaling pathway governs dendritic branching and tiling (Emoto, 2004).

A Hippo-like signaling pathway controls tracheal morphogenesis in Drosophila melanogaster

Hippo-like pathways are ancient signaling modules first identified in yeasts. The best-defined metazoan module forms the core of the Hippo pathway, which regulates organ size and cell fate. Hippo-like kinase modules consist of a Sterile 20-like kinase, an NDR kinase, and non-catalytic protein scaffolds. In the Hippo pathway, the upstream kinase Hippo can be activated by another kinase, Tao-1. This study delineate a related Hippo-like signaling module that Tao-1 regulates to control tracheal morphogenesis in Drosophila melanogaster. Tao-1 activates the Sterile 20-like kinase GckIII by phosphorylating its activation loop, a mode of regulation that is conserved in humans. Tao-1 and GckIII act upstream of the NDR kinase Tricornered to ensure proper tube formation in trachea. This study reveals that Tao-1 activates two related kinase modules to control both growth and morphogenesis. The Hippo-like signaling pathway delineated by this study has a potential role in the human vascular disease cerebral cavernous malformation (Poon, 2018).

Most organs are composed of epithelial or endothelial cells organized as tubes; junctional complexes maintain the intercellular or autocellular connections ('seams') that seal the cells into selectively permeable tubes. Should these connections be compromised, the consequences for organ function can be catastrophic. For example, individuals with the vascular disease, familial cerebral cavernous malformation (CCM), may suffer from seizures and strokes as a consequence of dilated leaky tubes (Poon, 2018).

Three architecturally distinct tube types have been described, and all three are found in the Drosophila tracheal system. These include multicellular seamed tubes (with intercellular junctions), seamed tubes formed by single cells (with autocellular junctions), and seamless tubes formed within single cells (no junctions). Most seamless tubes are thought to form intracellularly, although they may also form by fusion of membrane along autocellular junctions, converting autocellular tubes into seamless ones (Poon, 2018).

Tubes with single seams (autocellular) and seamless tubes form relatively late during tracheal development. The first tubes of the tracheal system are large multicellular sacs generated by invagination from the embryonic ectoderm. The tracheal epithelial cells are polarized along their apical-basolateral axis, with the apical membrane of each cell facing the lumen of the tracheal sac to which it belongs. Cells are next recruited to the distinct primary branches that migrate away from the sacs toward Branchless-FGF chemoattractant cues. Many of the primary branches initially form short wide tubes that lengthen and narrow over time, as the cells that comprise them intercalate, changing the underlying tubular architecture from multicellular to autocellular. Tip cells are required for, and lead, the migration of the new branches. Ultimately, tip cells assume specialized cell fates (terminal cell or fusion cell), and initiate secondary branch formation by targeting apical membrane internally to form seamless tube. The precise mechanism by which this occurs remains subject to debate and may differ between terminal and fusion cells (Poon, 2018).

Intriguingly, Drosophila terminal cells contain both autocellular and seamless tubes. Transition from one tube type (autocellular) to the other (seamless) occurs within the terminal cell at a location proximal to the terminal cell nucleus. This observation raises a number of questions including: how do the two tube types connect to each other, how do they match each other in diameter, and which pathways are required to regulate and execute these processes? To begin to address these questions, this study has taken a genetic approach and have screened through a large collection of terminal cell mutants to identify those that display tube morphogenesis defects within the region of the terminal cell wherein the autocellular-to-seamless tube transition occurs (hereafter, the 'transition zone') (Poon, 2018).

This study reports the identification and characterization of two mutants that disrupt lumen morphology in the transition zone in strikingly different ways. The first mutant, lotus, shows a transition zone gap in gas-filled tube, and more precise analysis shows that the gap corresponds to a local absence of tube, although tube is present proximal and distal to the gap. The second mutant, wheezy, shows dramatic tube dilation in the transition zone. These mutants may thus have opposing effects on the addition of apical membrane within the transition zone: too little, resulting in a gap, in lotus mutants; and too much, resulting in a dilation, in wheezy mutants. Lotus encodes N-ethylmaleimide sensitive factor 2 (NSF2), a protein required for SNARE recycling. This implies an especially stringent requirement for vesicle traffic in connecting the two tube types. Significantly, this study found that the second mutant, wheezy, encodes Germinal center kinase III (GCKIII). The human orthologs of GCKIII are effectors of the protein encoded by the human vascular disease gene, Cerebral cavernous malformation 3 (Ccm3), and are putative Golgi-resident kinases. Patients suffering from cerebral cavernous malformations show gross dilations of cerebral capillaries, thus suggesting that CCM3/GCKIII play a conserved role in tubulogenesis in limiting diametric tube expansion. Mutations in lotus and in wheezy both point to a crucial role of apical membrane delivery in tube morphogenesis (Poon, 2018).

The study goes on to show that Drosophila Ccm3 has a loss of function phenotype identical to GckIII, and that it interacts genetically with GckIII. Interestingly, the results stand in contrast to a previous report suggesting a requirement for the genes in tracheal lumen formation. Further, ectopic localization was found of junctional proteins in Ccm3-depleted or GckIII mutant cells, and an overaccumulation of the apical determinant, Crumbs, in GckIII mutant cells. In contrast, although Crumbs also accumulates in lotus/Nsf2 mutant cells, it does not appear to be enriched in the lumenal membrane. Overexpression of Crumbs by itself induces formation of multiple tiny dilations throughout the terminal cell, but not a large transition zone tube dilation. Nevertheless, knockdown of crumbs in GckIII mutants strongly suppresses the large local dilation defect. Similarly, mutations in lotus (Nsf2), that cause mislocalization of Crumbs, were found to epistatic to GckIII. It was also found that knockdown of the essential septate junction scaffold, Varicose (a MAGUK protein orthologous to human PALS-2), suppresses the transition zone dilation defect associated with GckIII mutants. These data suggest that it is the inappropriate increase of septate junctions in combination with overaccumulation of Crumbs that confers a transition zone dilation defect. These studies of transition zone tube morphogenesis have direct relevance to human development and disease, and provide important mechanistic insight into the basic biological question of how tubes of differing architecture connect to each other (Poon, 2018).

In the terminal cell 'transition zone,' seamed and seamless tubes must connect. This study found that during development, remodeling of cell junctions reduced tube type complexity in the transition zone, such that by third instar only seamless tubes are still present in the majority of terminal cells. How the tubes connect initially has been an outstanding question in the field for more than a decade, and whether, or how, transition zone tubes remodel over developmental time has not previously been addressed. This study describes several mutations that disrupt tube morphogenesis in the terminal cell transition zone. The mutants fall into two classes that appear to act in opposing manners: in the case of mutants like lotus, there appears to be a deficit of apical membrane addition to the growing tube within the transition zone, most frequently resulting in a failure to maintain a continuous tube. In the case of mutants such as wheezy, too much apical membrane is added to the transition zone tube, resulting in a grossly dilated appearance. Molecular genetic characterization of the genes is consistent with this hypothesis as lotus encodes a factor required for membrane traffic and wheezy encodes a putative Golgi-resident kinase that is required for restricting the accumulation of apical and junctional proteins. In this context, it is important to consider that secretion of specific cargo into the tube lumen during embryonic tracheal development is dependent upon septate junctions; perhaps an increase in transition zone septate junctions promotes enhanced targeting of vesicles to the lumenal membrane. However, the existence of septate junctions in the transition zone by itself does not appear to lead to tube dilation because ~4% of wild-type third instar terminal cells have septate junctions in the transition zone, but do not have dilations. This, together with the inability of Crumbs overexpression to cause transition zone dilations, and with the suppression of such dilations by knockdown of crumbs or of varicose, leads to the proposal that both ectopic septate junctions and increased accumulation of apical polarity proteins are required for transition zone tube dilation. This study provides genetic and molecular insight into the process of junctional remodeling and tubulogenesis in the transition zone (Poon, 2018).

This study has determined that wheezy encodes GCKIII, the only known downstream effector of CCM3, a protein mutated in human vascular disease characterized by grossly dilated, leaky capillaries. It therefore becomes important to ask whether the insights into the cellular basis of GCKIII/CCM3 deficiency are compatible with what is known from patient samples and vertebrate CCM models. In excised CCMs, staining of apical polarity proteins and junctional proteins has not been reported, but ultrastructural analyses suggest that endothelial cells lack functional tight junctions, and in mouse models, lymphatic vessels show gaps between endothelial cells or show shorter intercellular junctions. On the other hand, EM analysis of vessels from endothelial-specific Ccm3 knockout mice showed tight junctions were present and did not detect any gaps between endothelial cells. It is not clear how to reconcile these data, although it is possible that loss of CCM1 and CCM2 cause tube dilation by a mechanism that is distinct from that seen with loss of CCM3, as suggested by Chan (2011). In the Drosophila tracheal system, a clear and dramatic increase is seen in apical polarity protein accumulation, and a convincing defect in remodeling of septate junctions; this suggests that a reexamination of endothelial tight junctions and an initial study of apical polarity determinants in vertebrate CCM models should be a high priority. Indeed, extant data hint that such studies could be of great value. For example, mutations in the zebrafish orthologs of Stardust and Yurt (physical interactors and regulators of Crumbs in Drosophila) have cardiovascular defects. Moreover, remodeling of endothelial cell junctions during angiogenesis, as cells change neighbors and as the tubes they comprise change architectures, is likely to be critical to the formation of functional tubes. That terminal cells are the only tracheal cell type affected by loss of CCM3 or GCKIII activity is striking, and may be directly relevant to human disease given that ∼30% of cells comprising capillaries in the cerebral cortex have an analogous cellular architecture, the so-called 'seamless' endothelial cells). In addition, it is noted that the cellular architecture of endothelial tip cells that lead outgrowth of the zebrafish intersomitic vessels is dynamic: these cells often form seamless tubes, but sometimes appear to make tubes of mixed autocellular and seamless character (like fly terminal cells), and at other times appear to contribute to multicellular tubes. Given the dynamic nature of tight junctions during angiogenesis, defects in junction remodeling such as those seen with wheezy mutants in the Drosophila tracheal system, might be expected to give rise to leaky vessels composed of endothelial cells that are not properly attached to each other (Poon, 2018).

The precise function of CCM3 and GCKIII during angiogenesis is not clear. another study has suggested a requirement for the CCM3 pathway in lumen formation, whereas a third has suggested that the pathway regulates cell junctions via phosphorylation of Moesin. The current data conclusively show that neither model is consistent with the observed effects of loss of the CCM3/GCKIII pathway in the fly tracheal system; each model is discussed in the context of the data below (Poon, 2018).

Chan (2011) is built upon the outcome of two sets of experiments: tubulogenesis assays of CCM3- or GCKIII-depleted endothelial cells and RNAi studies in the Drosophila tracheal system. The tubulogenesis assays have been used to great effect, allowing identification of a number of genes required for endothelial lumen formation; however, it is important to appreciate that the endothelial cells in this assay must do more than simply lumenize, they must also migrate, remodel the matrix, and organize and reorganize cell:cell contacts. Disruption in any of these preconditions for lumenization would also compromise tubulogenesis. Thus, should the junctional remodeling defects observed in the fly tracheal system also pertain to endothelial cells depleted for CCM3 or GCKIII, those remodeling defects might be sufficient to block lumenization in the in vitro assays. Additionally, the fact that patient CCMs are dilated capillaries rather than lumenless capillaries would also seem to argue against this model (Poon, 2018).

It is noted that the data for GckIII and Ccm3 in the Drosophila tracheal system stand in contrast to the findings of Chan (2011). Unlike Chan, this study found tube dilation to be the primary consequence of loss of CCM3 or GCKIII function. The fully penetrant transition zone defectobserved in wheezy mutant flies is compelling, and is directly supported by RNAi experiments targeting either GckIII or Ccm3, including with RNAi transgenes employed by Chan. Consistent with the findings of Chan, no defects are observed in terminal cell gas-filling (but not in other tracheal cell tubes by mosaic analysis using a null allele of GckIII). However, the lack of observable gas-filled tubes is not due to failure in tube formation, as suggested by Chan, but rather due to a failure in liquid clearance. Indeed, liquid-filled tubes were readily detected in the knocked-down cells. Importantly, the data argue that Ccm3 and GckIII are not required for making a lumen, but instead are required to prevent inappropriate trafficking of junctional and apical proteins (Poon, 2018).

This study also addressed the model that phosphorylation of Moesin is the essential function of the CCM3/GCKIII pathway during tubulogenesis. Moesin is the only ERM (ezrin-radixin-moesin) family member in Drosophila, and this study found that phosphorylation of Moesin in the tracheal system is not compromised by loss of CCM3 or GCKIII (in fact it is elevated). These data suggest that the essential target of GCKIII kinase activity is not Moesin, but another protein yet to be identified. Going forward, it will be of great interest to determine what proteins are regulated by GCKIII kinase activity in the tracheal system, and to test whether vertebrate orthologs of these factors are required during angiogenesis (Poon, 2018).

REGULATION

Protein Interactions

Tricornered interacts with Rac

The RhoGTPase family, including Rho, Rac, and Cdc42, plays a crucial role in neuronal morphogenesis. In particular, proper activation of Rac in developing neurons is essential for establishing and maintaining their unique dendritic branching pattern. To test whether Trc signaling involves Rac regulation, it was first asked whether overexpression of wild-type and mutant Rac1 affects dendritic morphology, and then the effects of coexpressing wild-type or mutant Trc and Rac1 were examined. Overexpression of wild-type Rac1 (RacWT) in class IV neurons results in overbranching of dendrites but does not produce any obvious tiling phenotype. This overbranching phenotype is partially suppressed by coexpression of wild-type Trc. Importantly, whereas expressing the dominant-negative Rac1 (RacN17) alone does not cause a detectable dendritic phenotype, RacN17 significantly suppresses the overbranching phenotype but not the tiling phenotypes in neurons expressing the dominant-negative Trc(K112A) mutant. The involvement of Rac in Trc signaling appears specific; coexpression of the dominant-negative RhoL (RhoN25) did not result in a significant change of dendritic branching and tiling phenotypes in neurons expressing the K112A mutant. These results suggest that Trc/Fry may negatively regulate Rac signaling to control dendritic branching (Emoto, 2004).

To further test this possibility, coimmunoprecipitation experiments were carried out and Trc was found in a complex with Rac1 but not with Cdc42 in Drosophila S2 cells. Moreover, using a pull-down assay in which Rac-GTP (the activated form of Rac) but not Rac-GDP is isolated via the Rac-GTP binding domain of PAK conjugated to GST, it was found that overexpression of wild-type Trc in stably transfected cell lines causes a significant reduction of the amount of Rac1-GTP compared to control cells, whereas expression of the dominant-negative Trc(K112A) mutant increases Rac1-GTP level. Taken together, these findings suggest that the Trc/Fry signaling negatively regulates Rac activity to control dendritic branching whereas another, distinct pathway mediates the action of Trc in tiling (Emoto, 2004).

furry, a gene that interacts genetically with tricornered, is important for maintaining the integrity of cellular extensions during morphogenesis

The Drosophila imaginal cells that produce epidermal hairs, the shafts of sensory bristles and the lateral extensions of the arista are attractive model systems for studying the morphogenesis of polarized cell extensions. furry, an essential Drosophila gene, is involved in maintaining the integrity of these cellular extensions during morphogenesis. Mutations in furry result in the formation of branched arista laterals, branched bristles and a strong multiple hair cell phenotype that consists of clusters of epidermal hairs and branched hairs. By following the morphogenesis of arista laterals in pupae, it has been determined that the branched laterals are due to the splitting of individual laterals during elongation. In genetic mosaics furry acts cell autonomously in the wing. The phenotypes of double mutant cells argue that furry functions independently of the frizzled planar polarity pathway and that it probably functions in the same pathway as the tricornered gene. A P-element insertion allele was used as a tag to clone the furry gene, and it was found to be a large and complicated gene that encodes a pair of large conserved proteins of unknown biochemical function (Cong, 2001).

Fry is a member of a conserved family of proteins found in mammals, C. elegans, Arabidopsis and yeast. The similarity is concentrated in five regions separated by short regions of little or no similarity. The similarity is highest in the N-terminal most region, called the fry domain. In this 630 amino acid region, the Fry and the human CAB4244 protein are 63% identical and 79% similar. The fly, human and worm proteins contain all five of the similarity regions, suggesting these regions may represent functional domains. The Arabidopsis and yeast proteins only contain the first three similarity regions. Several additional database sequences share similarity to part of the C-terminal region of Fry. These sequences are likely to represent partial cDNAs. The Fry-S protein contains the Fry domain and about half of the second homology segment. It is unclear whether other organisms produce a protein that is equivalent to the Fry-S protein. The homolog from yeast (Tao3) has been found to be nonessential. Two phenotypes have been identified with the knockout of this gene. One is altered transcription of the OCH1gene and the second is the clumping of mutant cells (Cong, 2001 and references therein).

The detailed morphology of fry mutant cells is indistinguishable from trc mutant cells, suggesting they might function in the same cellular process. Consistent with this hypothesis, it was found that cells doubly mutant for fry and trc show a phenotype that is indistinguishable from either single mutant. Previous data has shown that trc encodes the Drosophila NDR kinase (Geng, 2000). This raises the possibility that fry might encode a substrate for this kinase or a protein involved in modulating trc activity. A target peptide sequence for the human NDR kinase has been reported (Millward, 1995), but this sequence is not found in Fry. However, as Millward noted, this synthetic peptide does not appear to be an optimal substrate for NDR (Cong, 2001).

Mutations in genes such as mwh, a downstream component of the fz pathway in the wing, also gives rise to multiple hair cells. The morphology of these differs from the fry mutant cells in a number of ways. Notably, in fry the multiple hairs are clustered more tightly: there is much more evidence of splitting. Furthermore, the prominent polarity abnormalities of genes such as mwh are missing in fry. mwh fry¹ cells have a much stronger phenotype than either single mutant, a result that stands in sharp contrast to the lack of additivity for the fry and trc. This result is interpreted as mwh causing the formation of multiple independent prehair initiation sites and each of these giving rise to a cluster of hairs due to the fry-dependent splitting. Indeed the phenotype of the doubly mutant cells approximates the multiplicative phenotype this model predicts. Similar results were also found for mwh trc⁶ mutant cells. Similar experiment were carried out where fry clones were induced in a fy or frtz mutant background. These two genes, which also appear to be components of the fz pathway in the wing have a much weaker multiple hair cell phenotype than mwh. In both of these cases the doubly mutant cells also have a stronger phenotype than either single mutant (Cong, 2001).

The fry phenotypes in wing hairs, bristles and arista laterals show striking similarities, suggesting a common mechanism is involved. Observations on developing pupal aristae show that in fry mutants, laterals can split at a variety of stages. The splitting can be early or late in lateral morphogenesis, near the distal tip or far from it. Observations on fixed pupal bristles and adult cuticular bristles suggest this is the case in this cell type as well. This argues that fry functions to maintain the integrity of these structures during their morphogenesis. The function of fry does not appear to be absolutely essential for their morphogenesis, since at least some bristles in fry mutants are indistinguishable from wild type. The situation is less clear for wing hairs, since many fry cells produce clusters of hairs with only a minority of hairs being obviously split. It is possible that a lack of fry function could cause the formation of independent hair initiation centers in wing cells or to hair initiation centers that are too large to ensure a single hair is formed. It would not be surprising if the assembly and crosslinking properties of actin and tubulin might function to reduce the size of an initiation center to insure that a single hair was formed. fry mutations could interfere with this process, leading to a larger center and hair clusters. Alternatively the clusters could be due to splitting that occurs early in hair morphogenesis or the splitting of the initiation center prior to actual hair outgrowth. This latter hypothesis provides a common explanation for the phenotypes seen in all three cell types, and hence, is in some ways more appealing (Cong, 2001).

The morphology of branched hairs, laterals or bristles is typically normal except for the region of the branch point. This suggests that fry does not encode an integral component of these cellular extensions. In this way, fry and trc differ in a fundamental way from mutations in actin cytoskeleton components such as crinkled, singed or forked, which result in abnormal morphology in all regions of the structure. It has been suggested that trc might encode a component of a system that either coordinates or organizes the growth of different subcellular components during morphogenesis (e.g., membrane, actin cytoskeleton, etc.), or monitors the 'quality' of the developing structure to insure its integrity (i.e., it functions in a pathway that is analogous to a morphogenetic checkpoint) (Geng, 2000). It would also be appealing to explain the function of fry in the same way (Cong, 2000).

The outgrowth of a cellular extension is a feature of many cells. In several systems, studies on the mechanisms of elongation have led to the conclusion that the polymerization of actin at the membrane drives extension. Indeed, the ultrastructure of actin bundles in developing bristles argues that the large bundles of actin filaments are assembled in smaller units at the distal tip of the elongating bristle. It is not clear, however, how other cellular components (e.g., plasma membrane) are added to such outgrowths. A model of growth only from the tip does not easily accommodate observations on split elongating laterals. If growth were only from the tip, then the branch-point would not be expected to move distally as growth proceeds; however this is a routine and prominent result. It is noted however, that a similar distal movement of 'blebs' was reported in a classic paper on the growth of the Thyone acrosomal process. This is an example where actin polymerization at the tip is believed to drive extension. In addition, the observations do not support a simple model of growth being restricted to the proximal base of the lateral. In this case the length of an arm distal to a branch-point would not be expected to grow but this is also routinely seen. These data could be explained by growth taking place at both the distal and proximal ends of the lateral; however, this model cannot explain an increase in the distance between branch-points on multiply branched laterals. Such multiply branched laterals were rare in the fry⁷/fry¹ mutants examined, but they are seen frequently in equivalent experiments on trc mutants where multiple branch-points are much more common. The data can most simply be explained by growth taking place at all locations along the proximal distal axis of the laterals. An alternative hypothesis is that growth is normally from the tip and that growth throughout the lateral is a consequence of the fry mutation. The examination of additional 'split lateral' mutants should determine if this latter hypothesis is tenable. It remains to be established whether all cellular components are added to a growing lateral (or bristle) in the same way. It should be possible to follow the addition of different cellular components by the induction of transgenes that encode tagged proteins during lateral elongation (e.g., GFP-actin). For example, if actin is added only at the tip it would be predicted that the tip will be the principal location of GFP fluorescence shortly after transgene induction. Such experiments are in progress (Cong, 2000).

Tricornered regulated by phosphorylation and interacts with Furry during Drosophila wing hair development

Tricornered is required for the normal morphogenesis of epidermal hairs, bristles, laterals, and dendrites. In vivo evidence has been obtained that Trc function is regulated by phosphorylation and that mutations in key regulatory sites resulted in dominant negative alleles. Wild-type, but not mutant Trc, is found in growing hairs, and no Trc has been detected in pupal wing nuclei, implying that in this developmental context Trc functions in the cytoplasm. The furry gene and its homologues in yeast and Caenorhabditis elegans have been implicated as being essential for the function of the Ndr kinase family. Drosophila furry (Fry) also is found in growing hairs, that its subcellular localization is dependent on Trc function, and it can be coimmunoprecipitated with Trc. These data suggest a feedback mechanism involving Trc activity regulates the accumulation of Fry in developing hairs (He, 2005a).

Tricornered-related human and yeast kinases contain a pair of conserved phosphorylation sites corresponding to Ser-281 and Thr-444 in Ndr (Millward, 1995). In vitro protein kinase activity assays on human Ndr1 and in vivo functional tests for Yeast Dbf2 have confirmed the critical importance of the two conserved sites (Millward, 1999; Mah, 2001). Mutations of these sites in Trc to alanine, which cannot be phosphorylated, results in a loss of rescue activity and in dominant negative proteins. It is hypothesized that the dominant negative behavior of these mutants arises from the nonproductive binding of targets to an inactive (or low-activity) mutant Trc protein competing with the productive binding of the endogenous Trc protein (He, 2005a).

Autophosphorylation at Ser281 (and to a lesser extent Thr444) in Ndr1 is thought to be important for kinase activity (Millward, 1995; Devroe, 2004; Stegert, 2004). Surprisingly, it was found that Ser292Ala and Thr453Ala act as dominant negative proteins, whereas Lys122Ala and Lys122Ala Thr453Ala do not. This argues that the dominant negative behavior of Ser292Ala and Thr453Ala is not simply due to their lacking activated kinase activity. One possibility is that a second autophosphorylation is important for Trc to be able to bind substrate. In the complete absence of Trc kinase activity (i.e., Lys122Ala), this site would not be phosphorylated and this would result in the inactive Trc not competing for substrate binding and hence being unable to act as a dominant negative protein. An additional Ca²⁺-dependent phosphorylation site (Thr74) has also been proposed to be important for Ndr1 regulation (Bhattacharya, 2003; Tamaskovic, 2003a; Tamaskovic, 2003b). It will be interesting to determine whether Drosophila Trc also is activated by Ca²⁺ in a similar way and whether Thr74 could be the hypothesized second autophosphorylation site (He, 2005a).

Several observations argue that Trc and Fry function in a common signaling pathway to regulate the actin cytoskeleton during hair, bristle, lateral, and dendrite morphogenesis. In both yeast and flies, Fry is essential for Trc/Cbk1 kinase activity (Nelson, 2003; Emoto, 2004). In vivo evidence for this is derived from the observation that a reduction in Fry dose enhances a trc dominant negative phenotype. In both flies and yeast, these genes seem to act in the same pathway because double null mutants have the same phenotype as each single mutant (Cong, 2001; Hirata, 2002; Nelson, 2003; He, 2005a and references therein).

In S. cerevisiae, Cbk1 is found in both the nucleus and cytoplasm, whereas Tao3 (the Furry homolog) is only cytoplasmic. Data indicate that these proteins behave differently in the fly system. In pupal wing cells, there is no evidence for the nuclear accumulation of either Trc or Fry. Interestingly, both proteins accumulate in growing hairs where they seem to be associated with vesicles or particles. This suggests the possibility that these proteins function to regulate or direct intracellular transport in growing hairs. In other cells types (e.g., salivary gland cells), evidence was seen for both nuclear and cytoplasmic accumulation of both Trc and Fry; hence, the subcellular localization of these two proteins is subject to cell type-specific regulation. There are a number of animal cell types where trc and fry function in the morphogenesis of polarized cell extensions (Zallen, 2000; Cong, 2001; Emoto, 2004). The finding that Trc and Fry accumulate in wing hairs suggests that that these proteins function locally within the cellular extension to regulate its outgrowth (He, 2005a).

In both the yeast systems and in Drosophila, Trc and Fry (and their yeast homologues) influence the subcellular localization of the other, although at least superficially there seem to be substantial differences. Decreased accumulation of Trc was found in hairs in fry mutant cells, suggesting the possibility that Fry serves to recruit Trc to the hair. In contrast, in S. cerevisiae, Tao3 mutants do not alter the bud neck accumulation of Cbk1 as would be expected if Tao3 helped recruit Cbk1 to the neck. Instead, in a Tao3 mutant the restriction of Cbk1 to the daughter cell nucleus is lost. Thus, it is not clear whether the mechanism used by Fry/Tao3 to regulate Trc/Cbk1 localization is conserved. However, given that differences are seen in the subcellular localization of Trc and Fry in different tissues in Drosophila it is perhaps not surprising that differences are seen between yeast and fly. Increased accumulation of Fry is seen in hairs in trc mutant cells, suggesting that Trc activity negatively regulates Fry recruitment to the hair. This is reminiscent of the finding in yeast that the amount of Tao3 in the bud neck increases slightly in Cbk1 mutants; this was taken as evidence of a feedback system linked to Cbk1 activity governing the accumulation of Cbk1/Tao3 at the bud neck (Nelson, 2003). However, both of these results are different from the decrease in polarized Mor2 accumulation in orb6 mutants in S. pombe (Hirata, 2002). Together, these results suggest that an interaction between trc and fry (and their homologues) is conserved over a wide phylogenetic range but that the interaction is modified in organism-, cell-, and development-specific ways (He, 2005a).

Previous observations showed that mutations in trc and fry cause cells to form ectopic wing hairs, implying that these genes function developmentally before hair initiation (Geng, 2000; Cong, 2001). Time-lapse observations on developing arista laterals in fry and trc mutants show the ectopic initiation of laterals and the splitting of laterals at a variety of developmental stages (Cong, 2001; He, 2001). These observations indicate that trc and fry act before lateral initiation and suggest they continue to function during the 18 h or so of lateral outgrowth (He, 2001). Several results argue that trc and fry function for a long period in wing hair differentiation. Before hair morphogenesis, trc and fry mutant cells have a greater than normal cross section and are less regular in shape. In addition, the level of F-actin below the apical membrane is often increased in mutant cells. These observations indicate that trc and fry function before hair initiation. Both Trc and Fry protein are observed in developing hairs and their accumulation in these locations is sensitive to trc and fry function, consistent with these proteins functioning during the process of hair elongation. The reorganization of the actin cytoskeleton is response to Trc and Fry is reminiscent of observations in S. pombe (Hirata, 2002), where Mor2 is important for the localization of F-actin at the cell end(s) (He, 2005a).

The observation that Trc accumulation in the growing hair is sharply reduced in phosphorylation site mutants suggests that the activity of Trc is related to its subcellular location. It is possible that the action of an upstream kinase plays a role in regulating Trc location and activity. A lack of Trc function, either from a mutation in the endogenous trc gene or from the directed expression of a dominant negative protein, increases the accumulation of Fry in the growing hair. It is hypothesized that Fry in the hair acts as a scaffold and recruits Trc to the hair. Trc in the hair would function to both phosphorylate substrates in regulating polarized growth and to limit the further recruitment of Fry to the hair. Such a feedback system could regulate both the activity and accumulation of these proteins in the hair (He, 2005a).

In all subcellular locations, both Trc and Fry were found in a punctuate pattern. This raises the possibility that Trc and Fry might be present in vesicles or other intracellular transport organelles. These putative vesicles did not stain for F-actin. The growth of polarized structures such as hairs, bristles, laterals, or dendrites requires the directed transport of cellular components from the central cytoplasm to the growing extension. To form a multiple hair cell requires that the cell transport material to several instead of to a single growing hair. It is suggested that Trc and Fry function to maintain hair integrity by regulating the transport of hair-forming components to a single targeted location on the apical surface. Wing cells begin to secrete large amounts of cuticulin early in the process of hair outgrowth. This raises the possibility that Trc and Fry could be present on secretory vesicles; however, it is not clear how to connect a defect in the deposition of cuticulin with the extreme multiple hair cell phenotype seen in trc and fry cells (He, 2005a).

In S. cerevisae, Cbk1 and Tao3 are part of the RAM pathway (Nelson, 2003). Another essential component of this pathway is Mob2p, which is known to bind to Cbk1p and to be important for Cbk1 activity (Colman-Lerner, 2001; Weiss, 2002; Nelson, 2003). Mutants of Cbk1, Tao3, and Mob2 share the same phenotype (Nelson, 2003). The related Dbf2 kinase and Mob1 function together as part of the mitotic exit network. Recently, evidence for an activating interaction between mammalian Ndr and Mob proteins was reported (Bichsel, 2004; Devroe, 2004). Given this conservation, it seems likely that Trc also will interact with a Mob (He, 2005a).

There are four Mob-like proteins encoded by the Drosophila genome (CG13852, CG4946, CG11711, and CG3403). A recent genomics scale two-hybrid screen of Drosophila proteins reported an interaction between Trc and CG13852. Further studies will be required to confirm this and to determine whether any of the other Dmob proteins can bind to Trc. The Drosophila warts/lats gene encodes a kinase that is related to the Ndr group, and it is also a candidate for binding to one or more of the fly Mob proteins (He, 2005a).

Drosophila wing hair development requires restriction of the initiation site to the apical-distal corner and the activation of actin assembly so that a single hair is formed. The Fz/Dsh planar cell polarity pathway has been shown to regulate the initiation site and hence the orientation of the hair. Rho1 and Drok activity regulates the number of hairs without affecting their orientation. This study provides evidence for a positive genetic interaction between Trc/Fry and Rho1/Drok. Drok is thought to regulate the cytoskeleton in a rather direct manner by phosphorylating Spaghetti Squash (Sqh) (Drosophila nonmuscle myosin regulatory light chain); thus, Trc is not likely to function downstream of Drok. It seems likely that Trc functions either upstream of Rho1/Drok or in parallel. Trc has been found to be upstream of and to negatively regulate DRac in sensory neurons (Emoto, 2004). Thus, Trc functioning upstream of Rho1/Drok seems feasible. However, evidence was also found for Trc interacting with other small GTPases, such as Drac1, Dcdc42, and Dral. It is possible that Trc is upstream of all of these, but the hypothesis that the proteins encoded by these genes function either in parallel or in a network that regulates the cytoskeleton is preferred (He, 2005a).

The genetic interaction between Trc/Fry and Drosophila Ral (Dral) is very strong. Dral has been implicated in regulating a number of different cellular processes. One of these is vesicle trafficking. This is intriguing given the finding that Trc and Fry are found in a punctuate pattern in wing hairs as if they are attached to vesicles or particles. It seems possible that the interaction between Dral and Trc/Fry could be functioning at this level (He, 2005a).

Drosophila Mob family proteins interact with the related Tricornered (Trc) and Warts (Wts) kinases

Individual Mob family proteins also interact with Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase that is required for the normal morphogenesis of a variety of polarized outgrowths including epidermal hairs, bristles, arista laterals, and dendrites. In yeast the Trc homolog Cbk1 needs to bind Mob2 to activate the RAM pathway. Genetic and biochemical data is provided that Drosophila Trc interacts with and is activated by Drosophila Dmob proteins, specifically Mats and Dmob2 (FlyBase terms the gene Dmob2 Mob1). Evidence is provided that Drosophila Mob proteins also interact with the related Warts/Lats kinase, which functions as a tumor suppressor in flies and mammals. In trc mutants the overall pattern of denticles is partly disorganized and many denticles are split. Split denticles are infrequent in wild-type larvae. The denticle pattern of mats mutant larvae is also disorganized and contains many split denticles. Interestingly, the overgrowth tumor phenotype that results from mutations in Dmob1 (mats) is only seen in genetic mosaics and not when the entire animal is mutant. Unlike in yeast, in Drosophila individual Mob proteins interact with multiple kinases and individual NDR family kinases interact with multiple Mob proteins; in particular, Mats interacts physically and genetically with trc and mats phenotype resembles that of trc. Notably, trc:mats double mutant larvae do not have a more severe phenotype than the single mutants. This lack of additivity argues that trc and mats function in a common pathway during denticle development. These observations also suggest that mats functions with both Trc and Wts (He, 2005b).

Previous genetic data pointed out the importance of tricornered (trc) and furry (fry), encoding a large conserved protein with multiple isoforms, for the morphogenesis of polarized cellular extension. Based on homology to yeast regulatory pathways involving homologs of trc, furry and mob, it seemed likely that one or more of the Drosophila mob genes would function along with trc and fry. Evidence was found supporting this hypothesis but the results are complicated by both pleiotropy and redundancy. This was illustrated most clearly in experiments with mats. Mutations in mats displayed phenotypes that were typical of both trc (split denticles and multiple hair cells) and of wts/lats (tumors, bulged cells, advanced hair differentiation). Mats can also interact with both Trc and Wts as detected using the yeast two-hybrid system. These observations stand in contrast to the situation in yeast, in which individual mob genes show specificity for individual Ndr family members. Further evidence for redundancy comes from the gene dosage interactions seen between trc and the other Dmobs (He, 2005b).

Evidence for a direct physical interaction has been reported for Ndr and Mob family members from yeast, flies and mammals. Previous yeast two-hybrid experiments showed evidence for a physical interaction between Trc and Mats and Mats and Wts. The results of this study extended these observations by showing a similar interaction between Trc and Dmob2 by both two hybrid and coimmunoprecipitation experiments and that Wts and Dmob2 interact in the two hybrid system. Residues known to be important for the interaction between yeast Mob1 and Dbf2 are also important for the interaction between Trc and Dmob2. The conservation of many of these residues in Dmob3 and Dmob4 suggests that these proteins will also interact with Trc. The genetic interactions seen between trc and Dmobs suggest that the binding of Dmobs to Trc is essential for in vivo function and activation of the protein. Consistent with this hypothesis it was found that Dmob2 and Trc colocalized to growing hairs in pupal wing cells (He, 2005b).

The observation that for two phenotypes (pupal wing cell cross section and time of hair initiation) mats and wts clone cells share a similar phenotype that is the opposite of trc is intriguing and needs to be reconciled with the positive gene dose interactions seen between mats and trc for the sensitized multiple wing hair cell assay and the similar denticle phenotype. Given this complexity it seems unlikely that a single simple mechanism is involved. Because Mats appears to function along with both Trc and Wts, some of the complexity may reflect interactions between these two kinase modules. Cells mutant for mats could have both modules inactive, although it is possible that the degree of possible mats redundancy might not be equivalent for the two modules. In principle these two modules could function in parallel or one could be upstream of the other. The observation that mats and wts clones have increased Fry accumulation in hairs is consistent with mats/wts being upstream of trc/fry/mats(mob). Because increased Fry accumulation in hairs is also seen in trc mutant cells, this hypothesis is also consistent with the positive gene dosage interactions. However, a different explanation is needed to explain the observation that with regard to cell size and the timing of hair initiation the mats/wts phenotype is opposite to that of trc/fry. If both modules are considered to be equally inactivated in a mats cell, then these latter observations suggest that trc/fry/mats could function antagonistically and upstream of mats/wts. In this situation a lack of trc function would result in increased wts function (and increased cell size and delayed hair formation). A lack of wts function would result in the reduced cross section and advanced hair morphogenesis. In a mats mutant a reduction is expected in both trc and wts function. This would result in a wts-like phenotype because a lack of trc inhibition of wts would be of no consequence in cells that already lack wts activity. However, this model does not explain the multiple hair cell interactions. Given the difficulties in any single model it is suggested that the interactions are context dependent and/or the two modules function entirely in parallel (He, 2005b).

The Drosophila Fry protein interacts with Trc and is highly mobile in vivo

Cell polarity is a common feature of eukaryotic cells. The NDR kinases have been found to regulate polarized growth in both animal cells and fungi. Drosophila Tricornered is an NDR kinase that is essential for the normal polarized growth of extensions of epidermal cells and for the tiling and branching of dendrites of da sensory neurons. Tricornered function requires interacting with the large Furry protein (3479 amino acid). A furry (fry) transgene was constructed and it was able to rescue the lethality of fry null mutations. The encoded protein was tagged at both its amino and carboxy termini and this allowed demonstration that the protein existed as an uncleaved protein in vivo. The C terminal GFP tag was used to follow the protein in vivo; it was found to be highly mobile. Interestingly Fry accumulated at the distal tip of growing bristles. It was established that Fry and Trc could be co-immunoprecipitated from wing discs. The mobility of Fry in both bristles and dendrites suggests that it could function in directing/mediating the intracellular transport needed for polarized growth. The observations that full length Fry and Trc show only partial co-localization in growing bristles while an amino terminal fragment of Fry shows close to complete co-localization with Trc suggests that the interaction between these proteins is transient and regulated (Fang, 2010).

Data from a number of systems from fungi to mammalian cells indicate that the Fry and NDR kinase family proteins interact directly in vivo. The data reported in this study provide further support. First it was established that Fry exists as a full-length protein in vivo in wing discs. It was also demonstrated that the full length Fry protein can be co-immunoprecipitated with Trc from wing disc cells and using the two-hybrid system a region in the Trc protein was mapped that is essential for the interaction. It was also found that a Trc protein that is missing its most C terminal region interacts more strongly with Fry than does the complete Trc protein. This suggests that the interaction is regulated and this could be an important regulatory step in vivo (Fang, 2010).

Although the Trc and Fry proteins appear to interact in vivo extensive co-localization of the two proteins was not seen by immunostaining. This was true for both the endogenous proteins and for transgene encoded proteins. In the cells both proteins examined were distributed in a punctate manner but relatively few of the puncta stained positively for both Trc and Fry. It was also found that in two cell types the two proteins had distinctly different protein accumulation patterns. In bristle forming cells the provocative distal tip accumulation of Fry was not seen with Trc. Further, in da sensory neurons Trc accumulated preferentially in nuclei, while Fry was excluded from the nucleus. The possibility cannot be excluded that there could be multiple protein complexes that contain Fry and Trc and that in some of these one or the other of the proteins is shielded from antibody reagents. However, the data did not support this possibility. Extensive co-localization was seen of the amino and carboxyl end tags of transgene encoded Fry. Thus, both ends of the Fry protein appear to be equally accessible. A high degree of co-localization between Trc and Myc-N-Fry was seen. Thus, in any puncta that contains these proteins either both or neither are accessible to antibody. It is suggested that the interaction between Fry and Trc is transient and regulated and that much of these proteins in cells are not in a complex together. The observation that Trc accumulated in da neuron nuclei while Fry was excluded is reminiscent of the observation that CBK1p accumulated in yeast daughter cell nuclei, while Tao3p is excluded from the nucleus. Similarly, the observations that Trc is primarily cytoplasmic in some cell types (e.g., pupal wing cells) and primarily nuclear in others (da neurons) mirror the observations that mammalian NDR1 is cytoplasmic in some cells and nuclear in others. The subcellular localization of Trc and Fry could be different in different cell types due to differential modification or to it being bound by different partners in different cell types. Trc is known to be phosphorylated and Fry protein was found to be multiply phosphorylated in embryos, and that seems likely in other cell types as well. Differential phosphorylation could regulate the subcellular localizations of these two proteins, although there is no direct evidence for this (Fang, 2010).

The generation of a transgene that encoded a functional Fry-GFP protein allowed assessment of the mobility of Fry in living cells. FRAP was used to test this in both developing bristles and in the dendrites of da neurons. In both of these cell types Fry-GFP was found to move rapidly. The data suggest that Fry-GFP moves both distally and proximally in developing bristles and dendrites of da neurons. These observations suggested that Fry os in some way involved in intracellular transport and in that way regulated polarized growth. In mammalian cells Fry has been found to bind to microtubules and such a molecular activity would nicely fit with it regulating intracellular transport (Fang, 2010).

DEVELOPMENTAL BIOLOGY

Larval

To examine the expression pattern of Trc and Fry, immunostaining analyses were performed on dissected larvae using polyclonal antibodies raised against Trc or Fry. Both Trc and Fry are widely expressed including all da neurons in third instar wild-type larvae. In da neurons, Trc and Fry are localized predominantly in the soma but are also detected in axons and dendritic branches. When Trc tagged with FLAG-epitope (FLAG-Trc) was expressed in ddaC neurons by a class IV neuron-specific Gal4 driver (Grueber, 2003b; Ye, 2004), FLAG-Trc was distributed in soma, axon, as well as dendritic branches, similar to the endogenous Trc localization (Emoto, 2004).

The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance

Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a 'like-repels-like' behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. The other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. Biochemical and genetic evidence is provided for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis (Emoto, 2006).

Dendritic arborization patterns are critical to a neuron's ability to receive and process impinging signals. Whereas neurons normally maintain the gross morphology of their dendrites, cortical neurons of Down's syndrome patients gradually lose dendritic branches after initially forming normal dendritic fields. Thus, neurons appear to have separate mechanisms for establishment and maintenance of their dendritic fields (Emoto, 2006).

Dendritic tiling is an evolutionarily conserved mechanism for neurons of the same type to ensure complete but non-redundant coverage of dendritic fields. In the mammalian visual system, for instance, dendrites of each retinal ganglion cell type cover the entire retina with little overlap, like tiles on a floor. In Drosophila, the dendritic arborization sensory neurons can be divided into four classes (I-IV) based on their dendrite morphology, and the dendritic field of class IV dendritic arborization neurons is shaped, in part, through a like-repels-like tiling behaviour of dendrite terminals. The NDR family kinase Trc and its activator Furry (Fry) has been identified as essential regulators of dendritic tiling and branching of class IV dendritic arborization neurons. These proteins are evolutionarily conserved and probably serve similar functions in neurons of different organisms (Emoto, 2006).

In addition to Trc, Drosophila has one other NDR family kinase, Wts, which is a tumour suppressor protein that functions in the coordination of cell proliferation and cell death in flies. To uncover the cell-autonomous functions of Wts in neurons, MARCM (mosaic analysis with a repressive cell marker) was ised to generate mCD8-GFP-labelled wts clones in a heterozygous background. Wild-type class IV neurons elaborate highly branched dendrites that cover essentially the entire body wall. Compared to wild-type ddaC (dorsal dendrite arborization neuron C) neurons, wts clones showed a severe and highly penetrant simplification of dendritic trees, with significantly reduced number (wild type, 575.1; wts, 255.6) and length (wild type, 1,457.0; wts, 590.4) of dendritic branches, and hence a greatly reduced dendritic field (Emoto, 2006).

In contrast to the severe dendritic defects caused by loss of Wts function, wts mutant ddaC axons entered the ventral nerve cord at the appropriate position and showed arborization patterns very similar to wild-type controls, with their axons terminating on the innermost fascicle and sending ipsilateral branches anteriorly and posteriorly and sometimes also a collateral branch towards the midline. Thus, Wts seems to have a crucial role in dendrite-specific morphogenesis in post-mitotic neurons (Emoto, 2006).

In proliferating cells, Wts is part of a signalling complex for tumour suppression that includes the adaptor protein Salvador (Sav) and the serine/threonine kinase Hpo. sav mutant ddaC MARCM clones were examined and dendritic defects were observed similar to wts MARCM clones. In severely affected clones (3 of 15 clones), most of the high-order branches were missing, whereas moderately affected clones (12 of 15 clones) exhibited a partial loss of their fine branches and major branches (Emoto, 2006).

To confirm that Wts and Sav function in the same pathway, genetic interaction between wts and sav in regulating dendrite morphogenesis was tested. Whereas heterozygous wts or sav mutants had no obvious dendritic phenotype, trans-heterozygous combinations of wts and sav alleles resulted in simplified dendrites similar to moderately affected sav clones. Furthermore, sav wts double mutant clones showed a severe dendrite defect comparable to wts mutant clones. Thus, Wts and Sav most probably function together in class IV neurons to regulate dendrite morphogenesis (Emoto, 2006).

The dendritic phenotypes of wts mutants and sav mutants might result from defects in branch formation and/or elongation, or loss of normally formed dendrites. Therefore ddaC dendrites were examined at different time points of larval development using the pickpocket-EGFP reporter, which is specifically expressed in class IV dendritic arborization neurons. Wild-type ddaC neurons elaborated primary and secondary dendritic branches by 24-28 h after egg laying, but large regions of the body wall were not yet covered by dendrites. By 48-52 h after egg laying, the major branches reached the dorsal midline, and the open spaces between major branches were filled with fine branches, resulting in complete dendritic coverage of the body wall. This tiling of dendrites persisted throughout the rest of larval development. In wts and sav mutants, ddaC dendrites were indistinguishable from those of wild-type controls at 24-28 h after egg laying. By 48-52 h after egg laying, wts and sav dendrites tiled the body wall as in wild type. During the next 24 h, however, dendrites of wts and sav mutants no longer tiled the body wall. Therefore, wts and sav seem to be required for maintenance of the already established tiling of dendrites (Emoto, 2006).

The loss of dendrites was further documented in live mutant larvae imaged for 30 h starting in early second instar larvae (48-50 h after egg laying). In wild-type larvae, ddaC dendrites grew steadily; the number of terminal branches increased by 23.0 over this time period. By contrast, dendrites of wts and sav mutants gradually lost their fine branches (decrease of 27.5 and 31.5, respectively) as well as some of the major branches by 78-80 h after egg laying (Emoto, 2006).

Class-IV-neuron-specific expression of wts and sav largely rescued the dendritic phenotype of wts and sav mutants, respectively, confirming that Wts and Sav act cell autonomously in class IV neurons. Furthermore, no detectable defect in patterning of the epidermis (anti-Armadillo antibody) or muscle (Tropomyosin::GFP reporter) was observed in wts or sav mutant third instar larvae. Taken together, these results indicate that the Wts/Sav signalling pathway functions in class IV neurons to maintain dendritic arborizations (Emoto, 2006).

Wts kinase activity is regulated, at least in part, by the Ste20-like serine/threonine kinase Hpo. Indeed, ddaC clones mutant for hpo exhibited simplified dendritic trees in third instar larvae, similar to wts or sav mutant clones, but showed more extensive dendritic arborizations in earlier larval stages (second to early third instar), consistent with the involvement of Hpo in the maintenance of dendrites. Notably, in hpo mutant clones at earlier developmental stages, dendritic branches were often found to overlap. Both the dendritic tiling and maintenance phenotypes were rescued by hpo expression in MARCM clones, consistent with the cell-autonomous function of Hpo in class IV neurons. Because this tiling defect in hpo mutant clones was similar to the tiling defects of trc mutant clones, whether hpo could genetically interact with trc to regulate dendritic tiling was tested. Compared with wild-type controls, trans-heterozygous combinations of trc and hpo exhibited obvious iso-neuronal as well as hetero-neuronal tiling defects, whereas wts and hpo trans-heterozygotes displayed simplified dendrites similar to wts mutants. These dendritic defects were consistently observed in multiple allelic combinations between hpo and trc or wts. In contrast, trans-heterozygous combinations of trc and wts showed no significant dendritic phenotypes. Furthermore, overexpression of wild-type Trc, but not Wts, in hpo MARCM clones partially rescued the dendritic tiling defects in class IV neurons. Thus, Hpo acts through Trc and Wts to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

Not only did Hpo interact genetically with Trc and Wts, its physical association with these NDR kinases could be detected in vivo. When Flag-tagged Trc was expressed using a nervous-system-specific Gal4 driver, anti-Flag antibodies immunoprecipitated Trc together with Hpo. Similarly, Myc-tagged Wts co-immunoprecipitated with Hpo expressed in embryonic nervous systems. Hpo co-immunoprecipitation appeared to be specific, because Misshapen, another Ste20-like kinase protein present in neurons, was not co-immunoprecipitated by anti-Flag or anti-Myc antibodies in similar experiments. These results suggest that Hpo associates with Trc and Wts in the Drosophila nervous system (Emoto, 2006).

To examine further the physical interaction between Trc and Hpo, analogous experiments were carried out in Drosophila S2 cells co-transfected with a haemagglutinin (HA)-tagged Trc construct and a Flag-tagged Hpo construct containing the full open reading frame, an amino-terminal fragment containing the kinase domain, or a carboxy-terminal fragment containing the regulatory domain. Full-length Hpo and the C-terminal portion of Hpo, but not the N-terminal fragment, were co-immunoprecipitated with Trc, suggesting that the C-terminal domain of Hpo is sufficient for Trc-Hpo complex formation (Emoto, 2006).

Hpo physically interacts with Wts and promotes Wts phosphorylation at multiple serine/threonine sites, including two sites, S920 and T1083 of Drosophila Wts, that appear to be necessary for Wts kinase activation. Indeed, Wts protein with mutations in the S920 and T1083 residues was unable to rescue the wts mutant dendritic phenotypes. Given that the corresponding phosphorylation sites in Trc are critical for Trc activation as well as control of dendritic tiling and branching, it was of interest to know whether Hpo may promote Trc phosphorylation at the critical serine and/or threonine residue. Wild-type Hpo, but not catalytically inactive Hpo or the Misshapen kinase, led to substantial incorporation of ³²P-labelled phosphate into recombinant Trc or Trc with a mutation at the S292 site (S292A), but not the T449A mutant form of Trc. Analogous results were obtained with Wts. These results support a model in which Hpo associates with and phosphorylates Trc and Wts at a critical threonine residue to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

Both genetic and biochemical evidence reveals that Hpo regulates complementary aspects of dendrite development through two distinct downstream signalling pathways: the Trc kinase pathway for tiling and the Wts kinase pathway for maintenance. These studies of class IV dendritic arborization neurons, together with the recent report that Wts signalling is required for cell fate specification of photoreceptor cells in Drosophila retina, demonstrate that the Wts signalling pathway is important for post-mitotic neurons. In proliferating cells, Wts phosphorylates Yorkie (Yki), a transcriptional co-activator, to regulate cell cycle and apoptosis in growing cells. However, Yki is dispensable for Hpo/Wts-mediated dendrite maintenance. Hpo probably functions as an upstream kinase for Trc, as well as Wts, in neurons by phosphorylating a functionally essential threonine, which may also be regulated by MST3, a Ste20-like kinase closely related to Hpo. Given the evolutionary conservation of known components in the Trc and Wts signalling pathways, it will be important to identify their relevant downstream targets and explore mechanisms that coordinate the establishment and maintenance of dendritic fields, and to determine the role of Trc and Wts signalling in the mammalian nervous system (Emoto, 2006).

Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw

The directional flow of information in neurons depends on compartmentalization: dendrites receive inputs whereas axons transmit them. Axons and dendrites likewise contain structurally and functionally distinct subcompartments. Axon/dendrite compartmentalization can be attributed to neuronal polarization, but the developmental origin of subcompartments in axons and dendrites is less well understood. To identify the developmental bases for compartment-specific patterning in dendrites, a screen was carried out for mutations that affect discrete dendritic domains in Drosophila sensory neurons. From this screen, mutations were identified that affected distinct aspects of terminal dendrite development with little or no effect on major dendrite patterning. Mutation of one gene, raw, affected multiple aspects of terminal dendrite patterning, suggesting that Raw might coordinate multiple signaling pathways to shape terminal dendrite growth. Consistent with this notion, Raw localizes to branch-points and promotes dendrite stabilization together with the Tricornered (Trc) kinase via effects on cell adhesion. Raw independently influences terminal dendrite elongation through a mechanism that involves modulation of the cytoskeleton, and this pathway is likely to involve the RNA-binding protein Argonaute 1 (AGO1), as raw and AGO1 genetically interact to promote terminal dendrite growth but not adhesion. Thus, Raw defines a potential point of convergence in distinct pathways shaping terminal dendrite patterning (Lee, 2015).

Although the concept of positional information was first applied to embryonic development, intracellular positional information governs morphogenesis of individual cells as well. For example, positioning the nucleus at the cell center and growth zones at the cell periphery depends on positional information from the microtubule cytoskeleton in Schizosaccharomyces pombe. Several lines of evidence support the existence of distinct subcompartments in axons and dendrites, but the forms of intracellular positional information and the coordinate systems that guide the development of these subcompartments have not been extensively characterized. Results from this screen and other studies suggest that at least two types of positional information govern C4da dendrite patterning. First, terminal branch distribution along the proximal-distal axis depends on microtubule-based processes; perturbing microtubule-based transport leads to a distal-proximal shift in the distribution of terminal dendrites in C4da arbors. Interestingly, modulating the activity of the F-actin nucleator Spire also affects terminal dendrite positioning along the proximal-distal axis, suggesting that multiple pathways contribute to the fidelity of branch placement. Second, terminal dendrites rely on dedicated programs that may act locally to regulate terminal dendrite patterning. The observation that different pathways regulate different aspects of terminal dendrite development suggests that multiple signaling systems exist for the local control of dendrite growth (Lee, 2015).

This study identified raw as a key regulator of terminal dendrite patterning. raw encodes a membrane protein that accumulates at branch-points and coordinately regulates terminal dendrite adhesion/stability via a pathway that involves Trc and terminal dendrite elongation via a pathway that is likely to involve cytoskeletal remodeling and AGO1. Raw therefore provides a potential point of integration for external signals that regulate these downstream growth programs. These pathways could be responsive to the same signal -- for example, Raw association with an extracellular ligand or a co-receptor -- or could be spatially/sequentially segregated. Identification of additional raw-interacting genes should help clarify the architecture of these signaling pathways (Lee, 2015).

Raw regulates cell-cell signaling, and in gonad morphogenesis Raw modulates Cadherin-based interactions between somatic gonadal precursor cells and germ cells, in part by localizing Armadillo to the cell surface. Likewise, the data support a role for Raw in promoting Trc activation by localizing Trc to the plasma membrane. Thus, one plausible model for Raw function in dendrite development is that it interacts with an extracellular signal, which might be a component of the ECM or a cell surface protein on epithelial cells, and signals together with a co-receptor to stimulate downstream pathways for adhesion and cytoskeletal remodeling. Several analogous signaling systems involving interactions with the epidermis that influence terminal dendrite or sensory axon patterning have been described, but how many of these signaling systems are at work in a given neuron, and how Raw interfaces with other signaling pathways, remain to be determined (Lee, 2015).

Although Raw has no obvious vertebrate counterpart, stretches of the ECD bear similarity to mucins and leucine-rich repeat proteins, one of which might serve an analogous function. Moreover, components of both downstream signaling pathways that this study identified are conserved in vertebrates and play known roles in dendrite patterning, including roles in the local control of dendrite growth: the Trc orthologs NDR1/2 (STK38/STK38L) regulate aspects of dendrite branch and spine morphogenesis, and Argonaute proteins mediate miRNA-mediated control of dendrite patterning, in part through local effects on translation. Additionally, dendrites contain structures related to P-granules, and Argonaute proteins may influence local translation in P-granules as well. Thus, versions of the Raw-regulated signaling pathways might control terminal dendrite patterning in vertebrates (Lee, 2015).

EFFECTS OF MUTATION

In a screen for enhancer trap lines showing expression in dendrite arborization (da) neurons, one insertion line, termed KY319, was found with high expression in da neurons throughout the larval stages. Genomic DNA flaking the P element was isolated by plasmid rescue and the P element was found to be inserted in the first intron of the fry gene. Because the fry mutants were known to have branched bristles and multiple wing hair phenotypes (Cong, 2001 and Adler 2002), fry's potential role in the control of the dendritic morphogenesis of da neurons was explored (Emoto, 2004).

To visualize dendrites in fry mutants, the pickpocket-EGFP reporter, which is specifically expressed in class IV sensory neurons (Grueber, 2003a), was introduced into each fry mutant allele. fry mutant neurons exhibit excessive dendritic branches. In the wild-type third instar larvae, class IV ddaC neurons in the dorsal cluster elaborate highly complex but stereotyped dendritic trees and extend a single axon ventrally -- the ddaC dendrites normally display a consistent number of terminal branches (Grueber, 2003b; Ye, 2004). In the fry null mutant (fry¹), both the terminal branch number and the total branch length are increased by a factor of two, whereas the major branch architecture appears normal. This branching phenotype of fry mutants is already apparent in early first instar larvae, and the terminal branch number of the mutants is almost twice that of wild-type in the first, second, and third instar larval stages (Emoto, 2004).

The fry gene functions together with the trc gene to control wing hair and bristle morphology (Geng, 2000; Cong, 2001). In addition, fry homologs show strong genetic interactions with trc homologs in a variety of species including S. cerevisiae (Du, 2002; Nelson, 2003), S. pombe (Hirata, 2002), and C. elegans (Zallen, 2000). To test whether trc is also involved in controlling dendritic branching in neurons, ddaC dendrites of a trc null mutant (trc¹) were examined; the trc dendrites display overbranching phenotypes similar to those of fry mutants. These observations suggest that both trc and fry function to regulate dendritic branching of class IV da neurons (Emoto, 2004).

Recent studies show that class IV da neurons exhibit dendritic tiling, presumably by exclusion between the terminal branches (Grueber, 2003a; Sugimura, 2003). Indeed, the terminal branches of the same ddaC neuron typically stop growing or turn away before crossing each other, resulting in minimum overlap. In contrast, the dendritic branches of fry¹ and trc¹ ddaC neurons often overlapped each other. Approximately 13% of dendritic terminal branches crossed one another in fry¹ and trc¹ ddaC dendrites, compared to ~1% of crossing in wild-type dendrites. The crossing branches of fry and trc mutants display rigid and straight trajectories, implying an impairment in like-repels-like navigation. Because these terminal branches were sandwiched between the epidermis and muscles, which were typically less than 1 microm apart in both mutant and wild-type larvae, the excessive overlap of mutant dendrites is unlikely to result from abnormal stratification of terminal branches. These findings suggest that trc and fry are involved in regulation of the dendritic tiling in class IV neurons (Emoto, 2004).

Since trc and fry null mutants display dendritic branching and tiling phenotypes, it was asked whether the tiling phenotype in mutants might simply arise from the excessive branching of terminal dendrites. To address this possibility, the number of dendritic crossings was normalized by the branch number and total dendrite length; the normalized dendritic crossings remained significantly increased in fry and trc mutants (Emoto, 2004).

The class IV dendrites of hypomorphic alleles and transheterozygotes of trc and fry were examined; robust tiling phenotypes were still observed in mild mutants with fry and/or trc function reduced to a level that caused no overbranching. For instance, the fry⁶ hypomorphic allele shows a reduced fry expression due to a P element insertion (Cong, 2001). The pickpocket-EGFP reporter revealed that the fry⁶ mutant exhibits a clear tiling defect whereas the terminal branch number appears unaffected. Indeed, normalized to the total branch length, the number of dendritic crossings in the fry⁶ mutant dendrites was slightly higher (~20%) than that in null mutants. Similarly, tiling defect was apparent in larvae transheterozygous for fry¹ and fry⁶ or fry¹ and trc¹ despite the normal dendritic length and branch points. Taken together, these observations suggest that the tiling phenotype in trc and fry mutants is not secondary to the overbranching phenotype and that trc and fry function together to ensure dendritic tiling of class IV neurons (Emoto, 2004).

To determine whether trc and fry act cell autonomously in neurons, the MARCM (mosaic analysis with a repressive cell marker) system was used to generate mCD8-GFP-labeled trc, fry, or trc fry double mutant clones in heterozygous background. Compared to the wild-type ddaC dendrites, trc and fry mutant ddaC dendrites display a 50% increase in the number of branches. Moreover, mutant clones exhibit a tiling defect. A similar phenotype was observed in two ventral class IV MARCM clones. The phenotypes of mutant clones are less severe than those seen in null mutant animals, presumably due to perdurance of wild-type proteins. The dendritic phenotypes of trc fry double mutant clones are indistinguishable from those of trc or fry single mutant clones. These results indicate that Trc and Fry act cell autonomously to regulate dendritic branching and tiling of class IV neurons (Emoto, 2004).

Similarly, mutant clones of da neurons of class I, class II, and class III also show a 2- to 3-fold increase in the number of terminal dendritic branches. In contrast to the drastic phenotypes in terminal branches, the major dendritic branch architecture including primary, secondary, and tertiary branches, as well as the cell body shape are not obviously affected. Furthermore, the trc, fry, and double mutant clones show no detectable defects in bipolar dendrite neurons, external sensory neurons, or chordotonal neurons. Thus, among sensory neurons, trc and fry specifically control the terminal branching of da neuron dendrites (Emoto, 2004).

Given that trc and fry mutations compromise tiling of terminal branches from the same neuron (iso-neuronal tiling), it was of interest to see whether trc and fry also control tiling of dendrites from different neurons (hetero-neuronal tiling). The dendrites of the three class IV neurons in each abdominal hemisegment, ddaC, v'ada, and vdaB, normally cover the whole epidermis with minimal overlap (Grueber, 2002; Grueber, 2003a; Sugimura, 2003). For example, the adjacent v'ada and vdaB neurons appear to respect the respective dendritic territories and rarely send their dendrites into the dendritic fields of their neighbors. In fry¹ and trc¹ null mutants, however, the v'ada and vdaB dendrites often invade neighboring fields, resulting in a partial overlap of the dendritic fields. Major branches as well as terminal branches overlapped extensively in both fry and trc mutants. The fry⁶ hypomorphic mutant also displays clear hetero-neuronal tiling defects. Similar but milder dendritic tiling defects are observed in transheterozygotes for fry¹ and fry⁶ and in transheterozygotes for fry¹ and trc¹. As observed in ddaC neurons, v'ada and vdaB dendrites also display iso-neuronal tiling defects with 12%-14% crossing in fry and trc mutants, compared to only 1% crossing in the wild-type control. There is a good correlation between the strength of the iso-neuronal and hetero-neuronal tiling phenotypes. These observations suggest that trc and fry regulate both iso-neuronal and hetero-neuronal tiling, presumably through the same mechanisms (Emoto, 2004).

How might trc and fry prevent overlap of like dendrites? A priori, it is conceivable that multiple dendritic branches initially coinnervate the same territory and have extensive crossings; tiling could result from retraction of some of these branches. Alternatively, in the like-repels-like scenario, dendrites interact with one another to avoid overlap and crossing throughout development (Emoto, 2004).

To distinguish between these possibilities, dendritic crossings were examined during development. Within a few hours around the time of hatching (AEL 20-23 hr), the territories of v'ada and vdaB neurons become defined. In wild-type control, no significant overlap was found between dendrites of v'ada and vdaB neurons throughout development, including in newly hatched larvae, in first instar larvae 3 or 6 hr after hatching, or in second or third instar larvae. In contrast, dendritic crossings were already evident in newly hatched fry mutant larvae, and the number of crossings increased continuously during larval development. Thus, consistent with previous studies (Grueber, 2003; Sugimura, 2003), dendrites normally avoid one another as they meet initially, without going through a noticeable period of coinnervation followed by pruning; fry mutations affect the mechanism underlying this avoidance (Emoto, 2004).

To further define the cellular functions of fry and trc in dendritic tiling, the dendrites of live wild-type and fry mutants were examined for 16 hr starting at the early second instar larval stage, when class IV neurons have stabilized their major arbors. Many of the terminal branches remain dynamic, however, from the beginning to the end of the 16 hr period. Nearly 75% of the dynamic branches displayed a net extension, whereas 25% showed a net retraction in both wild-type and fry mutants. In wild-type larvae, 73% of those branches that extended their tips close (< 5 microm) to other branches turned away, hence avoiding crossing, and only 1% extended beyond other branches. The percentage of branches making a turn was significantly reduced by a factor of three in both null and hypomorphic fry mutants. Instead of turning, about 45% of the mutant dendritic branches ran across other branches at many locations. These findings are consistent with the like-repels-like scenario. It appears that in fry mutants, dendrites can grow and retract normally, but their inability to turn in order to avoid like dendrites results in tiling defects (Emoto, 2004).

To investigate the function of the Trc kinase in neurons, wild-type and mutant Trc were expressed in neurons of trc mutants and whether the trc mutant phenotype could be ameliorated was tested. The kinase domains of Trc and its orthologs share 70%-80% amino acid identity (Tamaskovic, 2003b). In addition, Trc has conserved phosphorylation sites at Ser292 and Thr449; phosphorylation at these residues is essential for maximal activation for the human Trc kinase in vitro (Tamaskovic, 2003b). To test whether Trc kinase activity is required for proper dendritic branching and tiling in vivo, a kinase-dead mutant (K122A) was generated and a mutant was generated in which both Ser292 and Thr449 were replaced with alanine (S292AT449A) to prevent phosphorylation. Specific expression of wild-type Trc with a class IV neuron-specific Gal4 driver largely rescued both dendritic branching and tiling defects of the trc¹ mutant; however, neither the K122A nor the S292AT449A mutant could rescue these phenotypes. These results suggest that Trc kinase activity in class IV neurons is important for their proper dendritic branching and tiling in vivo; this Trc activity is sufficient even in animals lacking Trc in other cell types (Emoto, 2004).

Interestingly, overexpression of wild-type Trc in class IV neurons of wild-type larvae caused a slight reduction of branch number. Moreover, expression of the K122A in wild-type class IV neurons resulted in a highly penetrant increase in terminal branches of ddaC dendrites as well as tiling defects similar to the phenotypes of trc and fry mutants, indicating that the K112A acts as a dominant-negative mutant. Specific expression of the S292AT449A Trc mutant in wild-type class IV neurons also led to an increase of terminal dendrites, albeit milder than that induced by the K122A mutant, whereas the dendritic tiling defects seen in S292AT449A-expressing dendrites were as obvious as those induced by the K122A mutant. These results strongly suggest that both Trc phosphorylation and Trc kinase activity in neurons play an essential role in dendritic branching and tiling (Emoto, 2004).

Given the genetic interaction between trc and fry and their evolutionarily conserved function in controlling branching of cellular processes, their functional relationship was investigated by assessing the Trc kinase activity in trc and fry mutants. Trc immunoprecipitates from wild-type, trc, or fry embryos were assayed for kinase activity by using histone H1 as an artificial substrate. Although similar amounts of Trc protein were precipitated from wild-type and fry homogenates, kinase activity of precipitates from fry mutants was significantly reduced, to the level similar to that of trc mutants. Since the Trc protein was undetectable in immunoprecipitates from trc embryos, histone phosphorylation by precipitates from trc mutants is likely due to other kinases coprecipitated with the beads. These results indicate that the Trc kinase is inactive in fry mutants. Most likely, Trc kinase activity requires Fry and is a key component of a signaling pathway primed for restraining growth toward like structures and limiting branching (Emoto, 2004).

The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase

During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility, a weak tricornered mutant was found to be hypersensitive to cytochalasin D. The tricornered gene was cloned and found to encode the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation (Geng, 2000).

Mutations in trc result in the splitting of epidermal hairs, the shafts of sensory bristles, larval denticles, and the lateral branches of the arista. Mutations in trc do not however cause dramatic effects on hair or bristle shape or length. Nor is evidence seen for trc delaying prehair initiation or slowing prehair elongation. Based on the similar phenotypes it seems likely that the Trc protein has a similar target(s) in all of these cell types. In comparing the trc-induced phenotypes to the effects of various inhibitors the actin cytoskeleton was identified as a candidate target; the inhibition of actin polymerization with CD or LAT A results in frequent split hairs, bristles, and arista lateral branches. However, the CD- and LAT A-induced phenotypes differed substantially from those found in trc mutants in that the drug treatments had major effects on the shape and length of hairs and bristles. Because of this it is suggested that trc mutations do not inhibit actin polymerization. Consistent with this, trc mutant hairs, bristles, and arista laterals routinely stained strongly for F-actin. It was also found that trc prehairs in clones develop and grow at the same rate as their wild-type neighbors. The data also argue that trc is not needed for the bundling of actin filaments in developing bristles since these looked normal in trc⁸/Df pupae (Geng, 2000).

The split hairs and bristles that are found in trc mutants appear normal except where they are split. In this way the trc phenotype differs profoundly from the phenotypes found in cells mutant for genes such as f, sn, or ck, which encode proteins that are functional components of the actin cytoskeleton. In these cases the mutant phenotype is displayed all along the length of the bristle or hair. It is suggested that this is because these proteins are part of the machinery that is directly involved in the morphogenesis of hairs and bristles and hence are needed throughout morphogenesis. This suggests the possibility that trc has a regulatory as opposed to structural function (Geng, 2000).

A possible hypothesis is that trc functions to repress the initiation of outgrowths and that splitting is a consequence of ectopic outgrowths. This hypothesis can account for some of the data, but it does not easily accommodate the finding that in splitting bristles no evidence is seen for new ectopic large bundles of actin filaments. Rather, existing neighboring bundles appear to separate as the segments split apart. These observations on trc differ from those on bristles after CD treatment where evidence was seen for ectopic bundles of actin filaments (Geng, 2000).

It is suggested that what is important for maintaining the integrity of hairs and bristles is coordinating the growth of actin filaments and/or other cellular components. When some filaments or bundles grow more rapidly than others splitting might occur. It is suggested that this mechanism contributes to the split hairs and bristles seen after CD and LAT A treatment. If this is the case then developing bristle cells would be expected to monitor the polymerization of actin filaments and try to prevent splitting by slowing down the polymerization of filaments or bundles that are growing more rapidly than others. This could be thought of as being equivalent to a morphogenetic checkpoint. The trc gene could be a component of a signal transduction pathway that mediates the monitor function. Such a monitor function might involve regulating gene expression, which could explain the nuclear location of NDR (Millward, 1995). A hint that such a monitor/checkpoint might exist comes from the finding of a delay in bristle or hair morphogenesis after treatment with low doses of CD. Further experiments will be needed to determine if such a monitoring function exists and if trc encodes a component of it. Several other plausible models exist to explain trc function in maintaining the integrity of cellular extensions. The Trc protein could function in the assembly of the cellular components that organize the outgrowth of these cellular extensions. Defects in properly assembling an organizing center might result in the splitting of the extensions at later stages in elongation. The Trc protein could also function to mechanically strengthen cellular extensions in some way to insure their integrity, e.g., by increasing the degree of cross-linking of the membrane cytoskeleton (Geng, 2000).

Interactions between tricornered and mob genes

The directed expression of a dominant negative Trc protein provides a sensitized system for identifying interacting genes (He, 2005a). Deficiencies for each of the fly mob genes (see mats) enhances the wing hair phenotype that results from driving expression of UAS-trc^T453A using either ap-GAL4 or ptc-gal4. The strongest enhancement is seen with deficiencies for mats and Dmob2 (FlyBase terms the gene Dmob2 Mob1). These results suggested the possibility that all 4 Dmobs can redundantly interact with Trc, although it is possible that the interactions could be indirect or due to other genes in deleted regions. It is worth noting that such interactions are not common. When the Drosophila deficiency collection was screened for enhancement or suppression of ap-GAL4 UAS-trc^T453A, <10% of the Dfs showed an interaction (He, 2005b).

To confirm that the genetic interaction between trc and Df (mats) was due to the reduction in mats dose, two independent alleles were used. One was the null allele described by Lai (2005) (mats^e235), which is deleted for almost the entire coding region, and the other was a lethal PiggyBac insertion allele of mats [PBac{RB}CG13852^e03077] (this allele is referred to as mats^PB). Because this later allele has not been well characterized, the insertion was determined to be lethal over a deficiency for the region (Df(3R)Exel6191), and it failed to complement the recessive lethality of mats^e235 consistent with the lethality being due to the PB insertion. This mutation could be reverted using a source of PiggyBac transposase. It was found that both mats alleles dominantly enhance the trc dominant negative phenotype and this enhancement is lost in the PB revertant. It was also found that over expression of mats from a UAS-mats transgene (Lai, 2005; driven by ptc-Gal4) partially suppresses the multiple hair cell phenotype that results from driving expression of Trc-DN using ptc-Gal4. These dose responses argue that Mats activates Trc. Interestingly, it was found that heterozygosity for a wts mutation also enhances the Trc dominant negative phenotype, although somewhat less strongly (He, 2005b).

Evidence was also obtained for mats functioning with trc and fry using simple loss of function mutations. Wild-type flies or flies heterozygous for either trc, fry, or mats appear normal and only rarely (on fewer than 5% of wings) is even a single multiple hair cell seen. Flies that were heterozygous for two of these genes showed a slightly higher frequency of wings with one or a couple of multiple hair cells (often ~10%) but the increase was not routinely significant. However, almost half of the wings from flies that are heterozygous for all three genes (e.g., fry² trc¹+/+ + mats^PB) show a weak multiple hair cell phenotype, a significant increase. This genetic interaction is further support for the hypothesis that trc, fry, and mats function together in regulating wing hair development. In this assay no equivalent interaction with wts^3-17 was seen (He, 2005b).

Previous studies established that trc also has a larval denticle phenotype (Geng, 2000). In trc mutants the overall pattern of denticles is partly disorganized and many denticles are split. Split denticles are infrequent in wild-type larvae. The denticle pattern of mats^PB/mats^PB homozygous larvae is also disorganized and contains many split denticles. The number of split denticles is similar in mats^PB/Df larvae, suggesting that for this phenotype, mats^PB is a strong, near phenotypic null allele. The mats^e235 also showed a similar denticle phenotype. The phenotype of mats^PB homozygotes is slightly less severe than that of trc^P/trc^P larvae. Notably, trc^P mats^PB/trc^P mats^PB double mutant larvae do not have a more severe phenotype than the single mutants. This lack of additivity argues that trc and mats function in a common pathway during denticle development (He, 2005b).

Both mats alleles are larval lethals with death typically in the second or early third instar. To examine the phenotype of mats in wing cells, mosaics were generated using FLP/FRT. mats clones on the wing, leg, thorax, and head display two types of phenotype. The most notable is indistinguishable from those produced by clones of wts, suggesting that mats also functions with wts as has been shown by Lai (2005). On the wing, small clones produced bulges that can be seen at low magnification with a stereomicroscope. In mounted wings individual cell outlines are visible in the cuticle and the cells appear to have a bulging apical surface. The hairs are located on an elevated pedestal, a phenotype that is indistinguishable from those seen in wts clones. The hairs were often broader than normal. Particularly in other body regions clones were abnormally pigmented (either darker or lighter than normal, and there were outgrowths of clone tissue. In highly abnormal wing clones, evidence of clustered and split hairs were often seen that were typical of trc mutant clones. Some multiple hair cells were seen in very abnormal wts clones but this phenotype appears less severe (e.g., number of hairs per cell) than that seen with mats or trc. These observations suggest that mats functions with both Trc and Wts (He, 2005b).

mats^PB and mats^e235 clones in pupal wings were examined. Mutant mats cells are able to outcompete their neighbors and end up comprising most of the wing when clones are induced early. As was expected from the morphology of clones in adult wings, the pupal clones produce bulges in the wing and individual cells also often appear bulged. Clone cells stain more brightly for F-actin. This is true both in developing hairs and in the general apical cortex. This phenotype is clear-cut enough that it could be used as a convenient marker of mats mutant cells. These phenotypes were seen with both mats alleles tested. A similar, increase in actin staining was seen in wts clones. A similar, but perhaps less severe increase in staining, is seen in trc clones (He, 2005a). In some, but not all mats clones, large numbers of multiple hair cells can be seen. At later stages a circular pedestal of actin staining could be seen surrounding the base of the hair in mutant cells but not in surrounding wild-type cells. At still later stages the wild-type cells also had a circular pedestal of actin staining, suggesting that the mutant cells might be developmentally more advanced. Consistent with this possibility, in many clones hair initiation and outgrowth appear to be advanced in mats mutant cells compared with neighbors. This is also the case for wts clones, but it is the opposite of trc clones, in which hair development is often delayed (He, 2005a). Cells in mats clones have a smaller cross section so that the array of hairs appears denser, which is also the opposite of what is seen in trc clones, in which there is an increase in cross-sectional area (He, 2005a). Once again the phenotype of the wts clone cells resembles that seen for mats cells. Thus, for several wing phenotypes mats mutant cells resembled wts and not trc cells. Indeed, the mats phenotype is the opposite of trc for both cell area and the timing of hair morphogenesis (He, 2005b).

It has been found that the accumulation of Fry in wing cells is subject to feedback control that is dependent on Trc activity. Hence, in a trc mutant, increased Fry accumulation is found (He, 2005b). Several of the observations described above suggest the hypothesis that mats functions along with trc and is important for Trc activation. From this it is predicted that Fry accumulation would also be elevated in Dmob1 clones. Increased levels of Fry immunostaining were found in Dmob1 clone cells; this is consistent with the hypothesis. This is also seen in wts mutant clones, although the increase appears less dramatic (He, 2005b).

Tumorous overgrowth phenotypes are a consequence of mutations in a number of Drosophila genes. In several cases, such as lethal giant discs overgrown imaginal discs are found in late third instar larvae. To determine whether that is also the case for mats, mats/Df mutant larvae were examined. These larvae grow slowly and after 5 d of growth, when wild-type larvae begin to pupate, mats/Df larvae are the size of early third instar larvae. These larvae routinely die without growing substantially larger. When 5.5-5-d-old mats larvae were dissected, not evidence of tumors or overgrowth of imaginal or other tissues was found. Rather, the imaginal discs were approximately the size of those seen in 4-d larvae. However, the mats homozygous discs did not appear normal, since they were abnormally shaped and more folded than normal discs of this size (He, 2005b).

In a number of experiments involving mats or wts, what appeared to be spontaneous tumors or clones was observed. This was seen most often in flies that also carried reduced doses of Dmob3 and Dmob4. It is thought that these overgrowths are due to spontaneous mitotic recombination, because when the flies were also mutant for trc or fry, evidence of trc or fry clones was seen. The trc and fry clones were seen less frequently. This could be due to these genes being located more proximally on the chromosome than mats or wts, but it might also be due to the competitive advantage of mats and wts clones, resulting in these clones being larger and easier to detect. The basis for these clones is unclear but suggests genomic instability in mats and/or wts mutants (He, 2005b).

The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway

To cover the receptive field completely and non-redundantly, neurons of certain functional groups arrange tiling of their dendrites. In Drosophila class IV dendrite arborization (da) neurons, the NDR family kinase Tricornered (Trc) is required for homotypic repulsion of dendrites that facilitates dendritic tiling. This study reports that Sin1, Rictor, and target of rapamycin (TOR), components of the TOR complex 2 (TORC2), are required for dendritic tiling of class IV da neurons. Similar to trc mutants, dendrites of sin1 and rictor mutants show inappropriate overlap of the dendritic fields. TORC2 components physically and genetically interact with Trc, consistent with a shared role in regulating dendritic tiling. Moreover, TORC2 is essential for Trc phosphorylation on a residue that is critical for Trc activity in vivo and in vitro. Remarkably, neuronal expression of a dominant active form of Trc rescues the tiling defects in sin1 and rictor mutants. These findings suggest that TORC2 likely acts together with the Trc signalling pathway to regulate the dendritic tiling of class IV da neurons, and thus uncover the first neuronal function of TORC2 in vivo (Koike-Kumagai, 2009).

EVOLUTIONARY HOMOLOGS

Ndr kinase homologs in yeast and Neurospora

Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-1 is a temperature sensitive mutant of N. crassa that exhibits restricted colonial growth. At temperatures above 32°C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. A genomic cosmid clone containing the wild type allele of cot-1 was isolated by complementation. Sequence analyses suggested that cot-1 encodes a member of the cAMP-dependent protein kinase family. Strains in which cot-1 was disrupted are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly results in restricted colonial growth. These results suggest that an active COT1 kinase is required for one or more events essential for hyphal elongation (Yarden, 1992).

The DBF2 gene of the budding yeast Saccharomyces cerevisiae encodes a cell cycle-regulated protein kinase that plays an important role in the telophase/G1 transition. As a component of the multisubunit CCR4 transcriptional complex, DBF2 is also involved in the regulation of gene expression. MOB1, an essential protein required for a late mitotic event in the cell cycle, genetically and physically interacts with DBF2. DBF2 binds MOB1 in vivo and can bind it in vitro in the absence of other yeast proteins. Expression of MOB1 is also cell cycle regulated, its expression peaking slightly before that of DBF2 at the G2/M boundary. While overexpression of DBF2 suppressed phenotypes associated with mob1 temperature-sensitive alleles, it could not suppress a mob1 deletion. In contrast, overexpression of MOB1 suppresses phenotypes associated with a dbf2-deleted strain and suppresses the lethality associated with a dbf2 dbf20 double deletion. A mob1 temperature-sensitive allele with a dbf2 disruption was also found to be synthetically lethal. These results are consistent with DBF2 acting through MOB1 and aiding in its function. Moreover, the ability of temperature-sensitive mutated versions of the MOB1 protein to interact with DBF2 is severely reduced, confirming that binding of DBF2 to MOB1 is required for a late mitotic event. While MOB1 and DBF2 are capable of physically associating in a complex that does not include CCR4, MOB1 interacts with other components of the CCR4 transcriptional complex. Models concerning the role of DBF2 and MOB1 in controlling the telophase/G1 transition are discussed (Komarnitsky, 1998).

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. This process was investigated in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34(cdc2) mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. These results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle (Verde, 1998).

During the early stages of budding, cell wall remodeling and polarized secretion are concentrated at the bud tip (apical growth). The CBK1 gene, encoding a putative serine/threonine protein kinase, was identified in a screen designed to isolate mutations that affect apical growth. Analysis of cbk1Delta cells reveals that Cbk1p is required for efficient apical growth, proper mating projection morphology, bipolar bud site selection in diploid cells, and cell separation. Epitope-tagged Cbk1p localizes to both sides of the bud neck in late anaphase, just prior to cell separation. CBK1 and another gene, HYM1, have been identified in a screen for genes involved in transcriptional repression and proposed to function in the same pathway. Deletion of HYM1 causes phenotypes similar to those observed in cbk1Delta cells and disrupts the bud neck localization of Cbk1p. Whole-genome transcriptional analysis of cbk1Delta suggests that the kinase regulates the expression of a number of genes with cell wall-related functions, including two genes required for efficient cell separation: the chitinase-encoding gene CTS1 and the glucanase-encoding gene SCW11. The Ace2p transcription factor is required for expression of CTS1 and has been shown to physically interact with Cbk1p. Analysis of ace2Delta cells reveals that Ace2p is required for cell separation but not for polarized growth. These results suggest that Cbk1p and Hym1p function to regulate two distinct cell morphogenesis pathways: an ACE2-independent pathway that is required for efficient apical growth and mating projection formation and an ACE2-dependent pathway that is required for efficient cell separation following cytokinesis. Cbk1p is most closely related to the (1) Neurospora crassa Cot-1; (2) Schizosaccharomyces pombe Orb6; (3) Caenorhabditis elegans, Drosophila, and human Ndr, and (4) Drosophila and mammalian WARTS/LATS kinases. Many Cbk1-related kinases have been shown to regulate cellular morphology (Bidlingmaier, 2001).

In Saccharomyces cerevisiae, mothers and daughters have distinct fates. Cbk1 kinase and its interacting protein Mob2 regulate this asymmetry by inducing daughter-specific genetic programs. Daughter-specific expression is due to Cbk1/Mob2-dependent activation and localization of the Ace2 transcription factor to the daughter nucleus. Ectopic localization of active Ace2 to mother nuclei is sufficient to activate daughter-specific genes in mothers. Eight genes are daughter-specific under the tested conditions, while two are daughter-specific only in saturated cultures. Some daughter-specific gene products contribute to cell separation by degrading the cell wall. These experiments define programs of gene expression specific to daughters and describe how those programs are controlled (Colman-Lerner, 2001).

Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14. Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1. The direct biochemical interactions between the components of the MEN remain largely unresolved. This study investigates the mechanisms that underlie activation of the protein kinase Dbf2. Dbf2 kinase activity depends on Tem1, Cdc15, and Mob1 in vivo. In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf2 was bound to Mob1. Conserved phosphorylation sites Ser-374 and Thr-544 (present in the human, Caenorhabditis elegans, and Drosophila melanogaster relatives of Dbf2) were required for DBF2 function in vivo, and activation of Dbf2-Mob1 by Cdc15 in vitro. Although Cdc15 phosphorylates Dbf2, Dbf2-Mob1, and Dbf2(S374A/T544A)-Mob1, the pattern of phosphate incorporation into Dbf2 Is substantially altered by either the S374A T544A mutations or omission of Mob1. Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2. It is proposed that Mob1 promotes this activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and Thr-544 phosphoacceptor sites of Dbf2 (Mah, 2001).

Protein kinases in the Cot-1/Orb6/Ndr/Warts family are important regulators of cell morphogenesis and proliferation. Cbk1p, a member of this family in Saccharomyces cerevisiae and a homolog of Fry, has been shown to be required for normal morphogenesis in vegetatively growing cells and in haploid cells responding to mating pheromone. A mutant of PAG1 (a homolog of fry), a novel gene in S. cerevisiae, displayed defects similar to those of cbk1 mutants. pag1 and cbk1 mutants share a common set of suppressors, including the disruption of SSD1, a gene encoding an RNA binding protein, and the overexpression of Sim1p, an extracellular protein. These genetic results suggest that PAG1 and CBK1 act in the same pathway. Furthermore, Pag1p and Cbk1p localize to the same polarized peripheral sites and they coimmunoprecipitate with each other. Pag1p is a conserved protein. The homologs of Pag1p in other organisms are likely to form complexes with the Cbk1p-related kinases and function with those kinases in the same biological processes (Du, 2002).

Fission yeast cells identify growing regions at the opposite ends of the cell, producing the rod-like shape. The positioning of the growth zone(s) and the polarized growth require CLIP170-like protein Tip1 and the Ndr kinase Orb6, respectively. The mor2/cps12 mutation disrupts the localization of F-actin at the cell ends, producing spherical cells and concomitantly inducing a G(2) delay at 36°C. Mor2 is important for the localization of F-actin at the cell end(s) but not at the medial region, and is essential for the restriction of the growth zone(s) where Tip1 targets. Mor2 is homologous to the Drosophila Furry protein, which is required to maintain the integrity of cellular extensions, and is localized at both cell ends and the medial region of the cell in an actin-dependent fashion. Cellular localization of Mor2 and Orb6 was interdependent. The tyrosine kinase Wee1 is necessary for the G(2) delay and maintenance of viability of the mor2 mutant. These results indicate that Mor2 plays an essential role in cell morphogenesis in concert with Orb6, and the mutation activates the mechanism coordinating morphogenesis with cell cycle progression (Hirata, 2002).

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates events associated with the M to G1 transition. The function of two S. cerevisiae proteins related to the MEN proteins Mob1p and Dbf2p kinase has been investigated. Cells lacking the Dbf2p-related protein Cbk1p fail to sustain polarized growth during early bud morphogenesis and mating projection formation. Cbk1p is also required for Ace2p-dependent transcription of genes involved in mother/daughter separation after cytokinesis. The Mob1p-related protein Mob2p physically associates with Cbk1p kinase throughout the cell cycle and is required for full Cbk1p kinase activity, which is periodically activated during polarized growth and mitosis. Both Mob2p and Cbk1p localize interdependently to the bud cortex during polarized growth and to the bud neck and daughter cell nucleus during late mitosis. Ace2p is restricted to daughter cell nuclei via a novel mechanism requiring Mob2p, Cbk1p, and a functional nuclear export pathway. Furthermore, nuclear localization of Mob2p and Ace2p does not occur in mob1-77 or cdc14-1 mutants, which are defective in MEN signaling, even when cell cycle arrest is bypassed. Collectively, these data indicate that Mob2p-Cbk1p functions to (1) maintain polarized cell growth, (2) prevent the nuclear export of Ace2p from the daughter cell nucleus after mitotic exit, and (3) coordinate Ace2p-dependent transcription with MEN activation. These findings may implicate related proteins in linking the regulation of cell morphology and cell cycle transitions with cell fate determination and development (Weiss, 2002).

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. A set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which has been designated RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, genetic screens were conducted for bilateral mating and cell separation mutants and alleles of the PAK-related kinase Kic1p were identified in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene results in a loss of Ace2p function and causes cell polarity defects that are distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. A novel leucine-rich repeat-containing protein Sog2p was also identified that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibit the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localizes to cortical sites of growth during both budding and mating pheromone response. Hym1p is Kic1p- and Sog2p-dependent and Sog2p and Kic1p are interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p are detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex are dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. These data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes (Nelson, 2003).

C. elegans Ndr kinase homologs

The C. elegans sax-1 gene regulates several aspects of neuronal cell shape. sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth. The ectopic neurites in sensory neurons of sax-1 mutants resemble the defects caused by decreased sensory activity. However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation. sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation. These kinases have similarity to Rho kinases but lack consensus Rho-binding domains. Dominant negative mutations in the C. elegans RhoA GTPase cause neuronal cell shape defects similar to those of sax-1 mutants, and genetic interactions between rhoA and sax-1 suggest shared functions. These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading (Zallen, 2000).

Mechanosensory neurons provide accurate information about stimulus location by restricting their sensory dendrites to nonoverlapping regions, a pattern called tiling. C. elegans sax-1 and sax-2 regulate mechanosensory tiling by controlling the termination point of sensory dendrites. During development, the posterior PLM mechanosensory dendrite overlaps transiently with the anterior ALM mechanosensory neuron. This overlap is eliminated during a discrete period of paused or slowed PLM process growth, between an early period of rapid outgrowth and a later period of maintenance growth. In sax-2 mutants, the PLM sensory dendrite fails to slow between the active growth and maintenance growth phases, leading to sustained overlap of anterior and posterior mechanosensory processes. sax-2 encodes a large conserved protein with HEAT/Armadillo repeats that functions with sax-1, an NDR cell morphology-regulating kinase. High-level expression of sax-2 leads to premature neurite termination, suggesting that SAX-2 can directly inhibit neurite growth (Gallegos, 2004).

Genes that control ray sensory neuron axon development in the Caenorhabditis elegans male; the sax-2/Furry and sax-1/Tricornered pathway affects ectopic neurite outgrowth and establishment of normal axon synapses

A set of male-specific sensory neurons in Caenorhabditis elegans establish axonal connections during postembryonic development. In the adult male, 9 bilateral pairs of ray sensory neurons innervate an acellular fan that serves as a presumptive tactile and olfactory organ during copulation. Ray axon commissures were visualized with a ray neuron-specific reporter gene and both known and new mutations that affect the establishment of connections to the pre-anal ganglion were studied. The UNC-6/netrin-UNC-40/DCC pathway provides the primary dorsoventral guidance cue to ray axon growth cones. Some axon growth cones also respond to an anteroposterior cue, following a segmented pathway, and most or all also have a tendency to fasciculate. Two newly identified genes, rax-1 and rax-4, are highly specific to the ray neurons and appear to be required for ray axon growth cones to respond to the dorsoventral cue. Among other genes identified, rax-2 and rax-3 affect anteroposterior signaling or fate specification and rax-5 and rax-6 affect ray identities. A mutation was identfied in sax-2; the sax-2/Furry and sax-1/Tricornered pathway affects ectopic neurite outgrowth and establishment of normal axon synapses. Mutations in genes for muscle proteins that affect axon pathways by distorting the conformation of the body wall. Thus ray axon pathfinding relies on a variety of general and more ray neuron-specific genes and provides a potentially fruitful system for further studies of how migrating axon growth cones locate their targets. This system is applicable to the study of mechanisms underlying topographic mapping of sensory neurons into target circuitry where the next stage of information processing is carried out (Jia, 2006; full text of article).

In sax-2(bx130), ray neurons send out axons that grow normally and form commissures to the pre-anal ganglion (PAG). In addition, they grow ectopic neurites that extend for varying distances. These aberrant outgrowths continue to form during the adult stage, becoming continuously more abundant as the animals age. Ectopic extensions behave like axons in growing anteriorly, but they fail to fasciculate or turn toward the ventral side. Additional neurons were examined and it was found that the four male-specific CEM neurons of the head, visualized with pkd-2::GFP, also have several abnormal posterior processes. The mutation bx130 mapped to and failed to complement the previously described gene sax-2. bx130 is rescued by the predicted gene F21H11.2. F21H11.2 encodes a large conserved protein with HEAT/Armadillo repeats (Jia, 2006).

sax-2 functions with sax-1, an NDR ser/thr kinase, in the maintenance of amphid neuronal morphology and mechanosensory neurite termination and tiling. Their Drosophila homologs, trc and fry, regulate the dendritic branching and tiling of Drosophila sensory neurons. sax-1(ky211) causes ectopic processes of ray axons similar to those in sax-2 mutants, suggesting that sax-1 and sax-2 function together to regulate ray axon morphology as they do for other sensory neurons. Interestingly, the unassigned mutation bx141 also causes the production of ectopic processes at late stage, albeit weakly. The similarity of sax-2(bx130) and bx141 mutant phenotypes suggests that bx141 either is a weak allele of sax-1 or sax-2 or may define an additional gene in this pathway (Jia, 2006).

Previous studies found little or no apparent disruption of neuron function in sax-2 and sax-1 mutants, consistent with a role in a maintenance pathway with little consequence for neuron activity. However, sax-2(bx130) males are defective in mating, suggesting ray neuron function is compromised. Therefore the density of presynaptic vesicles of ray neurons in the PAG was examined by scoring the expression of a PKD-2 promoter-driven SNB-1::GFP fusion protein (bxEx94). SNB-1 encodes C. elegans synaptobrevin, a synaptic vesicle protein expressed at synaptic sites. The presence of the sax-2(bx130) mutation dramatically reduces the density of GFP puncta in the PAG in the adult, suggesting a reduced density of synaptic vesicles. Thus sax-2 gene function may be necessary for normal synaptogenesis of the ray neurons (Jia, 2006).

Interestingly, a significant percentage of sax-2 animals also exhibit abnormal gonad morphology in both males and hermaphrodites. This phenotype was present for both alleles. In affected animals, the normally two-armed hermaphrodite gonad has either only the anterior arm or only the posterior arm. In the male, the testis fails to extend and forms a large bulb in the middle of the body. These observations suggest that sax-2 may also play a role in gonad morphogenesis (Jia, 2006).

Mammalian Ndr kinase homologs

Human, Drosophila, and C. elegans cDNA clones encoding homologues of Ndr protein kinase have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues has revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr undergoes intramolecular autophosphorylation on serine and threonine residues in vitro but fails to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells results in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division (Millward, 1995).

Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases implicated in the regulation of cell division and cell morphology. This subfamily includes the kinases LATS, Orb6, Cot-1, and Dbf2. Ndr is potently activated when intact cells are treated with okadaic acid, suggesting that Ndr is normally held in a state of low activity by protein phosphatase 2A. The regulatory phosphorylation sites of Ndr protein kinase were mapped; active Ndr is phosphorylated on Ser-281 and Thr-444. Mutation of either site to alanine strongly reduces both basal and okadaic acid-stimulated Ndr activity, while combined mutation abolishes Ndr activity completely. Importantly, each of these sites (and also the surrounding sequences) are conserved in the kinase relatives of Ndr, suggesting a general mechanism of activation for kinases of this subfamily. Ser-281 and Thr-444 are also similar to the regulatory phosphorylation sites in several targets of the phosphoinositide-dependent protein kinase PDK1. However, PDK1 does not appear to function as an upstream kinase for Ndr. Thus, Ndr and its close relatives may operate in a novel signaling pathway downstream of an as-yet-unidentified kinase with specificity similar to, but distinct from, PDK1 (Millward, 1999).

NDR, a nuclear serine/threonine kinase, belongs to the subfamily of Dbf2 kinases that is critical to the morphology and proliferation of cells. The activity of NDR kinase is modulated in a Ca(2+)/S100B-dependent manner by phosphorylation of Ser281 in the catalytic domain and Thr444 in the C-terminal regulatory domain. S100B, a member of the S100 subfamily of EF-hand proteins, binds to a basic/hydrophobic sequence at the junction of the N-terminal regulatory and catalytic domains [NDR(62-87)]. Unlike calmodulin-dependent kinases, regulation of NDR by S100B is not associated with direct autoinhibition of the active site, but rather involves a conformational change in the catalytic domain triggered by Ca(2+)/S100B binding to the junction region. To gain further insight into the mechanism of activation of the kinase, studies have been carried out on Ca(2+)/S100B in complex with the intact N-terminal regulatory domain, NDR(1-87). Multidimensional heteronuclear NMR analysis shows that the binding mode and stoichiometry of a peptide fragment of NDR [NDR(62-87)] is the same as for the intact N-terminal regulatory domain. The solution structure of Ca(2+)/S100B and NDR(62-87) has been determined. One target molecule is found to associate with each subunit of the S100B dimer. The peptide adopts three turns of helix in the bound state, and the complex is stabilized by both hydrophobic and electrostatic interactions. These structural studies, in combination with available biochemical data, have been used to develop a model for calcium-induced activation of NDR kinase by S100B (Bhattacharya, 2003).

Nuclear Dbf2-related (NDR) protein kinases are a family of AGC group kinases that are involved in the regulation of cell division and cell morphology. The human and mouse NDR2, a second mammalian isoform of NDR protein kinase, has been cloned and characterized. NDR1 and NDR2 share 86% amino acid identity and are highly conserved between human and mouse. However, they differ in expression pattern; mouse Ndr1 is expressed mainly in spleen, lung and thymus, whereas mouse Ndr2 shows highest expression in the gastrointestinal tract. NDR2 is potently activated in cells following treatment with the protein phosphatase 2A inhibitor okadaic acid, which also results in phosphorylation on the activation segment residue Ser-282 and the hydrophobic motif residue Thr-442. Ser-282 becomes autophosphorylated in vivo, whereas Thr-442 is targeted by an upstream kinase. This phosphorylation can be mimicked by replacing the hydrophobic motif of NDR2 with a PRK2-derived sequence, resulting in a constitutively active kinase. Similar to NDR1, the autophosphorylation of NDR2 protein kinase is stimulated in vitro by S100B, an EF-hand Ca(2+)-binding protein of the S100 family, suggesting that the two isoforms are regulated by the same mechanisms. Further, a predominant cytoplasmic localization of ectopically expressed NDR2 is demonstrated (Stegert, 2004).

Human NDR1 (nuclear Dbf2-related) is a widely expressed nuclear serine-threonine kinase that has been implicated in cell proliferation and/or tumor progression. The human NDR2 serine-threonine kinase, which shares approximately 87% sequence identity with NDR1, has been characterized. NDR2 is expressed in most human tissues with the highest expression in the thymus. In contrast to NDR1, NDR2 is excluded from the nucleus and exhibits a punctate cytoplasmic distribution. The differential localization of NDR1 and NDR2 suggests that each kinase may serve distinct functions. Thus, to identify proteins that interact with NDR1 or NDR2, epitope-tagged kinases were immunoprecipitated from Jurkat T-cells. Two uncharacterized proteins that are homologous to the Saccharomyces cerevisiae kinase regulators Mob1 and Mob2 were identified. NDR1 and NDR2 partially colocalize with human Mob2 in HeLa cells and the NDR-Mob interactions were confirmed in cell extracts. Interestingly, NDR1 and NDR2 form stable complexes with Mob2, and this association dramatically stimulates NDR1 and NDR2 catalytic activity. In summary, this work identifies a unique class of human kinase-activating subunits that may be functionally analagous to cyclins (Devore, 2004).

NDR (nuclear Dbf2-related) kinase belongs to a family of kinases that is highly conserved throughout the eukaryotic world. NDR is regulated by phosphorylation and by the Ca(2+)-binding protein, S100B. The budding yeast relatives of Homo sapiens NDR, Cbk1, and Dbf2, interact with Mob2 (Mps one binder 2) and Mob1, respectively. This interaction is required for the activity and biological function of these kinases. In this study, hMOB1, the closest relative of yeast Mob1 and Mob2, is shown to stimulate NDR kinase activity and interacts with NDR both in vivo and in vitro. The point mutations of highly conserved residues within the N-terminal domain of NDR reduce NDR kinase activity as well as human MOB1 binding. A novel feature of NDR kinases is an insert within the catalytic domain between subdomains VII and VIII. The amino acid sequence within this insert shows a high basic amino acid content in all of the kinases of the NDR family known to interact with MOB proteins. This sequence is autoinhibitory: the data indicate that the binding of human MOB1 to the N-terminal domain of NDR induces the release of this autoinhibition (Bichsel, 2004).

A novel member of the Ndr subfamily of serine/threonine protein kinases, Ndr2, has been identified as a gene product that is induced in the mouse amygdala during fear memory consolidation, and a possible function of this kinase in neural differentiation was examined. Expression of Ndr2 mRNA was detected in various cortical and subcortical brain regions, as well as non-neuronal tissues. Its expression in the amygdala increases 6 h after Pavlovian fear conditioning training and returns to control levels within 24 h. To study intracellular localization and functions of Ndr2, EGFP::Ndr2 fusion proteins were expressed in rat pheochromocytoma (PC12) cells and acutely isolated cortical neurons, thereby revealing an association of Ndr2 with the actin cytoskeleton in somata, neurites and filopodia, in spines and at sites of cell contact. Co-precipitation and pull-down experiments support this finding. Further evidence for an involvement of Ndr2 in actin-mediated cellular functions comes from the observation of decreased cell spreading and changes in neurite outgrowth that were associated with protein serine phosphorylation in transfected PC12 cells. Together, these data suggest that Ndr2 is an interesting candidate gene for the regulation of structural processes in differentiating and mature neuronal cells (Stork, 2004).

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date revised: 25 July 2023

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