InteractiveFly: GeneBrief

furry: Biological Overview | References

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, but this sequence is not found in Fry. However, 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). 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).

Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons

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, 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. 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, S. pombe, and C. elegans. 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. 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. 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, 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. 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. 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).

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, 2005).

Tricornered-related human and yeast kinases contain a pair of conserved phosphorylation sites corresponding to Ser-281 and Thr-444 in Ndr. 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. 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, 2005).

Autophosphorylation at Ser281 (and to a lesser extent Thr444) in Ndr1 is thought to be important for kinase activity. 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. 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, 2005).

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, 2005 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, 2005).

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. 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, 2005).

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, 2005).

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, 2005).

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, 2005).

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. 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. Given this conservation, it seems likely that Trc also will interact with a Mob (He, 2005).

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, 2005).

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, 2005).

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, 2005).

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).

Search PubMed for articles about Drosophila Furry

Adler, P. N. (2002). Planar signaling and morphogenesis in Drosophila. Dev. Cell 2: 525-535. 1201596

Cong, J., Geng, W., He, B., Liu, J., Charlton, J. and Adler, P.N. (2001). The furry gene of Drosophila is important for maintaining the integrity of cellular extensions during morphogenesis. Development 128: 2793-2802. 11526084

Emoto, K., He, Y., Ye, B., Grueber, W. B., Adler, P. N., Jan, L. Y. and Jan, Y. N. (2004). Control of dendritic branching and tiling by the Tricornered-kinase/Furry signaling pathway in Drosophila sensory neurons. Cell 119: 245-256. 15479641

Fang, X., Lu, Q., Emoto, K. and Adler, P. N. (2010). The Drosophila Fry protein interacts with Trc and is highly mobile in vivo. BMC Dev. Biol. 10: 40. PubMed ID: 20406475

Geng, W., He, B., Wang, M. and Adler, P. N. (2000). The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase. Genetics 156: 1817-1828. 11102376

He, Y., Fang, X., Emoto, K., Jan, Y. N. and Adler, P. N. (2005). The Tricornered Ser/Thr protein kinase is regulated by phosphorylation and interacts with Furry during Drosophila wing hair development. Mol. Biol. Cell. 16(2): 689-700. 15591127

Hirata, D., Kishimoto, N., Suda, M., Sogabe, Y., Nakagawa, S., Yoshida, Y., Sakai, K., Mizunuma, M., Miyakawa, T., Ishiguro, J. and Toda, T. (2002). Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is essential for cell morphogenesis and its mutation induces Wee1-dependent G2 delay. EMBO J. 21: 4863-4874. 12234926

Nelson, B., et al. (2003). Ram: A conserved signaling network that regulates ace2p transcriptional activity and polarized morphogenesis. Mol. Biol. Cell 14: 3782-3803. 12972564

Zallen, J. A., Peckol, E. L., Tobin, D. M. and Bargmann, C. I. (2000). Neuronal cell shape and neurite initiation are regulated by the Ndr kinase SAX-1, a member of the Orb6/COT-1/warts serine/threonine kinase family. Mol. Biol. Cell 11: 3177-3190. 10982409

date revised: 15 December 2011

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