Fasciclin 2


Transcriptional Regulation

BarH1 and BarH2 play essential roles in the formation and specification of the distal leg segments of Drosophila. In early third instar, juxtaposition of Bar-positive and Bar-negative tissues causes central folding that may separate future tarsal segments 2 from 3, while juxtaposition of tissues differentially expressing Bar homeobox genes at later stages gives rise to segmental boundaries of distal tarsi including the tarsus/pretarsus boundary. Tarsus/pretarsus boundary formation requires at least two different Bar functions: early antagonistic interactions with a pretarsus-specific homeobox gene, aristaless, and the subsequent induction of Fas II expression in pretarsus cells abutting tarsal segment 5. Bar homeobox genes are also required for specification of distal tarsi. Bar expression requires Distal-less but not dachshund, while early circular dachshund expression is delimited interiorly by BarH1 and BarH2 (Kojima, 2000).

At late third instar, cells in the distalmost region of leg discs are densely packed at the apical surface and distinguishable from surrounding loosely packed cells. Double-staining with rhodamine-phalloidin and anti-BarH1 antibody reveal that the former corresponds to Bar-negative pretarsus cells, and the latter, Bar-positive tarsus cells. Proximalmost pretarsus cells (border cells) are frequently rectangular in apical shape. Staining for Fasciclin II (Fas II) shows that border cells prominently express Fas II at late third instar. Fas II expression is interrupted by Bar minus clones. Fas II misexpression is induced along Bar-misexpressing presumptive pretarsus, while endogenous Fas II expression is repressed. Thus, Bar upregulates and downregulates Fas II expression in Bar-negative border cells and Bar-positive non-border cells, respectively. It is concluded that Bar is essential for the establishment of the boundary between tarsal segment 5 and pretarsus. Thus, Bar may establish the boundary between the pretarsus (Bar-negative) and tarsal segment 5 (Bar-positive) by regulating the expression of cell adhesion molecules such as Fas II (Kojima, 2000). Interestingly, BarX2, a mouse gene encoding a Bar-related homeodomain protein, has been reported to regulate the expression of Fas II-like cell adhesion molecules (Kojima, 2000 and references therein).

During Drosophila leg development, the distal-most compartment (pretarsus) and its immediate neighbor (tarsal segment 5) are specified by a pretarsus-specific homeobox gene, aristaless, and tarsal-segment-specific Bar homeobox genes, respectively; the pretarsus/tarsal-segment boundary is formed by antagonistic interactions between Bar and pretarsus-specific genes that include aristaless. Drosophila Lim1 is involved in pretarsus specification and boundary formation through its activation of aristaless. Ectopic expression of Lim1 causes aristaless misexpression, while aristaless expression is significantly reduced in Lim1-null mutant clones. Pretarsus Lim1 expression is negatively regulated by Bar and is abolished in leg discs lacking aristaless activity, which is associated with strong Bar misexpression in the presumptive pretarsus. No Lim1 misexpression occurred upon aristaless misexpression. The concerted function of Lim1 and aristaless is required to maintain Fasciclin 2 expression in border cells and form a smooth pretarsus/tarsal-segment boundary. Lim1 is also required for femur, coxa and antennal development (Tsuji, 2000).

Mutually antagonistic interactions between al and Bar are essential for the strict separation of Al and Bar domains, leading to localized Fas2 induction by Bar in border cells. Although the absence of Lim1 shows little Bar misexpression in the pretarsus, increased Bar misexpression in Lim1;al leg discs could indicate the involvement of Lim1 in the repression of Bar expression in the pretarsus. Remarkable decrease in Fas2 expression in putative Lim1;al mutant border cells indicates that Fas2 expression requires al and Lim1 functions, in addition to cell non-autonomous functions of Bar. Lim1 may be involved in pretarsus specification and boundary formation only through its activation of al. Low al expression in Lim1 single mutants may still be sufficient for maintaining the normal expression of Bar and Fas2, but with further reduction in al expression in Lim1;al double mutants, Bar misexpression and loss of Fas2 expression may result. Alternatively, Lim1 may act independently of al, and simultaneous reduction in al and Lim1 expression may cause Bar misexpression and reduction of Fas2 expression in the double mutants. These considerations are not mutually exclusive (Tsuji, 2000).

The gene spalt is expressed in the embryonic central nervous system of Drosophila but its function in this tissue is still unknown. To investigate this question, a combination of techniques was used to analyse spalt mutant embryos. Electron microscopy shows that in the absence of Spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy has revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments have each confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the ß-catenin Armadillo, its binding partner N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organization of the central nervous system (Cantera, 2002).

A possible interpretation of sal phenotype would be that components of cell adhesion are seriously compromised in the CNS of sal embryos during early stage 16. To test this hypothesis specific antibodies and laser confocal microscopy were used to survey the expression of molecules known to be important for cell adhesion in embryonic CNS at early stage 16. All the markers are detectably expressed in Df(2L)32FP-5;sal445 mutant embryos at both stages, and their spatial patterns of expression in the CNS are normal, showing that sal is not essential for any of these proteins to be expressed. However, the quantification of fluorescence intensity revealed that most markers were present in abnormally high or low levels. In transheterozygous Df(2L)32FP-5;sal445 mutants at early stage 16, when the strong transmission electron microscopy TEM phenotype is manifest, lower fluorescence levels were measured for Armadillo, N-Cadherin, Neuroglian, Fasciclin 2 and Fasciclin 3; higher fluorescence levels were measured for Notch; and levels similar to wild type for Neurotactin, Neurexin IV and Faint Sausage. Comparison between wild-type, heterozygous and null sal mutant embryos revealed a stepwise decrease in the fluorescence levels for Armadillo and N-Cadherin, indicating that the effect of the mutation is dominant (Cantera, 2002).

Fluorescence levels were measured at the stage when the TEM phenotype is reverted (stage 17). The wild-type fluorescence for the three markers studied in this regard (Armadillo, Fasciclin 2, Neuroglian) changes between early stage 16 and stage 17, indicating that during this short developmental interval the levels of cell adhesion proteins are regulated. Relative to these new wild-type levels, the three proteins that are not affected during the expression of the TEM phenotype (Neurotactin, Neurexin IV and Faint Sausage) remain normal in the mutant. The levels of Notch switch from abnormally high to slightly lower than normal. All other markers still exhibit lower-than-normal fluorescence levels, with the exception of N-Cadherin, which exhibits a partial recovery. Taken together, these data led to the conclusions that the expression of sal is necessary to maintain correct dynamic levels of several adhesion molecules in the CNS and that sal exerts this function in a persistent and dominant fashion (Cantera, 2002).

The rapid recovery of sal CNS during the course of stage 16 could be explained by the robustness inherent to a system in which adhesion is mediated by a combination of proteins and the possible compensatory effect mediated by upregulation of other members of the system. However, an alternative view is proposed. The ultrastructural recovery may as well reflect the normal dynamics of combinations of adhesion proteins expressed successively along embryonic development. From this point of view, the rapid recovery from the adhesion phenotype will reflect the normal transition between two particular combinations of adhesion proteins expressed at early or late stage 16. For this to be valid, the expression levels of several adhesion proteins must change along this interval during normal development. Interestingly, the data do support this possibility, since the fluorescence levels for Armadillo, Fasciclin 2 and Neuroglian change between stages 16 and 17 in wild-type CNS. Whether sal is required for the regulation of a combination of cell adhesion and cytoskeletal proteins at a particular developmental stage could be tested by deleting the expression of Sal exclusively in CNS tissue within short developmental intervals. This approach could now be possible using techniques based on combinations of the GAL4-UAS system and RNA interference (Cantera, 2002).

In an attempt to identify gene targets of ash2, an expression analysis was performed by using cDNA microarrays. Genes involved in cell cycle, cell proliferation, and cell adhesion are among these targets, and some of them are validated by functional and expression studies. Even though trithorax proteins act by modulating chromatin structure at particular chromosomal locations, evidence of physical aggregation of ash2-regulated genes has not been found. This work represents the first microarray analysis of a trithorax-group gene (Beltran, 2003).

Genes involved in cell adhesion and/or development of the neural system (i.e., FasII, mfas, Ama, Lac, and shg) are two of the main classes regulated by ash2. Focus was placed on the up-regulated gene FasII, and clonal analysis was performed on a Minute background, to assess whether the behavior of the FasII transcript observed with this ash2 mutant was also kept at the protein level in wing imaginal discs. Homozygous mutant cells show a clear up-regulation of FASII, mainly in the wing pouch area further away from the dorsoventral margin, where FASII was found to be very slightly expressed in WT wing discs. The up-regulation of FasII and other cell adhesion molecules like mfas, together with the up-regulation of the transcription factor vri, could explain some of the phenotypes previously found by clonal analysis, such as disruption of vein-intervein patterning, because it is known that preferential accumulation of specific adhesion molecules characterizes the final stages of vein differentiation. Furthermore, because FASII is involved in the development of the neural system, its pattern of expression in ash2I1 mutant brains was compared with that of WT; they present a distorted phenotype in the optic lobes as well as fasciculation defects in the ventral ganglion, a process in which FASII plays a central role. A gain-of-function screen (Kraut, 2001) identified ash2 and FasII as genes involved in the development of the neural system, further supporting a relationship between them (Beltran, 2003).

Targets of Activity

FAS2 loss of function mutants fail to express the achaete proneural gene in antennal imaginal disc precursors of mechanosensory neurons, and fail to express atonal in antennal imaginal disc precursors of ocellar photoreceptors (Garcia-Alonso, 1995). Mutations in the Abelson tyrosine kinase gene show dominant interactions with Fas2 mutations, suggesting that ABL and FAS2 function in a signaling pathway that controls proneural gene expression (Garcia-Alonso, 1995).

Mutations in sec15 cause defects in synaptic specificity, axon targeting and localization of axon guidance components

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized exocytosis/secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. An exocyst component, sec15, has been isolated in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. The data suggest a role for Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions (Mehta, 2005).

Elevated levels of chaoptin in photoreceptor terminals have been described for another vesicle-trafficking mutant, the vesicle-SNARE neuronal-synaptobrevin (n-syb). This mutant also exhibits neuronal targeting defects. This observation raises the possibility that vesicle-dependent trafficking of transmembrane or other signaling molecules might be responsible for the neuronal targeting defects of sec15 mutant photoreceptors. Recently, Zhang (2004) identified Rab11 as an interacting partner of Sec15 in mammalian cell culture and proposed that Sec15 is an effector for some but not all Rabs. Indeed, an accumulation or upregulation of Rab11 immunoreactivity was seen in sec15 mutant photoreceptors, consistent with Rab11-positive vesicles failing to fuse with their target sites. To further test this hypothesis, the localization of cell adhesion and signaling molecules was examined in mutant photoreceptor cell bodies as well as terminals during photoreceptor development, precisely when target selection and cartridge formation occur (between P + 5% to P + 40% referring to time after pupation). Proteins were examined that have either been shown to be required for photoreceptor target selection, such as Dlar, N-cadherin, flamingo, and IrreC-rst, or that are likely to be required, based on work in other systems, such as Armadillo, Chaoptin, and Fasciclin II (Mehta, 2005).

Fasciclin II (Fas2) localization was examined in sec15 mutant photoreceptors, since chaoptin upregulation coincides with elevated levels of Fas2 in n-syb mutant photoreceptors. Fas2 appears to be present in aggregates in sec15 mutant photoreceptor cell bodies at P + 20%, in contrast to wild-type photoreceptors. In addition, the neuronal connections of the cell bodies exhibit Fas2 aggregated along the length of the mutant axons. Similarly, overexpression of Fas2 in photoreceptors causes neuronal targeting defects between P + 20% and P + 40%. In contrast to n-syb, however, no elevated levels of Fas2 are observed later in development. Hence, the data suggest that an aberrant localization of Fas2 in a specific developmental time window may at least partially underlie the observed phenotypes (Mehta, 2005).

Similar mislocalization phenotypes in photoreceptor cell bodies were also observed for other cell adhesion molecules such as Dlar and IrreC-rst during the developmental time window of photoreceptor target selection. Dlar is normally restricted apically in developing wild-type photoreceptors, at the center of the ommatidial array. In sec15 mutant photoreceptors it appears much more randomly distributed, such that a basal optical section through the eye shows Dlar at higher levels in mutant ommatidia. Although these results show mislocalization of cell adhesion molecules in the correct cell at the time when they are known to be required for proper target selection, no obvious defects were detected in the localization of Dlar or IrreC-rst in the developing lamina. This leaves open the question of whether mislocalization of Dlar and IrreC-rst beyond the resolution limit of confocal microscopy additionally contributes to the observed targeting defects (Mehta, 2005).

In vertebrates, Lar is known to localize to adherens junctions. Hence, a possible explanation for the mislocalization of Fas2, IrreC-rst, and Dlar in mutant photoreceptor cell bodies is a defect of adherens junctions. The subcellular localization of the adherens junction markers N-cadherin and armadillo was examined in the cell bodies as well as the terminals of mutant photoreceptors, but no mislocalization of N-cadherin was detected in either compartment. However, armadillo displayed localization defects selectively in the developing lamina, but not the photoreceptor cell bodies. Several other cell adhesion and signaling molecules, including flamingo, Crumbs, and Bazooka, were examined, all of which did not display aberrant localization at the level of light microscopy. It is concluded conclude that a specific subset of proteins is mislocalized in sec15 mutants (Mehta, 2005).

Protein Interactions

The cell adhesion molecule Fasciclin II (FASII) is involved in synapse development and plasticity. Proper localization of FASII at type I glutamatergic synapses of the Drosophila neuromuscular junction is mediated by binding between the intracellular tSXV bearing C-terminal tail of FASII and the PDZ1-2 domains of Discs-Large (DLG). Moreover, mutations in fasII and/or dlg have similar effects on presynaptic ultrastructure, suggesting their functional involvement in a common developmental pathway. DLG can directly mediate a biochemical complex and a macroscopic cluster of FASII and Shaker K+ channels in heterologous cells. These results indicate a central role for DLG in the structural organization and downstream signaling mechanisms of cell adhesion molecules and ion channels at synapses (Thomas, 1997).

Both the Fasciclin II (Fas II) cell adhesion molecule and the Shaker potassium channel are localized at the Drosophila neuromuscular junction, where they function in the growth and plasticity of the synapse. Both proteins contain -S/T-X-V sequences at their C termini, identifying them as proteins that could interact with PDZ domains. The GAL4-UAS system was used to drive expression of the chimeric proteins CD8-Fas II and CD8-Shaker. The C-terminal sequences of both Fas II and Shaker are necessary and sufficient to drive the synaptic localization of a heterologous protein. The PDZ-containing protein Discs-Large (Dlg) controls the localization of Fas II and Shaker, most likely through a direct interaction with their C-terminal amino acids. Transient expression studies show that the pathway these proteins take to the synapse involves either an active clustering or a selective stabilization in the synaptic membrane. Following a pulse of protein expression, the CD8-Shaker protein is initially distibuted uniformly on the muscle membrane, followed by concentration at the synapse. Thus the results are consistent with a model of uniform membrane targeting followed by active clustering or selective retention at the synapse. If interactions with Dlg stabilize FasII and Shaker at the synapse, one mechanism to regulate the levels of Fas II and Shaker could be through regulation of these interactions. Such a regulation has been demonstrated for the mammalian inwardly rectifying potassium channel Kir2.3, in which phosphorylation of its PDZ-interaction motif by cAMP-dependent protein kinase (PKA) inhibits its binding to PSD-95. A similar mechanism to regulate interaction between Dlg and FasII or Shaker could provide an activity-dependent mechanism for regulation of the levels of these proteins at the synapse (Zito, 1997).

Mutations in rho-type guanine exchange factor (rt/GEF), also called dpix, were recovered from a large-scale screen in Drosophila for genes that control synaptic structure. dPix/rtGEF is homologous to mammalian Pix. dPix plays a major role in regulating postsynaptic structure and protein localization at the Drosophila glutamatergic neuromuscular junction. dpix mutations lead to decreased synaptic levels of the PDZ protein Discs large, the cell adhesion molecule Fas II, and the glutamate receptor subunit GluRIIA, and to a complete reduction of the serine/threonine kinase Pak and the subsynaptic reticulum. The electrophysiology of these mutant synapses is nearly normal. Many, but not all, dpix defects are mediated through dPak, a member of the family of Cdc42/Rac1-activated kinases. Direct interaction of mammalian Pix with Pak has been detected. Thus, a Rho-type GEF (Pix) and Rho-type effector kinase (Pak) regulate postsynaptic structure (Parnas, 2001).

In mammals, the Pix family contains two members: alphaPix (Cool-2) and ßPix (Cool-1). Pix has an SH3 domain, a DBL-homology GEF domain, and a pleckstrin homology domain. The Cool (for cloned-out of library)/Pix (for PAK-interactive exchange factor) proteins directly bind to members of the PAK family of serine/threonine kinases and regulate their activity. In Drosophila, dPix is localized to the PSD: dpix mutations lead to the loss of synaptic Pak kinase. Paks are a family of Cdc42/Rac1-activated serine/threonine kinases important in regulating actin-containing structures. In the fly NMJ, Pak kinase is localized to the PSD. In mammals, Pak is recruited to focal complexes in a Cdc42-, Rac1-, and Pix- dependent manner (Parnas, 2001).

In addition to the absence of Pak kinase at the synapse, dpix mutations lead to the decrease in synaptic levels of the PDZ protein Discs-large (Dlg), the cell adhesion molecule Fasciclin II (Fas II), the glutamate receptor (GluR) subunit GluRIIA, and to the elimination of the subsynaptic reticulum (SSR). In Drosophila, the PSD-95 homolog Dlg has been shown to be directly responsible for the clustering of the Shaker potassium channel and to partially control the clustering of the cell adhesion molecule Fas II to the NMJ. Many, but not all, dpix defects are mediated through Pak kinase. Thus, the data suggest a pathway for synaptic clustering from dPix to Pak kinase to Dlg to Shaker and to Fas II (Parnas, 2001).

The dpix phenotype is consistent with at least two functions at the postsynaptic compartment: targeting and stabilization of postsynaptic components. In dpix mutants, Pak kinase is completely missing from the synapse. Since Pix is known to directly interact with Pak in mammals and target it to focal complexes, the data best fit with the model in which dPix targets Pak kinase to the synapse via a direct interaction. Furthermore, overexpressing either Pak kinase or a membrane-tethered gain-of-function form of Pak kinase does not result in any accumulation of Pak kinase at the synapse. Still, it is possible that Pak kinase is targeted to the synapse via a different mechanism and fails to stabilize in dpix mutants (Parnas, 2001).

In contrast to Pak kinase, Dlg and GluRIIA are not completely eliminated from the synapse in dpix mutants, but rather, their levels are reduced. In the case of Dlg, its localization pattern is also disrupted, indicating that dPix controls the postsynaptic targeting of Dlg at least to some extent, as well as its stabilization at the synapse. The localization pattern of GluRIIA (in subsynaptic domains opposite active zones) is intact. Thus, dPix is not necessary for the synaptic targeting of GluRIIA per se, but rather, it is important for maintenance of its levels and/or stabilization (Parnas, 2001).

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (Dlg), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (Fas2) and the position specific (PS) integrin receptors, including ßPS (Myospheroid) and its alpha partners (alphaPS1, alphaPS2, PS3/alpha Volado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that Dlg and Fas2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. ßPS integrin physically associates with the synaptic complex anchored by the Dlg scaffolding protein, which contains CaMKII and Fas2. This study demonstrates an alteration of the Fas2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory ßPS integrin mutations increase the expression and synaptic localization of Fas2. Synaptic structural defects in ßPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of Fas2 (distal in pathway). These studies demonstrate that ßPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology (Beumer, 2002).

ßPS integrin is a transmembrane receptor present in both pre- and post-synaptic membranes at the larval NMJ. Dlg is a synaptic scaffolding protein associated with both pre-and post-synaptic membranes and is also involved in the regulation of synaptic morphology, through the localization of diverse transmembrane proteins (including Fas2). Studies were undertaken to determine whether ßPS integrin associates with the DLG complex to tie together these disparate receptor components in a common molecular machine. In confocal analyses, ßPS integrin and Dlg co-localize at the larval NMJ. Dlg clearly has less extensive expression, more tightly localized at NMJ boutons, whereas ßPS is more extensive through the subsynaptic reticulum (SSR) and also localized at extrasynaptic sites in the muscle, including the muscle sarcomere and attachment sites. However, both proteins are most intensively expressed at the NMJ presynaptic/postsynaptic interface, where they co-localize. Therefore, tests were performed to determine if ßPS integrins form part of the synaptic complex linked by Dlg. Protein immunoprecipitation assays were performed using rabbit anti-DLG to probe Oregon R head extracts. Dlg antibodies clearly co-immunoprecipitate Dlg and ßPS integrin protein, consistent with co-localization observed in confocal analysis. Inspection of the ratio of both bound proteins and proteins not bound to beads (immunoprecipitation versus flow through lanes) indicates that a large portion of the ßPS integrin protein associates with a complex containing Dlg. The fact that ßPS was found to co-immunoprecipitate with the complex mediated by Dlg provides support for integrins existing in a synaptic complex with Fas2 and CaMKII at the synapse (Beumer, 2002).

To assay the mys requirement in synaptic development comprehensively, two regulatory alleles of mys with opposing structural phenotypes were compared and contrasted: mysb9, which causes NMJ undergrowth, and mysts1, which causes NMJ overgrowth. In both mutants, a correlation was observed between the morphological alterations and synaptic function, in contrast to the homeostasis seen in Fas2 mutant synapses but consistent with the parallel alterations seen in CaMKII-inhibited synapses. However, the correlation between synaptic structural and functional alterations in mys mutants is not striking, and it is clear that integrins primarily mediate architectural regulation. Therefore, the focus in this study was exclusively on the mechanism(s) by which integrins modulate NMJ structural development. This article presents molecular and genetic evidence that strongly support the hypothesis that synaptic integrin receptors containing ßPS modulate CaMKII activation on both sides of the synaptic cleft and, through CaMKII, control both the expression and the synaptic localization of Fas2 at the synapse. The primary experimental results supporting these conclusions are detailed below (Beumer, 2002).

(1) Transgenic expression of CaMKII is sufficient to completely rescue all synaptic structure defects in mys mutants. Genetically increasing CaMKII in postsynaptic muscle, but not presynaptic neuron, completely rescues the structural undergrowth of the mysb9 integrin mutant, whereas it is necessary to elevate CaMKII both pre- and post-synaptically to rescue structural overgrowth in the mysts1 mutant. These results are consistent with the postsynaptic mislocalization of ßPS integrin in the mysb9 mutant, as opposed to the global loss of synaptic integrin in the mysts1 mutant. These data indicate that coordinate regulation of CaMKII in the muscle and motoneuron is necessary for proper development of synaptic architecture (Beumer, 2002).

(2) At the distal end of the cascade, both the expression and synaptic localization of Fas2 are increased in mys mutants, although the extent of Fas2 misregulation is significantly different between the two regulatory mutants. Importantly, both the NMJ overgrowth (mysts1) and undergrowth (mysb9) phenotypes are rescued towards wild-type structure by genetically reducing the amount of Fas2 available at the synapse to near normal levels. In mysts1 mutants, correcting for Fas2 level suppresses the synaptic overgrowth, while in mysb9 mutants, correcting the Fas2 level suppresses the synaptic undergrowth. These results support the conclusion that Fas2 is centrally involved in mediating synaptic growth, but suggest that the Fas2-mediated mechanism is more complex than previously thought (Beumer, 2002).

The results demonstrate that integrins regulate morphological growth at the postembryonic NMJ through activation of CaMKII in both pre- and post-synaptic compartments. One important target of CaMKII is Fas2 and it is clear that regulation of Fas2 expression and localization is an important component of integrin regulation. However, the modulation of Fas2 levels alone is unlikely to account fully for the alterations in synaptic architecture in integrin mutants. In particular, both mysts1 and mysb9 upregulate synaptic Fas2 levels, albeit to different degrees, yet show opposite alterations in synaptic growth. Moreover, reducing Fas2 levels rescues both under- and overgrowth defects, but the rescue is not perfect (Beumer, 2002).

Precise control of Fas2 levels finely tunes morphological development at the NMJ. Different degrees of reduced Fas2 expression can either facilitate or inhibit the growth/maintenance of the NMJ, and reduced Fas2 expression has been demonstrated in other overgrowth mutants. However, overexpression of Fas2 in specific muscles drives increased NMJ elaboration/bouton differentiation and selective preference for a high-expressing muscle over a low-expressing muscle. How can this complexity be interpreted? One likely explanation is interaction between Fas2 and other developmental pathways regulated by CaMKII. To date, the only known Fas2-independent regulation of synaptic morphology involves a deubiquitinating protease encoded by fat facets (faf), and a putative ubiquitin ligase encoded by highwire (hiw), which have been shown to work together to modulate the degree of NMJ growth. Loss-of-function hiw mutants display NMJ structural overgrowth, importantly without a concomitant decrease in Fas2. Indeed, overexpression of Fas2 cannot suppress the overgrowth seen in hiw mutants. These observations are consistent with the overgrowth combined with overexpression of Fas2 observed in mysts1 mutants in this study. However, any putative interaction between Fas2-dependent and -independent mechanisms of morphological growth regulation at the Drosophila NMJ synapse remain to be elucidated. It is expected that further study will reveal an additional structural control mechanism regulated by CaMKII, acting in parallel to and interacting with Fas2. The ubiquination mechanism discussed here is one candidate mechanism, but as CaMKII is known to have many synaptic targets, it is clearly not the only candidate (Beumer, 2002).

In summary, it is concluded that architectural developmental defects observed in NMJ synapses mutant for ßPS integrin are due to the loss of the ability to regulate synaptic CaMKII properly. One function of CaMKII is to phosphorylate the scaffolding protein Dlg, and thus regulate the proteins synaptically localized by this scaffold. Fas2 is the central known output of this regulatory cascade. Loss of this regulation is central to the mys mutant defects in the postembryonic elaboration of NMJ structure. It is clear from this study, however, that regulation of Fas2 localization via CaMKII is only one facet of how integrins function at the synapse (Beumer, 2002).

Regulation of Fasciclin II and synaptic terminal development by the splicing factor Beag

Pre-mRNA alternative splicing is an important mechanism for the generation of synaptic protein diversity, but few factors governing this process have been identified. From a screen for Drosophila mutants with aberrant synaptic development, beag, a mutant with fewer synaptic boutons and decreased neurotransmitter release, was identified. Beag encodes a spliceosomal protein similar to splicing factors in humans and Caenorhabditis elegans. Both beag mutants and mutants of an interacting gene dsmu1 have changes in the synaptic levels of specific splice isoforms of Fasciclin II (FasII), the Drosophila ortholog of neural cell adhesion molecule. Restoration of one splice isoform of FasII can rescue synaptic morphology in beag mutants while expression of other isoforms cannot. It was further demonstrated that this FasII isoform has unique functions in synaptic development independent of transsynaptic adhesion. beag and dsmu1 mutants demonstrate an essential role for these previously uncharacterized splicing factors in the regulation of synapse development and function (Beck, 2012).

Although important roles of FasII in synapse growth and plasticity are well established, the expression and function of individual Drosophila FasII splice isoforms had not previously been systematically analyzed. This study demonstrates that the transmembrane isoforms of FasII are essential for synaptic development. In contrast, FasII-C cannot rescue synaptic development in fasII mutants. These results are consistent with prior results showing that expression of transgenic FasII-A-PEST+ (containing an exon that encodes a PEST domain), in contrast to FasII-C, can alter synapse function in the CNS. Previous studies have also shown that reducing levels of all FasII isoforms by 90%-100% causes a large decrease in NMJ bouton number. However studies of more modest reductions of fasII in heterozygote mutants have been inconsistent. This study finda that the bouton number is unchanged compared with wild type in larvae heterozygous for either of two fasII alleles or for a deficiency that removes the entire fasII gene. It was confirmed that these fasII manipulations have a ~50% decrease in synaptic FasII levels. The contrast between fasII heterozygotes and beag mutants suggests that a change in the relative FasII isoform levels and the presynaptic to postsynaptic FasII ratio, rather than a uniform reduction in FasII levels, can produce aberrant synaptic morphology (Beck, 2012).

The importance for normal NMJ development of the relative levels and presynaptic and postsynaptic distribution of individual FasII isoforms is supported by several lines of evidence. Simultaneous presynaptic and postsynaptic overexpression of FasII-A-PEST+ was shown previously to induce synaptic overgrowth, while in contrast this study found that solely presynaptic overexpression of FasII-A-PEST+ does not alter NMJ morphology. In addition, this study showed that neuronal overexpression of FasII-A-PEST− can induce synaptic overgrowth while overexpression of FasII-C, like FasII-A-PEST+, does not, revealing unique effects of each isoform on synapse growth. Furthermore, expression of FasII-A-PEST+ but not FasII-A-PEST− can rescue the beag NMJ morphology defects. These data suggest that these two isoforms have distinct activities and that a disruption in the presynaptic balance of transmembrane FasII isoforms at the NMJ in beag mutants leads to a decrease in bouton number, even while overall total synaptic FasII levels are maintained (Beck, 2012).

These results also show that the intracellular domain of FasII-A-PEST+ alone is sufficient to rescue beag mutant synapse morphology defects, revealing an important function for this domain that is independent of transynaptic adhesion. It is noteworthy that differences have been described in the downstream signaling of transmembrane isoforms of mammalian NCAM. For example, the src family kinase Fyn interacts with NCAM 140, but not 180, and this interaction is essential for neurite outgrowth. In contrast, the scaffolding protein Spectrin has a higher affinity for NCAM 180 than 140, which is required for NCAM-induced recruitment of synaptic proteins to the postsynaptic density. Little is known about the cytoplasmic signaling functions of either of the Drosophila FasII transmembrane isoforms. Comparison of NCAM 180 and 140 with FasII-A-PEST+ and PEST− shows little homology between their intracellular domains. The full cytoplasmic domain of FasII-A-PEST+ is sufficient for postsynaptic localization at the NMJ, and this study shows that this domain is also sufficient for NMJ presynaptic localization. The cytoplasmic domains of both FasII-A isoforms contain a C-terminal PDZ-binding sequence. This domain is not absolutely required for presynaptic localization of FasII-A-PEST+ and is also not required for rescue of beag mutants by FasII-A-PEST+. The only difference between FasII-A-PEST+ and FasII-A-PEST− is one exon encoding 29 aa within the intracellular region. These 29 aa contain a PEST sequence (rich in proline, glutamic acid, serine, and threonine residues) that could preferentially target FasII-A-PEST+ for proteasomal degradation. It is difficult, however, to relate enhanced degradation to the specific synaptic activity of FasII-A-PEST+ in beag mutants. Nonetheless, analysis of beag and dsmu1 mutants has revealed unique roles for transmembrane FasII isoforms at the synapse and an important future goal will be to determine the nature of these distinctions (Beck, 2012).

Tao controls epithelial morphogenesis by promoting Fasciclin 2 endocytosis

Regulation of epithelial cell shape, for example, changes in relative sizes of apical, basal, and lateral membranes, is a key mechanism driving morphogenesis. However, it is unclear how epithelial cells control the size of their membranes. In the epithelium of the Drosophila melanogaster ovary, cuboidal precursor cells transform into a squamous epithelium through a process that involves lateral membrane shortening coupled to apical membrane extension. This paper reports a mutation in the gene Tao, which resulted in the loss of this cuboidal to squamous transition. The inability of Tao mutant cells to shorten their membranes was caused by the accumulation of the cell adhesion molecule Fasciclin 2, the Drosophila N-CAM (neural cell adhesion molecule) homologue. Fasciclin 2 accumulation at the lateral membrane of Tao mutant cells prevented membrane shrinking and thereby inhibited morphogenesis. In wild-type cells, Tao initiated morphogenesis by promoting Fasciclin 2 endocytosis at the lateral membrane. Thus, this study identified a mechanism controlling the morphogenesis of a squamous epithelium (Gomez, 2012).

This study reports a novel mechanism regulating the morphogenesis of a squamous epithelium. The data show that timely controlled removal of the adhesion molecule Fas2 from the lateral membrane is critical for the stretching of cuboidal precursor cells. In this process, Tao initiates morphogenesis by promoting Fas2 endocytosis from the lateral membrane, which reduces adhesive forces and thereby provides plasticity for the cuboidal to squamous cell shape change. The results are consistent with a model in which homophilic Fas2 interactions act as a glue preventing shortening of the membrane (Gomez, 2012).

Tao function is essential for the process of stretching of epithelial cells. However, the data indicate that Tao uses none of its known pathways to control morphogenesis. Similarly, no evidence was found that Tao regulates cell stretching via the cytoskeleton. Moreover, the results argue against a role for Tao in transcriptional repression of Fas2, indicating a function in posttranslational regulation. Indeed, quantitative analysis of Fas2 vesicles in wild-type follicles indicates that membrane shrinking is initiated by enhanced endocytosis. Tao mutant cells have a reduced ability to internalize Fas2 vesicles, contain tubular Fas2 structures typical for endocytosis defects, and show fewer endocytotic Fas2 vesicles in the cytoplasm. This indicates that the overaccumulation of Fas2 at the lateral membrane is caused by defective Fas2 internalization, suggesting a role for Tao in promoting endocytosis at the lateral membrane. A similar function of Tao in triggering endocytosis of homophilic adhesion proteins has been identified in cultured hippocampal neurons (Yasuda, 2007). In contrast to the follicular epithelium, however, Tao is acting via the p38 MAP signaling pathway in this cell type (Gomez, 2012).

Tao promotes Fas2 endocytosis not only in the anterior squamous epithelium but acts also in the posterior epithelium, whose cells become columnar by elongating their lateral membrane. Although it is surprising that these cells down-regulate a protein that is able to induce cell elongation, it appears that during stage 7/8, all epithelial cells clear their lateral membrane from Fas2 to allow morphogenesis. This suggests that other proteins than Fas2 are responsible for cell elongation in the posterior epithelium (Gomez, 2012).

Is Tao an endocytosis promoter specific for the lateral membrane? Endocytosis is only impaired but not blocked in Tao mutant cells, as they are still able to internalize reduced levels of dextran and Fas2. Consistent with this, the Tao phenotype differs from mutants affecting endocytosis in general. Although cell clones mutant for Rab5, the syntaxin family member Avalanche, and Dynamin/Shibire show severe polarity defects and massive overproliferation in the follicular epithelium, the Tao phenotype is restricted to overaccumulation of membrane proteins and cell shape defects. It is therefore concluded that Tao is not a general endocytosis factor but enhances endocytosis at a critical developmental stage to allow epithelial morphogenesis (Gomez, 2012).

This timely controlled requirement of Tao function raises the question whether Tao expression starts just before the onset of morphogenesis or whether the protein is present also in early stages and only activated when Fas2 has to be removed from the lateral membrane. Endogenous Tao protein could not be detected, as antibodies showed no specific signal in the follicular epithelium. Moreover, analysis of Tao mRNA expression does not allow unambiguous conclusions about the onset of Tao transcription in the follicular epithelium as a strong signal from the germline cyst covers the signal in the surrounding epithelium (Gomez, 2012).

Using a HA fusion protein, Tao protein was detected at the lateral membrane and in the basolateral cytoplasm of the follicular epithelium. Interestingly, Tao protein accumulation appears as a gradient with the lowest levels in the apical and the highest levels in the basal region of the follicular epithelium. Since rescue experiments demonstrate the functionality of the fusion protein, a gradient of endogenous Tao activity might also exist. This raises the possibility that Tao promotes endocytosis more strongly in basal regions of the lateral membrane. It is speculated that there is a switch in Tao activity at the border between the lateral and the basal membrane and that the basal membrane domain is protected from Tao function. A spatial restriction of Tao function to the lateral membrane could be achieved by the localization of a yet unidentified target of Tao kinase. Such a Tao mediator could exclusively localize to the lateral membrane (Gomez, 2012).

The finding that not only Fas2 but also apical and apicolateral proteins, such as DE-cadherin and Crb, overaccumulate in Tao mutants might argue against a restriction of Tao function to the lateral membrane. However, in the light of the dramatic change in the geometry of stretching cells, an alternative explanation for the accumulation of these proteins seems more likely. Squamous cell morphogenesis involves a 15-fold surface expansion, which is accompanied by a strong increase in the amounts of proteins determining the apical membrane and forming the adherens junctions. Interestingly, also, posterior cells expand their surface, albeit in less dramatic manner. Tao mutant cells are unable to increase their surface area, but they nevertheless produce the same amount of apical and apicolateral proteins, which then concentrate within a restricted area. It is therefore proposed that the accumulation of Arm, DE-cadherin, and apical proteins in Tao mutant cells is not caused by impaired endocytosis but a result of the inability of these cells to increase their surface area. Consistent with this, it was found that cells whose stretching is prevented by Fas2 overexpression (and which are wild type for Tao function) also concentrate Arm within their restricted apical surface (Gomez, 2012).

A membrane domain-specific endocytosis function for Tao is also supported by experiments, which deplete Fas2 in Tao mutant cells. If Tao would act all along the plasma membrane, Fas2 removal from the lateral membrane should selectively rescue the lateral defect (i.e., Fas2 accumulation) but should not suppress the accumulation of apical and apicolateral proteins (e.g., Arm). However, Fas2 Tao double mutant cells do suppress Arm accumulation. Thus, the primary defect of Tao mutants is Fas2 accumulation, which prevents stretching and expansion of the apical surface. The concentration of Arm is only a secondary effect of the cell shape defect. In conclusion, the data favor a model in which Tao promotes endocytosis specifically at the lateral membrane to relieve Fas2-mediated cell adhesion (Gomez, 2012).

The identification of a mechanism initiating epithelial morphogenesis in the follicular epithelium allows the current model of squamous cell morphogenesis in the follicular epithelium to be updaed. Before morphogenesis, all epithelial cells are cuboidal and adhere apicolaterally via DE-cadherin and more basolaterally via Fas2 interactions. In this period, Fas2 is endocytosed and recycled back to the membrane. Morphogenesis is primed by the Tao kinase, which promotes the endocytosis of Fas2 vesicles. It is assumed that a large proportion of the Fas2 vesicles enter the lysosomal pathway, leading to Fas2 degradation. The removal of Fas2 from the lateral membrane is a critical first step, which allows morphogenesis to occur. Once Fas2 has disappeared, the cuboidal shape of the epithelial cells is solely stabilized by the zonula adherens. Cell flattening requires a tensile force, and morphometric analysis indicates that this force originates from the continuously growing germline cyst, which stretches cells in the anterior epithelium. Flattening correlates well with the local breakdown of the zonula adherens, suggesting an important role for adherens junctions remodeling in cell stretchin. Although the exact molecular mechanism controlling remodeling has still to be elucidated, the data indicate that before cell stretching, lateral cell adhesion has to be reduced to provide morphogenetic plasticity to the lateral membrane (Gomez, 2012).

Given the critical role of Fas2 down-regulation for epithelial stretching, it is counterintuitive that Fas2 mutant cells undergo normal morphogenesis. However, a similar scenario has been shown for the morphogenesis of the peripheral nervous system (PNS) in Drosophila. Here, Fas2 has to be endocytosed in axons to allow glia cell migration. Despite this important function for Fas2 down-regulation, Fas2 mutants show no gross morphological defects in the PNS. Thus, although Fas2 down-regulation is critical for epithelial and PNS morphogenesis, both tissues can compensate for the complete loss of the protein. It is proposed that Fas2 helps to stabilize a morphological state and allows development to proceed only after its timely controlled endocytosis. This mechanism guarantees the correct timing of morphogenesis and thereby helps to coordinate the development of different cell types and tissues. In the follicular epithelium, Fas2 might collaborate with the zonula adherens in counteracting the pressure from the growing germline cyst. As a result, the cuboidal cell shape is stabilized and premature, and uncontrolled stretching is prevented. Subsequently, Fas2 internalization overcomes this control mechanism, and epithelial morphogenesis proceeds (Gomez, 2012).

Fasciclin 2: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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