BIOLOGICAL OVERVIEW

One of the main ways that signals are transduced between proteins is by the addition or removal of a phosphate group from the amino acid tyrosine. The addition of a phosphate group is carried out by kinases while the removal of phosphate is carried out by phosphatases. Leukocyte-antigen-related-like/Dlar influences the process of axon guidance by removal of phosphate groups from phosphotyrosine residues. Examples of receptors that act as kinases include the EGF-R and Sevenless. Each of these interact with an extracellular ligand and transduce the extracellular signal to the inside of the cell resulting in the activation of the kinase associated with the intracellular domain of the receptor.

Receptor tyrosine phosphatases likewise receive extracellular signals (in most cases from unknown ligands), and transduce those signals to the inside of the cell, activating the phosphatase enzymatic function associated with the intracellular domain of the receptor. Phosphatases remove phosphate groups from proteins, instigating a signal that can be passed from protein to protein, ultimately changing the behavior of the cell.

A family of protein receptor tyrosine phosphatases exists in Drosophila as well as in vertebrates that share adhesion-like domains in the extracellular region and phosphatase domains associated with the intracellular region. The receptor tyrosine phosphatases in both insects and vertebrates are found associated with the nervous system. In Drosophila they are involved in axon guidance.

In each segment of the fly, specific motor axons upon leaving the CNS diverge from a common motor pathway and divide into five major peripheral nerve branches that project to different groups of muscles. In Dlar mutant embryos, the common motor pathway appears normal, but two of the nerves that branch from the intersegmental nerve, carrying information between segments, are abnormal. In one of these two nerves, the SNb axons bypass their normal entry point to the ventral muscle region (at a defined choice point) and instead continue to extend distally as a distinct fascicle (bundle), following the intersegmental nerve further along its dorsal trajectory to a more lateral region of muscles. In such mutants, ventral muscles are completely devoid of innervation. In some cases nerves terminate without leaving the dorsal axon pathway, and in other cases inappropriate muscles become innervated (Krueger, 1996).

It is believed that each SNb growth cone may have an independent ability to navigate the choice point; that is, no individual axon is required to pioneer the SNb pathway, with the rest relegated to the status of faithful followers. This conclusion is based on studies in which the aCC motoneuron (whose axon pioneers the intersegmental nerve) is ablated. Later growth cones are capable (with some delay) of independently pioneering the intersegmental nerve (Lin, 1995).

The ability of motoneuron growth cones to both recognize and enter their correct target appears to be dependent on the phosphorylation state of key intracellular proteins. A dynamic balance of kinase and phosphatase activities at the leading edge of the growth cone endows it with the ability to integrate signals and translate them into appropriate directional choices. Two mechanisms for axon guidance are involved. At the choice point, SNb axons defasciculate from the intersegmental nerve axons and form a separate bundle. This bundle of axons is then directed or steered into the ventral muscle region. Genes other than Dlar produce subtly different phenotypes capable of altering defasciculation and/or steering, suggesting a complex genetic basis for axon pathfinding in the fly (Krueger, 1996).

The tyrosine kinase Abl and its substrate enabled collaborate with the receptor phosphatase Dlar to control motor axon guidance

Genetic analysis of growth cone guidance choice points in Drosophila has identified neuronal receptor protein tyrosine phosphatases (RPTPs) as key determinants of axon pathfinding behavior. The Drosophila Abl tyrosine kinase functions in the intersegmental nerve b (ISNb) motor choice point pathway as an antagonist of the RPTP Dlar. The function of Abl in this pathway is dependent on an intact catalytic domain. The Abl phosphoprotein substrate Enabled (Ena) is required for choice point navigation. Both Abl and Ena proteins associate with the Dlar cytoplasmic domain and serve as substrates for Dlar in vitro, suggesting that they play a direct role in the Dlar pathway. These data suggest that Dlar, Abl, and Ena define a phosphorylation state-dependent switch that controls growth cone behavior by transmitting signals at the cell surface to the actin cytoskeleton (Wills, 1999).

The reciprocal catalytic activities of a tyrosine kinase and phosphatase predict that a reduction in kinase activity within the Dlar pathway might suppress the Dlar motor axon phenotype. In Dlar mutant embryos, subsets of axons derived from the intersegmental nerve route (ISN), called ISNb and ISNd, fail to enter adjacent muscle target domains just outside the ventral nerve cord. Instead, Dlar mutant ISNb and ISNd axons follow the ISN toward dorsal targets (the bypass phenotype. Since abl loss of function is known to disrupt the outgrowth of ISNb, the Abl tyrosine kinase is an excellent candidate for a role in Dlar signaling. Therefore, various genetic backgrounds were examined in which homozygous Dlar mutations were combined with mutations in a single allele of abl. Reduction of abl of up to half the normal gene dose has a profound effect on the penetrance of the Dlar motor axon guidance phenotype, suppressing the Dlar phenotype up to 10-fold; for example, ISNb bypass in Dlar mutants is reduced from 38% to 4% in abl heterozygote mutants (Wills, 1999).

Western blot analysis shows that endogenous Abl protein binds specifically to the full-length Dlar cytoplasmic domain (GST-Dlar D1-D2). The association of Dlar and Abl in cell extracts is consistent with a direct functional relationship between the two proteins. However, the binding could depend on other factors present in the crude extract. Therefore, the association of purified recombinant Abl protein with Dlar fusion proteins was examined in the absence of other Drosophila proteins. Recombinant Abl binds to Dlar with somewhat less specificity than does the Abl endogenous to S2 cells. Purified mammalian v-Abl binds to Dlar under the same conditions, with a profile of specificity very similar to that of Drosophila Abl. Since v-Abl represents only the kinase and SH2 domains of Abl, these domains appear sufficient to mediate Dlar binding. As further evidence of direct physical interactions between Abl and the Dlar D2 domain, kinase assays reveal that Drosophila Abl phosphorylates GST-Dlar D2 in vitro. In addition to the Dlar D2 domain, Drosophila Abl can weakly phosphorylate the D2 domain of another receptor tyrosine kinase, DPTP69D; this is interesting, since DPTP69D is tyrosine phosphorylated in S2 cells. The physical interactions between Abl and Dlar support a model whereby both proteins function in the same signaling pathway. Furthermore, the phosphorylation of the D2 domain in vitro raises the intriguing possibility that d-Abl activity regulates Dlar function in vivo (Wills, 1999).

The contrast between the abl and Dlar phenotypes and the suppression of the Dlar phenotype by abl alleles suggest that Abl and Dlar play functionally antagonistic roles in ISNb development. This hypothesis makes a simple prediction: gain of function in Abl should result in a phenotype similar to loss of Dlar. Therefore, the GAL4 expression system was used to target high-level expression of wild-type Abl to postmitotic neurons and then the development of motor axon pathways was examined. With three independent neural specific GAL4 drivers, in combination with an abl cDNA under the control of the GAL4 upstream activator sequence (UAS), GAL4-dependent phenotypes were observed. When wild-type Abl is overexpressed, ISNb axons bypass their ventral target muscles in a manner indistinguishable from that of the ISNb phenotype observed in Dlar mutants. The kinase activity of Abl has been shown to be necessary for its role in ISNb neurons (Wills, 1999).

Since Ena acts as a genetic antagonist of Abl, it was reasoned that loss of Ena should resemble gain of Abl. ISNb bypass phenotypes are seen in all ena mutant combinations. Two types of ISNb phenotypes are observed in ena mutants: (1) failure of ISNb to enter the ventral muscles after a successful defasciculation (characteristic of embryos lacking Dlar alone), and (2) failure of ISNb axons to defasciculate from the ISN pathway (characteristic of embryos lacking multiple phosphatases. In addition, the frequency of ISNb bypass in strong ena mutants is twice that observed in the strongest Dlar alleles. These observations may indicate that Ena acts as a point of convergence for multiple inputs in the ISNb guidance mechanism. Ena family members share a conserved domain structure, including an N-terminal EVH1 domain that mediates binding to Zyxin and Listeria ActA, a proline-rich region that supports associations with Profilin and SH3 domains, and a C-terminal EVH2 domain that promotes multimerization. Mutations are available that specifically disrupt either the EVH1 or the EVH2 domains of Ena. Mutations in either domain display highly penetrant ISNb bypass, demonstrating a requirement for both domains in the guidance mechanism. Although Ena is restricted to axons in the developing nervous system late in embryogenesis, it is expressed broadly prior to germ band retraction. To confirm that neuronal Ena function is necessary for ISNb choice point navigation, wild-type ena cDNA was expressed under neuronal GAL4 control in an ena mutant background. Neural specific ena expression attenuates the ISNb phenotype significantly. If the quantity of Ena protein is rate limiting in wild-type ISNb axons, one might expect Ena overexpression to disrupt ISNb guidance. However, no ISNb phenotypes are observed, even when UAS-ena is combined with the strongest neural driver P[elav-GAL4] (Wills, 1999).

The genetic relationship between Abl and Dlar and the requirement of Ena function for ISNb target entry suggest that Ena might act in the Dlar signaling pathway. To test this model, it was asked whether Ena associates with the cytoplasmic domain of Dlar. Endogenous Ena protein associates with a Dlar full-length cytoplasmic domain (GST-Dlar D1-D2) or with D2 alone but not comparably with wild-type D1. Since Abl is known to associate with Ena, and since binding between Abl and Dlar has been demonstrated, it is possible that Ena binding to Dlar requires Abl or additional proteins. Purified Ena has been shown to bind to the Dlar cytoplasmic domain. In both extract and recombinant protein binding assays, Ena shows only weak association with DPTP10D. However, Ena binds effectively to the D2 domain of DPTP69D. The preferential binding of Ena to the D2 domains of Dlar and DPTP69D, as compared with the D1 domains of the same RPTPs, suggests that these interactions are specific. The parallel between Dlar and DPTP69D binding is interesting, given the published observation that DPTP69D is required for ISNb guidance and can partially substitute for Dlar in vivo. Furthermore, the nature and penetrance of ISNb defects in ena mutants suggest that Ena may function downstream of multiple inputs (Wills, 1999).

The relationships between Abl, Ena, and Dlar in motor axon guidance suggest a model whereby Abl and Dlar compete for shared substrates to regulate growth cone behavior. Although the Dlar cytoplasmic domain was previously shown to encode an active PTP domain, using artificial phospho-peptide substrates in vitro, no physiological substrates have been identified. Since nearly all of the tyrosine phosphatase activity of LAR family RPTPs resides in the D1 domain, the ability of the GST-Dlar D1 fusion protein to dephosphorylate purified Drosophila Abl or Ena proteins after these proteins have been phosphorylated with recombinant d-Abl was examined. Incorporated ³²P is rapidly released from both Abl and Ena after addition of wild-type GST-Dlar D1 but not after addition of the catalytically inactive C-to-S mutant GST-Dlar D1 fusion protein. These results suggest that the bacterially expressed GST-Dlar protein is correctly folded and that Drosophila Abl and Ena are both potential Dlar substrates. However, because PTPs are known to be promiscuous in vitro, additional experiments will be necessary to determine whether Abl and/or Ena are targets for Dlar activity in vivo (Wills, 1999).

The receptor tyrosine phosphatase Lar regulates adhesion between Drosophila male germline stem cells and the niche

The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. This study shows that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions (Srinivasan, 2012).

This work identifies a role for the transmembrane receptor tyrosine phosphatase Lar, acting cell-autonomously to maintain attachment of Drosophila male GSCs to the hub. Lar function appears to promote the maintenance of robust adherens junctions between GSCs and hub cells and to localize and/or retain E-cadherin at the hub-GSC interface. Consistent with the recently demonstrated requirement for E-cadherin to polarize GSCs by localizing Apc2 at the hub-GSC interface and to establish centrosome orientation in GSCs (Inaba, 2010), Apc2 was often mislocalized around the GSC cortex and centrosomes were often misoriented in Lar mutant GSCs (Srinivasan, 2012).

Lar may function in parallel with other cell signaling pathways that are important for maintaining attachment of GSCs to the hub. Activation of the JAK-STAT pathway in Drosophila male GSCs maintains GSCs at the hub. However, Stat92E protein levels appeared normal in Lar mutant GSCs, suggesting that Lar function is not required for activation of the JAK-STAT pathway. The Rap1 GTPase/Rap1 guanine nucleotide exchange factor (Rap-GEF) signaling pathway also regulates hub-GSC adhesion. Like Lar mutants, Rap-GEF mutants have impaired adherens junctions at the hub- GSC interface resulting in GSC loss. However, Rap-GEF function is required in hub cells, whereas Lar functions in GSCs to promote hub-GSC adhesion. Interestingly, expression of E-cadherin-GFP in either hub cells or GSCs of Rap-GEF mutants resulted in wild-type numbers of GSCs and restored E-cadherin localization at the hub- GSC interface, whereas expression of Ecadherin-GFP in Lar mutant GSCs did not rescue the loss of GSCs, suggesting that the Rap-GEF and Lar signaling pathways might use different mechanisms to build and/or maintain adherens junctions between the hub cells and GSCs (Srinivasan, 2012).

The ability of some GSCs to persist next to the hub in Lar mutant testes might be due to partial redundancy between Lar and other tyrosine phosphatases such as the type IIA family receptor tyrosine phosphatase Ptp69D, which has overlapping functions with Drosophila Lar in the central nervous system and the visual cortex and shares common signaling mechanisms. Alternatively, weak hub-GSC adhesion in Lar mutant testes might enable CySCs to compete for attachment to the hub, displacing some, but not all, GSCs from the hub. CySCs normally have smaller regions of contact with the hub than do GSCs but can outcompete GSCs from the hub when provided with an advantage. For example, overexpression of components of the integrin-based adhesion system in CySCs resulted in displacement of GSCs from the hub by CySCs (Srinivasan, 2012).

In wild-type testes, Lar localizes to the hub-GSC interface, which is the region of cell cortex where localized adherens junctions anchor GSCs to their niche. Adherens junctions are formed by extended clustering of transmembrane cadherin proteins that form homotypic interactions with cadherins on opposing cell membranes. The highly conserved cytoplasmic tail of E-cadherin acts as an anchor for β-catenin and p120-catenin and indirectly for α- catenin through its interaction with β-catenin. Lar also localizes to adherens junctions in epithelial cells and in neuronal synapses that are enriched in cadherin-catenin complexes. Lar physically associates with the cadherin-catenin complex in cultured cells and with N-cadherin in Drosophila embryos (Srinivasan, 2012).

Adherens junctions are associated with underlying arrays of cortical F-actin, organized by the high local concentration of β-catenin dimers. F-actin filaments, in turn, regulate the stability and strength of adherens junctions. Biochemical and genetic analyses of Lar indicate a role in regulating the actin cytoskeleton. Loss of Lar function in Drosophila oocytes results in defects in follicle formation, egg elongation and anterior-posterior polarity that are correlated with defects in actin filament organization. Lar might help to maintain hub- GSC adhesion by interacting with and modulating the function of regulators of F-actin. Drosophila Lar and its homologs physically and genetically interact with Ena, a member of the Ena/VASP family of actin regulators. Drosophila Ena and its mammalian homologs localize to adherens junctions and have been implicated in the formation and strengthening of adherens junctions in several cell types. However, although Ena localized to the hub-GSC interface, where adherens junctions are present, its function was not absolutely required for GSC maintenance, suggesting that other F-actin regulators in addition to Ena function to maintain hub-GSC adhesion (Srinivasan, 2012).

Lar protein localized to the hub-GSC interface might instead, or in addition, regulate the tyrosine phosphorylation state of components of the adherens junctions to maintain strong adhesion between hub cells and GSCs. Regulation of the tyrosine phosphorylation of components of adherens junctions plays an important role in modulating the adhesive state of cells. Tyrosine phosphorylation of E-cadherin in epithelial cells induces loss of cell-cell contacts and the endocytosis of E-cadherin. A possible role of Lar is to maintain adherens junctions by dephosphorylating E-cadherin. Alternatively, Lar might target E-cadherin to the membrane to build adherens junctions, as has been shown for the mammalian homolog of Lar in cultured hippocampal neurons, where it promotes the accumulation of cadherin-catenin complexes at the synapse to enhance cell adhesion. Alternatively, or in addition, Lar might regulate tyrosine phosphorylation of the catenins associated with E-cadherin at the hub-GSC interface. Tyrosine phosphorylation of β-catenin leads to loss of cadherin-β-catenin interaction and to internalization of Ecadherin, reducing the strength of adherens junctions. Mammalian Lar has been shown to dephosphorylate β-catenin in vitro, suggesting that in vivo Lar might promote cell adhesion by regulating the phosphorylation of β-catenin (Srinivasan, 2012).

In addition to GSCs, Lar protein was also detected in two-, four- and eight-cell transit-amplifying spermatogonial cysts, which have the ability to dedifferentiate and reoccupy the hub to replace lost GSCs. Under conditions that promote dedifferentiation, spermatogonial cells send out dynamic, actin-rich, thin protrusions, suggesting acquisition of motility. An intriguing possibility is that Lar might facilitate the ability of dedifferentiating spermatogonial cells to reorganize their actin cytoskeleton to recognize and build adherens junctions with the hub, similar to the role of Lar in the nervous system, where it promotes axonal migration, possibly by facilitating reorganization of the actin cytoskeleton. One of the ligands of Lar identified in the nervous system, the heparan sulfate proteoglycan Dally-like (Dlp), is expressed by hub cells and helps maintain GSCs in their undifferentiated state. At Drosophila neuromuscular junctions, Dlp interacts with and inhibits the phosphatase activity of Lar to regulate active zone morphology and function at synapses. Similarly, in dedifferentiating spermatogonial cells, interaction of Lar in GSCs with Dlp on hub cells might inhibit cell motility and promote the formation of adherens junctions between hub cells and the dedifferentiating germ cells (Srinivasan, 2012).

Serial synapse formation through filopodial competition for synaptic seeding factors

Following axon pathfinding, growth cones transition from stochastic filopodial exploration to the formation of a limited number of synapses. How the interplay of filopodia and synapse assembly ensures robust connectivity in the brain has remained a challenging problem. This study developed a new 4D analysis method for filopodial dynamics and a data-driven computational model of synapse formation for R7 photoreceptor axons in developing Drosophila brains. Live data support a 'serial synapse formation' model, where at any time point only 1-2 'synaptogenic' filopodia suppress the synaptic competence of other filopodia through competition for synaptic seeding factors. Loss of the synaptic seeding factors Syd-1 and Liprin-alpha leads to a loss of this suppression, filopodial destabilization, and reduced synapse formation. The failure to form synapses can cause the destabilization and secondary retraction of axon terminals. This model provides a filopodial 'winner-takes-all' mechanism that ensures the formation of an appropriate number of synapses (Ozel, 2019).

After pathfinding, axon growth cones transition to become terminal structures with presynaptic active zones. How axon terminals form a defined number of synaptic contacts with a specific subset of partners is a daunting problem in dense brain regions. Stochastically extending and retracting filopodial extensions occur during both pathfinding and synapse formation and are thought to facilitate interactions between synaptic partners. However, little is known about the role of stochastic filopodial dynamics for robust synapse formation (Ozel, 2019).

Presynaptic active zone assembly is a key step in synapse formation and regulated by a conserved set of proteins. An early active zone 'seeding' step has been defined through the functions of the multidomain scaffold proteins Syd-1 and Liprin-α in C. elegans and Drosophila neuromuscular junction (NMJ). Syd-1 is a RhoGAP-domain-containing protein that recruits Liprin-α to the active zone. Liprin-α is an adaptor protein named after its direct interaction with the receptor tyrosine phosphatase Leukocyte common antigen-related (LAR). The Liprin-α and LAR interaction has been directly implicated in active zone assembly across species. Downstream, Liprin-α and Syd-1 recruit core active zone components and ELKS/CAST family protein Brp. Finally, the RhoGEF Trio has been proposed to function downstream of the Lar/Liprin-α/Syd-1 and has recently been suggested to regulate active zone size (Ozel, 2019).

Remarkably, the proposed Lar-Liprin-α-Syd-1-Trio pathway has been characterized in parallel for its role in axon guidance, independent of active zone assembly. In the Drosophila visual system, mutants in all four genes have been implicated in the layer-specific targeting of photoreceptor R7 axons in the medulla neuropil. It is unclear whether any of the four mutants affect active zone assembly in R7 neurons. Dual roles in axon pathfinding and synapse formation have been shown or proposed for all four genes. Independent implications in active zone assembly and axon pathfinding raise the question what functions are primary or secondary (Ozel, 2019).

This study investigated the relationship between filopodial dynamics and synapse assembly in the presynaptic R7 terminal. The early synaptic seeding factors Liprin-α and Syd-1 accumulate in only a single filopodium per terminal at any given point in time. Consequently, only 1-2 filopodia per terminal are stabilized, suggesting that only 1-2 filopodia are synaptogenic at any time. A data-driven computational model shows that this 'serial synapse formation model' is supported by the measured dynamics and could be tested in mutants for liprin-α, syd-1, lar, and trio. Specific defects in filopodial dynamics precede all other defects, including axon terminal retractions. A quantitative 'winner-takes-all' model, from stochastic filopodial dynamics to the formation of a limited number of synapses, is presented as well as a model for axon terminal stabilization based on filopodia and synapses (Ozel, 2019).

The data link bulbous filopodia to synapse formation based on three findings: (1) in the wild type, these are the only filopodia that specifically occur during the time window of synapse formation and do not exhibit stochastic dynamics, and wild-type R7 photoreceptor axons stabilize 1-2 bulbous filopodia at a time; (2) the synaptic seeding factors Liprin-α and Syd-1 non-randomly localize to 1-2 bulbous filopodia at a time; and (3) loss of liprin-α or syd-1 selectively affects the stabilization of bulbous filopodia. Loss of the upstream receptor lar similarly selectively affects bulbous filopodia but, in addition to bulb destabilization, also strongly affects bulb initiation. Together, these findings support a model whereby stochastic filopodial exploration leads to bulb stabilization and synapse formation one at a time. In this model, restrictive synaptogenic filopodia formation 'paces' the formation of ~25 synapses over 50 h, effectively controlling synapse numbers within the available developmental time window (Ozel, 2019).

The key mechanism of this model is the inhibitory feedback of synaptogenic filopodium formation. In contrast to all other filopodia, the dynamics of bulbous filopodia are not independent events. How are synaptic seeding factors competitively distributed between these filopodia? Live-imaging data suggest that Liprin-α or Syd-1 can traffic in and out of filopodia but overexpressed proteins accumulate in the axon terminal trunk and do not enter to more than 1-2 filopodia, indicating that trafficking into filopodia is restricted. Morphologically, filopodia are very thin structures that may not provide much space for freely diffusing proteins or organelles. On the other hand, the bulbous tip provides a much larger volume that may be required for sufficient amounts of synaptic seeding factors and other building material to initiate synapse formation. Furthermore, computational tests show that 'winner-takes-all' dynamics can arise from the dynamic distribution of a limited resource that confers a competitive advantage (longer lifetime, which leads to further accumulation) without the need for active filopodial communication (Ozel, 2019).

Since Syd-1 and Liprin-α are not required for bulb initiation, it is speculated that filopodial contact with a synaptic partner may initiate the bulb and precede active zone formation. The data suggest that Lar is a good candidate for a presynaptic receptor with such a role, but it is unlikely to be the sole upstream receptor. Neurexin and PTP69D, for example, are other known candidates. In the absence of an upstream receptor or the seeding factors themselves, synapse assembly fails and bulbs destabilize. New bulb generation following loss of bulbs in the absence of seeding factors can also be explained with seeding factors as a limiting resource with a competitive advantage. This is reminiscent of other competitive processes that shape neuronal morphology, e.g., the restricting role of building material in the competitive development of dendritic branches in a motorneuron (Ryglewski, 2017). Mutant analyses suggest that stable bulbs are linked to negative feedback on other bulbs via the function of the RhoGEF Trio. While the exact mechanism is unclear, it is tempting to speculate about a role of actin-dependent signaling downstream of synaptic seeding (Ozel, 2019).

The current model only considers the presynaptic axon terminal. The main postsynaptic partner of R7 are amacrine-like Dm8 cells, whose elaborate dendritic processes are present in direct vicinity to the R7 filopodia throughout the developmental period of synapse formation. Currently the dynamics of the postsynaptic processes and whether they restrict availability or are 'easily found' as postsynaptic partners are unknown. The presynaptic model presented in this study could explain the observed slow, serial synapse formation even in the presence of abundant postsynaptic partner processes (Ozel, 2019).

Mutations in the proposed pathway components Lar, Liprin-α, Syd1, and Trio have been independently characterized for their roles in active zone assembly (mostly at the larval neuromuscular junction) and axon targeting, in large part in the visual system . It is likely that all four genes exert more than one function in different contexts. Defects in synapse formation and retraction are captured by the measured parameters and the current model. However, some differences in overall morphology, including overextensions in the syd-1 mutant, may be described by parameters not considered in the model, e.g., filopodial length, and due to some differences in their molecular function. Similarly, for Lar, independent context-dependent functions have been characterized based on different downstream adaptors (Ozel, 2019).

It was asked to what extent a primary role for lar, trio, syd-1, and liprin-α in synapse formation could explain previously observed phenotypes. All filopodial defects in the four mutants occur independently and prior to possible retraction events. Combined live imaging and computational modeling suggests that defects in the syd-1 and liprin-α mutants are consistent with a primary defect in bulb stabilization and synapse formation. These defects may in turn lead to axon destabilization or represent independent functions; lar may have an additional earlier adhesion function and trio does not play a critical role in the formation of the correct number of synapses, while its effect on general filopodial dynamics may sensitize mutant axons to other changes (Ozel, 2019).

The conclusion that Lar, Liprin-α, and Syd-1 have a primary function in synapse formation is based on three pieces of evidence: (1) all mutants initially target correctly and exhibit normal filopodial dynamics prior to synapse formation; (2) the mutants start retracting only when synaptic contacts initiate, in the order and severity from the receptor to the downstream elements; and (3) all three mutants exhibit the loss of competitive bulb stabilization. Taken together, these observations support a direct role in synapse formation following bulb stabilization, but other molecular functions cannot be excluded. For example, in both C. elegans and Drosophila, Lar has been shown to function independently in axon guidance and synapse formation. This study found that syd-1ΔRhoGAP mutants have normal terminal morphology and only a mild decrease in the number of BrpD3-puncta. This is consistent with recent findings that a RhoGAP-deficient Syd-1 fragment is sufficient to rescue early active zone seeding events at the Drosophila neuromuscular junction but not the recruitment of Brp as the active zones mature (Spinner, 2018). However, since homozygous syd-1ΔRhoGAP flies have no obvious connectivity defects, synapse numbers are apparently sufficient for axon terminal stabilization (Ozel, 2019).

Finally, these observations suggest that loss of the primary functions of these proteins in filopodial dynamics and synapse formation are sufficient to cause axon retractions. The phenotypes observed in this study for lar, liprin-α, and syd-1 are somewhat similar but in contrast to cadN only occur at or after the time of synaptic partner identification. While filopodia continuously decrease, synapses continuously increase, thereby allowing a takeover of the axon terminal stabilization function. The modeling fits the wild type, liprin-α, syd-1, and trio remarkably well. In contrast, while retractions in the lar mutant are qualitatively predicted, the model fails to explain retractions quantitatively. A partial explanation may be that the model was parameterized only based on the lar mutant axon terminals that are still unretracted at P60. These are only 30% of terminals by that time, and this study has effectively selected for terminals with dynamics that prevented retractions thus far. It is likely that earlier retractions are caused by defects in filopodial adhesion or synaptic contacts. In sum, the data and modeling support a role for synapses in the stabilization of R7 axon terminals, which can lead to probabilistic axon retractions in mutants affecting synapse formation (Ozel, 2019).

Lar maintains the homeostasis of the hematopoietic organ in Drosophila by regulating insulin signaling in the niche

Stem cell compartments in metazoa get regulated by systemic factors as well as local stem cell niche-derived factors. However, the mechanisms by which systemic signals integrate with local factors in maintaining tissue homeostasis remain unclear. Employing the Drosophila lymph gland, which harbors differentiated blood cells, and stem-like progenitor cells and their niche, this study demonstrates how a systemic signal interacts and harmonizes with local factor/s to achieve cell type-specific tissue homeostasis. Genetic analyses uncovered a novel function of Lar, a receptor protein tyrosine phosphatase. Niche-specific loss of Lar leads to upregulated insulin signaling, causing increased niche cell proliferation and ectopic progenitor differentiation. Insulin signaling assayed by PI3K activation is downregulated after the second instar larval stage, a time point that coincides with the appearance of Lar in the hematopoietic niche. It was further demonstrated that Lar physically associates with InR and serves as a negative regulator for insulin signaling in the Drosophila larval hematopoietic niche. Whether Lar serves as a localized invariable negative regulator of systemic signals such as insulin in other stem cell niches remains to be explored (Kaur, 2019).

An effort to understand the maintenance of the hematopoietic niche led to the discovery of the role of Lar in regulating insulin signaling in the niche, which is crucial for lymph gland homeostasis. Lar in the hematopoietic niche acts as a rheostat, restricting excessive insulin signaling to limit proliferation in later developmental stages. A physiological consequence of insulinemia in the niche is upregulated ROS. As a result, the ROS/Spitz/EGFR/ERK circuit that is evoked during an immune response gets activated during normal development. Lar abrogation from the niche also activates JNK to bolster niche cell proliferation. In addition to Dpp, insulin signaling is also known to stimulate cell proliferation via Myc in the hematopoietic niche. It is, therefore, also possible that the InR/Pi3K activation and immense proliferation observed upon Lar loss from the hematopoietic niche might also involve Myc activation (Kaur, 2019).

Lar is a transmembrane type IIA receptor protein tyrosine phosphatase, which has two intracellular phosphatase domains (D1 and D2) and extracellular immunoglobulin (Ig) and fibronectin type III (FNIII) domains. The different domains of Lar provide this single molecule the ability to carry out diverse functions. A major interactor of Lar in Drosophila is the actin cytoskeleton. This interaction is often encountered in the developing nervous system, in which it plays a significant role in axonal migration and synapse morphogenesis (Kaur, 2019).

In addition, in oocytes, Lar is implicated in follicle cell development and patterning through actin organization. A study in Drosophila germline stem cells (GSCs) has demonstrated that Lar can act as a cell adhesion molecule by localizing E-cadherin at the GSC-hub interface, thereby maintaining the attachment between male GSCs and hub cells. Moreover, evidence of the physical interaction of Lar with N-cadherin in the Drosophila embryo further endorses the interaction of Lar with cell adhesion molecules (Kaur, 2019).

Many in-vitro studies in the mammalian system have revealed that Lar interacts with various tyrosine kinases, thereby modulating different signaling pathways. In vitro studies using mammalian cell lines demonstrated that InR physically associates with Lar. SPR has shown that the most preferred substrate for Drosophila Lar is InR, but the evidence for physical interaction remains to be demonstrated. The current study provides the first in vivo physical association of Lar with InR in Drosophila (Kaur, 2019).

Besides the evidence of physical interaction, genetic data demonstrate that loss of Lar in the hematopoietic niche results in hyperactivated insulin signaling. Upregulated PI3K expression suggests that Lar directly acts on InR and not on any other regulators of InR signaling such as Pten or Tsc1/2 in the hematopoietic niche. No alteration in InR expression upon Lar loss from the niche further confirms that Lar-InR interaction impinges on PI3K-Akt-insulin signaling and not on InR expression. Furthermore, the results show that the catalytic function of Lar protein that resides in the PTPD1 domain is crucial for Lar-InR interaction (Kaur, 2019).

LAR can modulate multiple tyrosine kinases; it appears that the spatial distribution of LAR gives it specificity for its cell-type tyrosine kinase receptor. Equivalent evidence comes from the current in vivo study, in which it was successfully demonstrated that, in hematopoietic tissue, wherever there is a high membranous tGPH (reporting insulin signaling), Lar expression is low, and vice versa. This reciprocal expression, coupled with the genetic analyses, projects a mechanism underpinning the activation of receptor tyrosine kinases by RPTPs in a cell-type-specific manner (Kaur, 2019).

Insulin signaling helps to coordinate nutritional status with systemic growth control both in invertebrates and in the mammalian system. Through its receptor, insulin is now known to have a much broader pleiotropic role, controlling several physiological processes. Therefore, inappropriate activation of insulin signaling has been linked with various aberrant scenarios such as infection, cancer and diabetes (Kaur, 2019).

The hematopoietic niche cells can sense the systemic insulin level, which is essential for their proliferation. Overactivation of InR increases the niche cell number, whereas downregulation causes it to decline. The systemic insulin level is also directly sensed by the hemocyte progenitors. Knockdown of insulin signaling in the progenitors results in their precocious differentiation, demonstrating that physiological levels of insulin signaling are essential for their maintenance. The expression data and genetic analyses unravel a differential requirement of insulin signaling in the niche compared with progenitor cells (Kaur, 2019).

This study further illustrated how the pleiotropic effect of excessive insulin signaling in the niche affects the homeostasis of the organ. The hyperactivated insulin signaling in the niche generates excessive ROS. This high level of ROS in the niche has a two-prong effect in two different cell types of the developing lymph gland. First, within the niche, it evokes JNK to provide a thrust to the ongoing proliferation of the niche cells. Previous literature has demonstrated that oxidative stress leads to the activation of JNK, which is known to have both pro- and anti-proliferative functions. Thus, by the stimuli, strength and duration of the JNK activation, diverse responses ranging from apoptosis, survival and altered proliferation can be evoked. The current study demonstrates that the ectopic activation of JNK due to the gradual accumulation of ROS collaborates with hyperactivated insulin signaling to boost cell proliferation. The second effect of the elevated ROS in the niche is the activation of ERK in the hemocyte progenitors. Activation of the Spitz/EGFR pathway by ectopic ROS in the niche is known to activate ERK in the progenitors. The activated ERK causes ectopic differentiation and lamellocyte generation (Kaur, 2019).

The cumulative effect of deregulated signals disturbs the homeostasis of the organ Interestingly, hyperactivation or hypoactivation of insulin signaling is also associated with deregulated hematopoiesis in the vertebrate system. For example, altered insulin signaling in diabetic mice affects the composite microenvironment of the bone marrow leading to compromised function of the hematopoietic niche. This work provides a strong genetic link between Lar and Insulin signaling, which should be tested in vertebrates. Although LAR is expressed in T-cell lineages in vertebrates, it remains to be seen whether Lar is also present in vertebrate hematopoietic niche/s and functions similarly. It is also intriguing to observe that, similar to the vertebrate system, a low level of ROS is present in the Drosophila hematopoietic niche. It will be fascinating to see whether the hyperactivation of insulin signaling also generates ROS in the vertebrate niche and affects cell fate specification via the same mechanism that is elucidated in this study (Kaur, 2019).

This study unravels a check on insulin signaling by Lar that authorizes the hematopoietic niche to act as the 'interlocutor', evaluating the physiological state of an organism and thereby relaying it to the hemocyte progenitors for their homeostasis (Kaur, 2019).

Fat2 polarizes Lar and Sema5c to coordinate the motility of collectively migrating epithelial cells

Migrating epithelial cells globally align their migration machinery to achieve tissue-level movement. Biochemical signaling across leading-trailing cell-cell interfaces can promote this alignment by partitioning migratory behaviors like protrusion and retraction to opposite sides of the interface. However, how signaling proteins become organized at interfaces to accomplish this is poorly understood. The follicular epithelial cells of Drosophila melanogaster have two signaling modules at their leading-trailing interfaces-one composed of the atypical cadherin Fat2 and the receptor tyrosine phosphatase Lar, and one composed of Semaphorin5c and its receptorPlexin A. This study shows that these modules form one interface signaling system with Fat2 at its core. Trailing edge-enriched Fat2 concentrates both Lar and Semaphorin5c at cells' leading edges, but Lar and Semaphorin5c play little role in Fat2's localization. Fat2 is also more stable at interfaces than Lar and Semaphorin5c. Once localized, Lar and Semaphorin5c act in parallel to promote collective migration. It is proposed that Fat2 serves as the organizer this interface signaling system by coupling and polarizing the distributions of multiple effectors that work together to align the migration machinery of neighboring cells (Williams, 2023).

Epithelial cells migrate collectively during animal development, wound healing, intestinal turnover and cancer metastasis. To do so, they must polarize within the epithelial plane at both the individual and tissue scales. At the individual scale, cells polarize along a leading-trailing axis. Protrusion and adhesion formation are biased to the leading edges of cells, and contractility and adhesion removal to their trailing edges, much as in cells migrating solo. At the tissue scale, cells throughout the epithelium are polarized, such that their leading edges preferentially point in the direction of migration, and trailing edges in the opposite direction, a form of planar cell polarity. At the intersection of these scales are the cell–cell interfaces that link the trailing edge of one cell to the leading edge of the cell behind. These leading-trailing interfaces can act as sites of biochemical or mechano-chemical signaling that polarize motility behaviors across the interface. However, little is known about how signaling proteins become organized along interfaces to accomplish this feat (Williams, 2023).

The rotational migration of the follicle cells in Drosophila melanogaster has proven to be a fruitful system for identifying signaling mechanisms that coordinate epithelial cell movements. Follicle cells are somatic cells of the egg chamber, the multicellular structure within the ovary that gives rise to an egg. They form a continuous monolayer epithelium around a central cluster of germ cells, and they are surrounded in turn by a basement membrane extracellular matrix that encapsulates the entire egg chamber. The apical surfaces of the follicle cells adhere to the germ cells, and their basal surfaces adhere to and crawl along the basement membrane. Migration in this topologically closed configuration causes the entire egg chamber to rotate within the stationary basement membrane. This motion changes the structure of the basement membrane, ultimately helping give the egg its elongated shape. Follicle cell migration requires WAVE complex-dependent lamellipodia, which are polarized to the leading edge of each cell and planar-polarized across the epithelium. Polarity emerges tissue-autonomously, without input from extrinsic directional cues. This simplifying feature allows more easy isolation od the contribution of within-group coordination to collective migration. It likely also makes these cells particularly reliant on such coordination for movement (Williams, 2023).

Two biochemical signaling modules operate at leading-trailing interfaces, where they coordinate the migratory behaviors of neighboring follicle cells. The first module is composed of the atypical cadherin Fat2 (also known as Kugelei) and the receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar). Fat2 is enriched along the trailing edge of each cell, where it acts in trans to concentrate Lar and the WAVE complex across the cell–cell interface, at the leading edge of the cell behind. Lar also contributes to WAVE complex localization, but not as strongly as Fat2, implying the existence of additional unidentified Fat2 effectors. Together, these proteins restrict cell protrusive activity to a single leading-edge domain and orient the protrusions from all the cells in a uniform direction across the tissue. The second module is composed of a transmembrane semaphorin (ligand) and plexin (receptor) pair, Semaphorin 5c (Sema5c) and Plexin A (PlexA), which are enriched at leading and trailing edges respectively. In other contexts, semaphorin–plexin signaling can lower integrin-based adhesion and/or inhibit protrusivity on the plexin-containing cell side. Similarly, overexpression of Sema5c in one follicle cell reduces the protrusivity of its neighbors in a PlexA-dependent manner. This led to the model that Sema5c signals through PlexA to maintain a non-protrusive state at the trailing edges of cells (Williams, 2023).

Despite their distinct depletion and overexpression phenotypes, several lines of evidence suggest that the Fat2–Lar and Sema5c–PlexA modules function within one interface-polarizing signaling system. A series of pairwise comparisons show that Fat2, Lar and Sema5c all colocalize with the WAVE complex in interface-spanning puncta that sit at the tips of filopodia within a broader lamellipodium. For reasons that are not yet clear, PlexA only rarely colocalizes with the other proteins. Loss of Lar also reduces the enrichment of Sema5c at leading edges, implying functional interaction between the two modules in addition to their shared spatial organization. This study investigated the hierarchy of interactions between Fat2, Lar, and Sema5c by which they form interface-spanning puncta, and asked how the three proteins work together to promote collective migration (Williams, 2023).

Fat2 was found to form the core of both the Lar and Sema5c-containing signaling modules, concentrating Sema5c at leading edges in trans as it was previously shown to do for Lar. Conversely, Lar and Sema5c play little or no role in the localization of Fat2. Using fluorescence recovery after photobleaching (FRAP) and acute inhibition experiments, it was shown that Fat2 resides more stably at trailing edges than do Lar or Sema5c at leading edges, and that Fat2 is likely continuously required to maintain the enrichment of Lar and Sema5c at leading edges in the face of their ongoing turnover. It was further found that Lar and Sema5c act in parallel to promote collective migration. From these data, it is proposed that Fat2 acts as a central organizer of the follicle cell interface-polarizing signaling system, serving to couple and polarize the distributions of multiple effectors that together align the motility machinery of neighboring cells (Williams, 2023).

The follicle cells use biochemical signaling across their leading-trailing interfaces to polarize their migration machinery at interface, cell and tissue scales. This study has shown that Fat2 is a central organizer of this signaling system. Fat2 acts at the trailing edge of each cell to concentrate both Lar and Sema5c at the leading edge of the cell behind. By contrast, Lar and Sema5c play at most minor roles in the localization of Fat2. In this way, Fat2 coordinates the activities of two effector proteins with distinct functions, allowing them to work synergistically to promote highly persistent collective migration (Williams, 2023).

One defining feature of this Fat2-based signaling system is that most of the component proteins colocalize in interface-spanning puncta. Cadherins often self-organize into clusters, making it likely that this punctate organization stems from Fat2. However, whether Fat2 concentrates Lar and Sema5c in the puncta through direct binding or through intermediary proteins is unknown. An ectopic Fat2 pool did not cause redistribution of Lar, suggesting that additional inputs also contribute to the localization of Lar. Also it is not known known how the receptor for Sema5c, PlexA, fits into this model. Like Fat2, PlexA is enriched at the trailing edges of cells and helps to localize Sema5c, and yet antibody staining indicates that PlexA only partially colocalizes with the Fat2-based puncta. The biggest open question is how Fat2 becomes localized to the trailing edge, as this appears to be the key event that polarizes the entire signaling system. Mechanical feedback from collective migration itself is required for the polarization of Fat2 to trailing edges, but the nature of this feedback, and whether Fat2 has a trans binding partner that further stabilizes its localization, will be important areas for future investigation (Williams, 2023).

This study includes the first measurements of the dynamics of planar signaling proteins in follicle cells, which is an important step towards understanding their polarization mechanism. Using FRAP, it was found that Fat2 is a more stable resident of the leading-trailing interface-spanning puncta than are Lar or Sema5c, consistent with its more central role in maintaining these structures. Based on these FRAP data, as well as the rapid redistribution of Lar and Sema5c upon extracellular Ca2+ chelation, it is hypothesized that Fat2 maintains the leading edge enrichment of Lar and Sema5c by slowing their turnover within the puncta, thereby concentrating them at the leading edge. However, the continuous requirement of Fat2 for the maintenance of polarization of Lar and Sema5c awaits confirmation with a more specific method of acute Fat2 inhibition, as extracellular Ca2+ chelation is a blunt tool, and it is possible that Fat2-independent effects, such as the disruption of another cadherin, contributed to the rapid localization changes of Lar and Sema5c. Further comparison of the dynamics of Lar and Sema5c with and without Fat2 will also be needed to determine whether Fat2 concentrates them through local stabilization (for example through direct or indirect binding) or by a different mechanism, such as increasing their rate of arrival at leading edges (Williams, 2023).

This work also sheds light on how Lar and Sema5c work together to promote collective migration. Previously work has shown that loss of either protein alone impairs migration but does not stop it. By contrast, it is now found that removing both proteins together fully blocks migration in a way that is indistinguishable from loss of Fat2. These data suggest that once Fat2 concentrates Lar and Sema5c to the leading edges of cells, they then act in parallel to promote collective migration, likely by polarizing distinct aspects of the migration machinery (Williams, 2023).

What aspects of the migration machinery do Lar and Sema5c each control? In the case of Lar, it seems to be part of the bridge between Fat2 and WAVE complex-dependent protrusions — both Fat2 and Lar increase protrusive F-actin enrichment at leading edges (Fat2 in trans and Lar in cis), and both also help polarize protrusions in the direction of migration. In addition, Lar acts in trans to promote retraction of the trailing edge of the cell ahead, but the mechanistic basis for this trans function and the degree to which it is separable from the cis function of Lar, remain undetermined. In the case of Sema5c, cell-scale loss-of-function phenotypes have proven more elusive, but enrichment of PlexA at trailing edges and the ability of overexpressed Sema5c to suppress protrusion in trans both point to the trailing edge as the likely site of regulation (Williams, 2023).

By positioning both Lar and Sema5c, Fat2 integrates two features of interface signaling systems known to operate in other collectively migrating cell types but not yet seen together. The first feature is the use of a trailing edge-associated mechanical cue to orient protrusions in the cell behind. Mechanical localization of Fat2 to trailing edges polarizes protrusions in the following cell, in part by localizing Lar. The second feature is the use of contact inhibition of locomotion, which causes cells to polarize away from one another by suppressing protrusion and/or increasing contractility at the point of contact. By localizing Lar and Sema5c to leading edges, Fat2 positions them to enforce trailing edge behavior at the contacting edge of the cell ahead. Therefore, Fat2 translates a mechanical cue into bi-directional signaling across leading-trailing interfaces to coordinate cell migratory behaviors for collective migration (Williams, 2023).

GENE STRUCTURE

Nearly half the protein coding sequence is present in exon 14, a single, large (3.3 kb) exon. Exons 1 through 4, which code for a total of less than 900 bp, are spread over 50 kb of genomic DNA. The screw gene is nested entirely between exons 4 and 5 of Dlar (Krueger, 1996 and Arora, 1994).

Exons - 17

PROTEIN STRUCTURE

Amino Acids - 1997

Structural Domains

DLAR has an N-terminal signal sequence, an extracellular domain of 1345aa, a transmembrane domain of 25aa and a 627aa cytoplasmic region. The cytoplasmic region of DLAR has 72% identity to vertebrate LAR, with two repeated phosphatase domains. Whereas LAR has eight FNIII repeats, DLAR has nine (Streuli, 1989).

The structure of DLAR is quite different from the structure of its mammalian relatives, LAR and HPTP delta. LAR has a modular gene structure, in which individual exon boundaries correspond to structural domains (such as the Ig-like domains and FNIII repeats). In contrast, the boundaries of exons in the Dlar gene show virtually no correspondence to structural domains of DLAR. The mammalian proteins are alternatively spliced to produce multiple isoforms, while it is unlikely that DLAR possesses multiple isoforms (Krueger, 1996).

DLAR has three N-terminal Ig domains, nine fibronectin III domains, a transmembrane domain and two C-terminal protein tyrosine phosphatase domains. Sequence comparison of the PTP-like domains indicates that DLAR is closely related the the mammalin PTPs, having 77% identity to LAR (Krueger, 1996 and Streuli, 1989).

date revised: 18 February 2024

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