BIOLOGICAL OVERVIEW

Given the genetic interaction between Abl and Disabled, and the potential for a physical interaction between Abl and Dbl, the physical interaction of Disabled with the intracellular domain of Notch (Giniger, 1998) might provide a basis for understanding the developmental role of Abl and Dbl. Described below is evidence that Notch and Abl mutations interact synergistically to produce synthetic lethality and defects in axon extension. These axonal aberrations cannot be accounted for on the basis of changes in cell identity because the Notch/Abl interaction has not been shown to cause neurogenic or anti-myogenic phenotypes. Notch is present in the growth cones of extending axons, and the Abl accessory protein Disabled binds to a signaling domain of Notch in vitro. It is therefore speculated that Disabled and Abl may play a role in Notch signaling in Drosophila axons, perhaps by binding to the Notch intracellular domain (Giniger, 1998).

The gross morphology of the nervous system is typically normal in N/Abl embryos, but specific axon tracts fail to develop. Axonal defects are observed in all of the nerve tracts that are known to require Notch, i.e., the CNS longitudinal tracts between neuromeres and the lateral portion of the ISN. In contrast, longitudinal tracts within each neuromere and commissural tracts appear normal, as do the dorsal and ventral portions of the ISN. The penetrance (fraction of embryos affected) and expressivity (number of affected hemisegments per affected animal) of the N/Abl axonal phenotype depend on the particular combination of alleles used (Giniger, 1998).

In principle, the axonal defects observe in mature N/Abl embryos could reflect a failure either to form axon tracts or to maintain them. Moreover, if the defect is in the initial development of the axon, it could be due to the absence of required substratum cells, the absence or improper identity of the neurons themselves, or else the failure of the actual guidance machinery of the growth cone. To discriminate among these possibilities, the development of pioneer neurons and substratum cells were examined directly for affected axon tracts. The initial extension of pioneer axons was examined in N/Abl embryos. Consistent with the terminal phenotype, the combined MP fascicle, the first to form between successive neuromeres, is obviously aberrant from a very early stage (st 13). In contrast, both the anterior and posterior commissures appear to develop normally, as do the longitudinal tracts within the neuromeres. The substratum cells for affected axon tracts were examined. The MP fascicle projects between neuromeres on a specific Fasciclin II-expressing glial cell, LG5, and this is present in affected hemisegments. In the PNS, the direct cellular substratum for ISN extension in the lateral part of the embryo is a cluster of lateral peritracheal cells that lie along the trachea. Examination of stalled motor axons in an N/Abl embryo shows that the nerve frays and stalls precisely as it attempts to grow along the trachea. Since substratum cells for affected axon tracts are present in N/Abl embryos, the pioneer neurons themselves were examined. The positions and cell body morphologies of the sensory neurons in the PNS provide sensitive assays for the identities of these cells, and these typically appear to be wild type. The neuron aCC that pioneers the ISN and innervates the most dorsal muscle (muscle 1) is readily apparent in N/Abl embryos. The neurons that pioneer the MP fascicle within the CNS are MP1, pCC, dMP2, and vMP2, and cells whose positions and axonal morphologies are appropriate for these cells can be seen in affected hemisegments of N/Abl embryos (Giniger, 1998).

The observation of morphologically normal pioneer neurons and substratum cells in N/Abl embryos is surprising, since perturbation of cell identity seems a priori to be the simplest explanation for the axonal defects in these embryos. Molecular markers for the development of affected pioneer neurons were therefore examined to determine whether their identities were disturbed in some more subtle way. This analysis suggests that most of the axonal defects in Notch/abl embryos cannot be accounted for on the basis of observed transformations of pioneer neuron identity. The converse question was therefore asked: whether Notch-dependent transformations of pioneer neuron identity are sufficient to produce axonal defects like those observed in N/Abl embryos. Indeed they are not. The data show directly that the Notch-dependent perturbations of cell identity induced in temperature-shifted N^ts embryos are not sufficient to produce the axonal defects observed in these embryos. These results therefore provide strong evidence that the requirement for Notch in axon patterning reflects a function of the protein at the time of axon outgrowth, genetically separable from the role of Notch in the establishment of cell identity (Giniger, 1998).

Abl is localized to developing axons: it is thought that Abl works in the axon directly to control cytoskeletal organization and function. Might Notch also act in the axon to control axon extension directly? Notch is known to be present in mature nerves, but its presence in developing nerves, and specifically in growth cones, has not been investigated. Since Notch expression in substratum cells interferes with visualizing growth cones in situ, the localization of Notch protein was examined in primary Drosophila neurons cultured in vitro. Primary fly embryo neurons were differentiated in culture and analyzed either by indirect immunofluorescence with anti-HRP, to characterize neuronal morphologies, or with anti-Notch. All samples were also labeled with anti-Elav, to verify that the cells being examined were neurons. Notch protein is clearly detected on the entire cell surface, including extending axons, and on a variety of bulbous, spiked, and flattened structures at the tips of axons, which have the appearance of growth cones. To test further whether the Notch-containing structures at the ends of axons are bona fide growth cones, cell preparations were double labeled for Notch and for a known growth cone marker, kinesin-ß-galactosidase. Notch protein is present on the growth cones of axons extending in culture (Giniger, 1998).

What might be the physical basis of the N/Abl genetic interaction? It is unlikely that the absence of Abl is affecting Notch protein levels, since Western analysis of extracts from homozygous abl^- females detects wild-type amounts of Notch protein. Such a mechanism would be expected to alter Notch-dependent cell identities as well as cell morphologies, and this does not generally occur. Might Abl bind Notch directly? This seems unlikely. While Abl contains a variety of protein interaction domains, Notch does not resemble its known ligands. It has recently been shown that the Drosophila Numb protein includes a PTB domain that binds two sites in the intracellular domain of Notch, even when Notch is not phosphorylated. Recalling that the Abl-interacting gene disabled includes a PTB domain closely related to the Numb PTB, and which like Numb can bind to nonphosphorylated targets, a test was performed to discover whether Dab can bind the intracellular domain of Notch in vitro (Giniger, 1998).

Three experiments demonstrate that the PTB domain of Drosophila Disabled binds directly to the intracellular domain of Notch in vitro. (1) Initially, beads bearing a glutathione S-transferase (GST) fusion of the Dab PTB domain were incubated in an extract of total embryo protein. Western analysis of the protein bound by Dab shows that GST-Dab selects Notch protein out of an embryo lysate, whereas GST alone binds only a small amount of Notch nonspecifically. (2) It was next asked what portion of Notch is recognized by Dab. Four protein fragments, each of which represents a distinct functional domain from the intracellular tail of Notch, were expressed. These are the RAM23 region (amino acids 1766-1896); the ankyrin repeats (amino acids 1896-2109); the PEST/OPA region (amino acids 2262-2606), and the notchoid region (amino acids 2612-2703). The four proteins were translated in vitro in reticulocyte lysates and assayed for binding to GST-Dab as above. Of the four Notch domains, only the RAM23 peptide binds to GST-Dab, while none of the four bind to GST alone. This pattern is similar but not identical to the pattern of Notch binding to the Numb PTB: like Dab, the Numb PTB binds to the Notch RAM23 domain but not to the ankyrin repeats or PEST/OPA region. Unlike Dab, Numb does bind to the notchoid domain. (3) Finally, to determine whether the Dab-Notch interaction is direct, a stable and soluble N-terminal fragment of the Notch intracellular domain (amino acids 1767-2235) was purifed from bacteria and its binding to the purified Dab PTB domain was assayed. The Notch intracellular domain is precipitated by GST-Dab beads but not by GST alone, demonstrating that the purified Dab PTB domain can bind directly to purified Notch intracellular domain in vitro (Giniger, 1998).

The data above demonstrate that Notch interacts genetically with Abl and biochemically with Disabled. These results beg the question whether Notch interacts genetically with disabled. Since isolated dab alleles were not available, their genetic interactions with Notch could not be tested directly. It was asked, however, whether flies that are triply heterozygous for all three mutations, Notch, abl, and dab, display any synthetic phenotypes. Flies were constructed that were both heterozygous for a strong Notch allele (N⁸ or N^55e11) and for one of two unrelated chromosomes that bear strong mutations of both Abl and dab. All pairwise combinations cause defects in eye development, giving rise to flies with rough eyes reminiscent of the defective eyes observed in Abl homozygotes (Giniger, 1998).

Given the genetic and biochemical evidence for Abl-Dab interaction, it is attractive to speculate that Dab may act as an adaptor protein that links Notch to Abl in response to a signal from Delta. Recruitment of Abl by Notch would in turn engage the actin cytoskeleton via mechanisms similar to those that have been studied in vertebrate systems. The notion that Notch may use distinct signaling pathways to control different downstream events is consistent with analysis of other signaling receptors. For example, receptor tyrosine kinases typically bind and activate a complex array of intracellular signaling proteins upon ligand induction, and different downstream signaling pathways are often responsible for different aspects of the induced phenotype. Finally, there is extensive precedent for receptors that control cell fate in some developmental contexts and cell motility or axon extension in others (Giniger, 1998 and references).

The interaction of Disabled and Notch might not be the last word in the complex signaling functions involved in axon guidance. Disabled has also been shown to interact with Drk, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2. Drk is required during signaling by the Sevenless receptor tyrosine kinase (Sev). One role for Drk is to provide a link between activated Sev and the Ras1 activator Sos. The ability of activated Ras1 to bypass the requirement for Sev function during R7 development has suggested that the primary function of Sev is to activate Ras. However, the model suggesting that the sole function of activated Sev is to bind Drk-Sos has been questioned by genetic studies that suggest the existence of multiple intracellular signaling pathways downstream of Sev. For example, although the association of Drk and Sos does not depend on the carboxy (C)-terminal SH3 domain of Drk, mutations that affect this domain partially compromise Sev signaling. Furthermore, a C-terminal SH3 domain-truncated Drk cannot rescue the lethality associated with homozygous drk mutations. These data suggest that Drk-binding proteins other than Sos may play important roles in signaling by Sev and other RTKs. The possibility that Drk performs functions other than binding to Sos has been investigated by identification of additional Drk-binding proteins. The phosphotyrosine-binding (PTB) domain-containing protein Disabled binds to the Drk SH3 domains. Taken together, these results indicate that Dab is a novel component of the Sev signaling pathway (Le, 1998).

Disabled has been implicated in other RTK signaling pathways. A murine DAB-related protein, mDAB1, has been identified as a tyrosine-phosphorylated protein that binds to the non-receptor protein tyrosine kinase Src. Recently, several reports have shown that mice lacking mDAB1 function have neuronal defects similar to those seen in reeler mice, including abnormal cortical lamination resulting from disruptions of neuronal migration processes. These results suggest that mDAB1 might participate in a signaling pathway triggered by REELIN, a secreted protein released near the targets of migrating neurons. The neuronal defects associated with Drosophila and mouse dab mutations and the identification of DAB as a putative adaptor protein acting downstream of the receptor tyrosine kinase Sev suggest that Dab may function downstream of many RTKs, including ones required for proper development of the Drosophila central nervous system (Le, 1998).

The Disabled protein functions in Clathrin-mediated synaptic vesicle endocytosis and exoendocytic coupling at the active zone

Members of the Disabled (Dab) family of proteins are known to play a conserved role in endocytic trafficking of cell surface receptors by functioning as monomeric Clathrin-associated sorting proteins that recruit cargo proteins into endocytic vesicles. This study reports a Drosophila disabled mutant revealing a novel role for Dab proteins in chemical synaptic transmission. This mutant exhibits impaired synaptic function, including a rapid activity-dependent reduction in neurotransmitter release and disruption of synaptic vesicle endocytosis. In presynaptic boutons, Drosophila Dab and Clathrin are highly colocalized within two distinct classes of puncta, including relatively dim puncta that are located at active zones and may reflect endocytic mechanisms operating at neurotransmitter release sites. Finally, broader analysis of endocytic proteins, including Dynamin, supported a general role for Clathrin-mediated endocytic mechanisms in rapid clearance of neurotransmitter release sites for subsequent vesicle priming and refilling of the release-ready vesicle pool (Kawasaki, 2011).

These results reveal a function for the Disabled family of Clathrin-associated sorting proteins (CLASPs) in synaptic vesicle endocytosis and further define the molecular basis for a rapid role of endocytic mechanisms in sustaining neurotransmitter release during synaptic activity (Kawasaki, 2011).

By revealing a function for Dab proteins in synaptic vesicle endocytosis, the present study has implicated a novel molecular component as well as an established set of Dab protein interactions in this process. dDab function in synaptic vesicle endocytosis appears to involve interactions with Clathrin, and possibly AP-2, which are likely to be mediated by conserved binding motifs. In addition, the PTB/DH domain of Dab proteins binds phosphoinositides, which are known to play an important role in synaptic vesicle trafficking. Finally, and importantly, the CLASP function of Dab proteins involves PTB/DH domain binding to a sorting motif (NPxY) in the cytosolic domain of cargo proteins (Yun, 2003). An initial survey of Drosophila synaptic vesicle proteins revealed an NPxY motif in the cytoplasmic domain of Drosophila Synaptotagmin 1 (dSyt1). Syt1 proteins play an important role in synaptic transmission by serving as a calcium sensor for neurotransmitter release and also function in synaptic vesicle endocytosis. The dSyt1 NPxY motif (residues 387-390; NPYY), which is identical in mammalian Syt1 proteins and conserved in other Syts, is located within the C2B domain near a basic region previously implicated in SYT1-AP-2 interactions. This motif is of great potential interest because no classic endocytosis signals have been identified previously in Syts. Finally, it is of interest to consider the roles of co-active-zone and non-active-zone populations of dDab (and Clathrin). The rapid-onset synaptic phenotypes observed are likely to reflect localization and function of dDab and Clathrin at the active zone. Further study is required to address whether non-AZ domains may mark an endosomal compartment from which Clathrin-mediated vesicle formation may occur. Thus far, immunocytochemistry with markers for several endosomal membrane compartments did not show strong colocalization with dDab and Clathrin (Kawasaki, 2011).

The preceding considerations raise interesting questions about how the loss of specific dDab molecular interactions may contribute to the resulting synaptic phenotype. It seems unlikely that the dab synaptic phenotype reflects simple mis-sorting of a synaptic vesicle protein, given the similar phenotypes associated with loss of function for several different endocytic proteins. Rather, as described in the following section, it appears that common features of these phenotypes, including a rapid activity-dependent reduction in neurotransmitter release, slowed recovery in paired-pulse depression (PPD), enlarged membrane cisternae, and persistence or accumulation of AZ-associated and docked synaptic vesicles, reflect a general loss of endocytic function. Consistent with this interpretation, dSyt1 exhibited a WT distribution at dab mutant synapses. Thus, either dDab does not participate in dSyt1 sorting or sufficient redundancy is provided by interactions of Syt1 with at least two other Clathrin-associated adaptor proteins, AP-2 and Stonins. Furthermore, the distributions of other synaptic vesicle proteins at dab mutant synapses, including neuronal Synaptobrevin and the vesicular glutamate transporter, were similar to those of WT. Thus, synaptic vesicle composition appears to be preserved in dab. These findings are consistent with the WT excitatory postsynaptic currents observed in response to the first stimulus and complete recovery in PPD, as expected for the presence and recovery of a fully functional release-ready vesicle pool (Kawasaki, 2011).

Finally, dab mutant synapses exhibit strong depression during prolonged stimulation but sustain a reduced steady-state level of neurotransmitter release. This is in contrast to the Dynamin mutant, shi^TS1, in which EPSC amplitudes progressively decline to zero. In light of the severe nature of the molecular lesion in dab^EC1, these findings suggest persistence of residual synaptic vesicle endocytosis in the absence of dDab. This likely reflects redundancy in the mechanisms of synaptic vesicle endocytosis, as shown for several other endocytic proteins (Kawasaki, 2011).

A previous analysis in the shibire (Dynamin) mutant demonstrated a rapid activity-dependent reduction in neurotransmitter release that could not be explained simply by the classic role of Dynamin in recycling synaptic vesicles (Kawasaki, 2000). Rather, it was suggested that accumulation of endocytic intermediates at release sites may occlude fast refilling of the release-ready vesicle pool and that their rapid clearance contributes to maintenance of neurotransmitter release during synaptic activity. Recent studies at the Calyx of Held have further established a rapid role for Dynamin and AP-2 in maintaining neurotransmitter release, which preceded formation of endocytic vesicles, and directly demonstrated its requirement for fast refilling of the release-ready vesicle pool. The present study provides several unique insights into the mechanisms by which endocytic processes regulate exocytosis. First, localization of both dDab and Clathrin at the AZ suggests a local role for endocytic mechanisms near release sites. Second, persistence or accumulation of the docked synaptic vesicle pool at AZs indicates a postdocking role for endocytic mechanisms in synaptic vesicle fusion. Third, the similar electrophysiological and ultrastructural phenotypes observed following loss of Dynamin, dDab, or Clathrin function strongly support a general role for Clathrin-mediated endocytic mechanisms in this process. Finally, comparing and combining endocytic loss of function with a SNAP-25 TS mutant suggests that rapid endocytic mechanisms are required for t-SNARE-mediated synaptic vesicle priming (Kawasaki, 2011).

Together with previous work, the results reported here support a working model in which components of the Clathrin-mediated endocytic machinery first interact at the AZ to clear neurotransmitter release sites and subsequently mediate vesicle formation in the PAZ. Key features of this model are discussed in the following text, including the spatial distribution of endocytic proteins and Clathrin-coated vesicle intermediates with respect to neurotransmitter release sites (Kawasaki, 2011).

The observation of dDab, Clathrin, and AP-2 localization to the AZ was greatly facilitated by the ability to examine isolated AZs at DLM neuromuscular synapses and by the restricted spatial distributions of these proteins. Note that these features are distinct from those of larval neuromuscular synapses and that previous studies suggest no rapid role for Dynamin in exoendocytic coupling in this preparation (Wu, 2005). At adult neuromuscular synapses, the rapid functional roles of Dynamin and DAP160 suggest these proteins are also present at the AZ and participate in early stages of Clathrin-mediated endocytosis. However, their broader distribution within boutons makes it more difficult to confirm localization to the AZ. With respect to Dynamin, which is known to complete vesicle formation through membrane fission (Mettlen, 2009), previous studies have shown it is also present and functionally important at early stages of Clathrin-mediated endocytosis (Evergren, 2004; Loerke, 2009). AZ localization of endocytic proteins might suggest a mechanism of release site clearance involving rapid formation of Clathrin-coated vesicles directly from the AZ; however, this mechanism is not favored, primarily because ultrastructural studies indicate that Clathrin-coated vesicles form at PAZ, rather than AZ, regions of the plasma membrane. Although elongated membrane invaginations occur at the AZ in the shibire (Dynamin) mutant during recovery from massive synaptic vesicle depletion, these do not appear to have Clathrin coats. In the present study, smaller membrane invaginations (Ω-structures) observed at the AZ were not Clathrin-coated but often exhibited filamentous connections with the presynaptic dense body (t-bar). These structures occurred at low frequencies that were not significantly different among all genotypes. It remains unclear whether or not they are related to rapid synaptic vesicle endocytosis and how they may contribute to neurotransmitter release (Kawasaki, 2011).

Regarding the spatial distribution of endocytic intermediates relative to AZs, remarkable ultrastructural analysis has defined a sequence of events in synaptic vesicle exocytosis and endocytosis with high time resolution. Vesicle fusion was observed within several milliseconds after a stimulus and appeared to deposit clusters of large particles into the AZ region of the plasma membrane. Particle clusters were maximally abundant at 20 ms after stimulation and disappeared rapidly over the following 200 ms, consistent with a role for rapid clearance of release sites in synaptic vesicle priming and short-term depression. Particle clusters were thought to dissipate after vesicle fusion, and further analysis suggested they may reassemble within Clathrin-coated pits at the PAZ. In contrast, recent superresolution light microscopy studies showed that native synaptic vesicle proteins deposited in the plasma membrane from different vesicles do not mix before endocytosis but, instead, remain in distinct clusters and are retrieved separately. Moreover, plasma membrane-resident synaptic vesicle proteins may tend to be excluded from the AZ, as observed in conventional confocal imaging of the native synaptic vesicle protein Synaptobrevin. In this current working model, endocytic mechanisms facilitate rapid clearance of synaptic vesicle proteins from release sites either within larger assemblies corresponding to single vesicles or within smaller protein complexes that subsequently assemble in the PAZ as Clathrin-coated pits. A parallel process was described in recent work at DLM neuromuscular synapses demonstrating activity-dependent redistribution of t-SNARE proteins from AZ to PAZ regions, which likely reflects their participation in plasma membrane cis-SNARE complexes (Kawasaki, 2009). In comparing these studies, it is of interest to consider the relative contributions of release site clearance and t-SNARE availability to synaptic vesicle priming and whether AZs maintain their distinctive protein composition, as well as functionality of neurotransmitter release sites, through sorting mechanisms that distinguish among different functional or biochemical states of a protein (Kawasaki, 2011).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 2411

Structural Domains

Amino acids 462 - 673 are removed by alternative splicing. There are 10 potential sites for tyrosine phosphorylation. The 1521-amino acid Abl protein contains one match to the tyrosine phosphorylation consensus (residue 543), which corresponds to the in vivo autophosphorylation site for vAbl. Of the 10 potential tyrosine phosphorylation sites in Dab, 8 are clustered within a 170-amino acid interval that also contains the three 11-amino-acid stretches of alternating acidic and arginine resides (Gertler, 1993). Like the known vertebrate Disabled homologs, Dab is a novel protein (Gertler, 1999).

date revised: 22 November 2022

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