Disabled

DEVELOPMENTAL BIOLOGY

Embryonic

The embryonic expression pattern of Dab mRNA, examined by in situ hybridization to whole-mount embryos, shows uniform expression from blastoderm through gastrulation. At the end of germ-band extension, higher levels of Dab mRNA are detected in the mesoderm and in the CNS. Polyclonal rabbit antibody to Dab detects a similar distribution. In the cellular blastoderm, immunohistochemical staining for Dab protein is detected in the cytoplasm and not the nucleus. During gastrulation, Dab protein is broadly distributed, but is higher in the CNS. Later in development, the signal in the CNS is concentrated in axon bundles. Dab protein is also detected in PNS cell clusters and the body wall musculature (Gertler, 1993).

Effects of Mutation or Deletion

During Drosophila embryogenesis, Abl is localized in the axons of the central nervous system (CNS). Mutations in Abl have no detectable effect on the morphology of the embryonic CNS; the mutant animals survive to the pupal and adult stages. However, in the absence of Abl function, heterozygous mutations or deletions of Disabled (Dab) exert dominant effects, disrupting axonal organization and shifting the lethal phase of the animals to embryonic and early larval stages. Embryos that are homozygous mutant for both Abl and Dab fail to develop any axon bundles in the CNS, although the peripheral nervous system and the larval cuticle appear normal. The genetic interaction between these two genes begins to define a process in which both the Abl tyrosine kinase and Enabled participate in establishing axonal connections in the embryonic CNS of Drosophila (Gertler, 1989).

In the absence of the Drosophila Abl protein-tyrosine kinase (PTK), loss-of-function mutations in either Disabled or prospero have dominant phenotypic effects on embryonic development. Molecular and genetic characterizations indicate that the products of these genes interact with the Abl PTK by different mechanisms. The interaction between Abl and prospero, which encodes a nuclear protein required for correct axonal outgrowth, is likely to be indirect. In contrast, the product of Disabled may be a substrate for the Abl PTK. The Disabled protein is colocalized with Abl in axons; its predicted amino acid sequence contains 10 motifs similar to the major autophosphorylation site of Abl, and the protein is recognized by antibodies to phosphotyrosine (Gertler, 1993).

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, Disabled, reveals that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase (Hill, 1995).

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).

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date revised: 5 August 2011

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