D a similar structure in wild-type axons. In this study, we performed electron microscopy on five wild-type animals, analyzing 150 sections from the segmental nerves. None of these sections showed evidence of electron-dense aggregates. We performed electron microscopy on nine srpk79D mutant animals, analyzing 325 sections from segmental nerves. Sections from every mutant animal showed evidence of electrondense plaques. Nearly every section from an individual mutant showed evidence of electron-dense plaques, consistent with the highly penetrant phenotype observed at the light level. The dimensions of these electron-dense structures, the prevalence of SRPK-Dependent Control of T-Bar Assembly 8 SRPK-Dependent Control of T-Bar Assembly these structures in our electron microscopy sections and the similarity of their shape to T-bars present at the AZ strongly suggest that these structures represent the large Brp aggregates that we observe at the light level in the srpk79D mutant background. Finally, similar to T-bars found at AZs, these electron-dense structures were surrounded by a filamentous matrix. 
Although vesicles were also observed in these areas, we believe that they are molecularly distinct from synaptic vesicles because synaptic vesicle markers do not colocalize with Brp in the srpk79D mutant axons. In contrast, synaptic ultrastructure in srpk79D mutants is identical to wild type. Thus, loss of srpk79D leads to the SB-1317 biological activity formation of T-barlike superassemblies in axons. Since we have never observed T-barlike structures in wild-type axons, we propose that SRPK79D is required as part of a mechanism that normally suppresses premature T-bar assembly in the axon. caused by BRP overexpression are not dramatically enhanced by mutating one copy of the srpk79D gene. To further address this issue, we asked whether Brp aggregates form in homozygous srpk79Datc mutants in which we decrease total Brp levels by removing one copy of the brp gene . We found that large axonal Brp aggregates persisted even when one copy of the brp gene is removed in the background of the srpk79Datc 
homozygous mutant. Taken together, these experiments indicate that an SRPK79D-dependent elevation in Brp protein is not the direct cause of premature Tbarlike assembly formation in the axon. We therefore favor an alternative model based upon the observation that SRPK79D PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19858302 colocalizes with Brp and speculate that SRPK79D could sequester or inhibit the function of axonal T-bar proteins and thereby prevent the formation of axonal T-barlike superassemblies. SRPK79D Colocalizes with Brp in the Axon and at the Active Zone To gain insight into the subcellular distribution of SRPK79D, we generated Venus-tagged srpk79D transgenes under UAS control and expressed these transgenes in Drosophila neurons. We found that neuronally expressed VenusSRPK79D-RD, which rescues axonal Brp accumulations, precisely colocalizes with Brp in both the nerve and at each Amezinium metilsulfate presynaptic AZ. The voltage-gated calcium channel Cacophony is among very few other proteins that have been demonstrated to colocalize with Brp at the presynaptic AZ. Furthermore, Venus-SRPK79D-RD is highly unusual in that this protein has been shown to colocalize with Brp in a wildtype axon. These data suggest that SRPK79D closely associates with Brp during axonal transport of the Brp protein to the presynaptic nerve terminal. It is possible that the distribution of the tagged-SRPK79D protein does not reflect the wild-type SRPK79D protein distri.D a similar structure in wild-type axons. In this study, we performed electron microscopy on five wild-type animals, analyzing 150 sections from the segmental nerves. None of these sections showed evidence of electron-dense aggregates. We performed electron microscopy on nine srpk79D mutant animals, analyzing 325 sections from segmental nerves. Sections from every mutant animal showed evidence of electrondense plaques. Nearly every section from an individual mutant showed evidence of electron-dense plaques, consistent with the highly penetrant phenotype observed at the light level. The dimensions of these electron-dense structures, the prevalence of SRPK-Dependent Control of T-Bar Assembly 8 SRPK-Dependent Control of T-Bar Assembly these structures in our electron microscopy sections and the similarity of their shape to T-bars present at the AZ strongly suggest that these structures represent the large Brp aggregates that we observe at the light level in the srpk79D mutant background. Finally, similar to T-bars found at AZs, these electron-dense structures were surrounded by a filamentous matrix. Although vesicles were also observed in these areas, we believe that they are molecularly distinct from synaptic vesicles because synaptic vesicle markers do not colocalize with Brp in the srpk79D mutant axons. In contrast, synaptic ultrastructure in srpk79D mutants is identical to wild type. Thus, loss of srpk79D leads to the formation of T-barlike superassemblies in axons. Since we have never observed T-barlike structures in wild-type axons, we propose that SRPK79D is required as part of a mechanism that normally suppresses premature T-bar assembly in the axon. caused by BRP overexpression are not dramatically enhanced by mutating one copy of the srpk79D gene. To further address this issue, we asked whether Brp aggregates form in homozygous srpk79Datc mutants in which we decrease total Brp levels by removing one copy of the brp gene . We found that large axonal Brp aggregates persisted even when one copy of the brp gene is removed in the background of the srpk79Datc homozygous mutant. Taken together, these experiments indicate that an SRPK79D-dependent elevation in Brp protein is not the direct cause of premature Tbarlike assembly formation in the axon. We therefore favor an alternative model based upon the observation that SRPK79D PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19858302 colocalizes with Brp and speculate that SRPK79D could sequester or inhibit the function of axonal T-bar proteins and thereby prevent the formation of axonal T-barlike superassemblies. SRPK79D Colocalizes with Brp in the Axon and at the Active Zone To gain insight into the subcellular distribution of SRPK79D, we generated Venus-tagged srpk79D transgenes under UAS control and expressed these transgenes in Drosophila neurons. We found that neuronally expressed VenusSRPK79D-RD, which rescues axonal Brp accumulations, precisely colocalizes with Brp in both the nerve and at each presynaptic AZ. The voltage-gated calcium channel Cacophony is among very few other proteins that have been demonstrated to colocalize with Brp at the presynaptic AZ. Furthermore, Venus-SRPK79D-RD is highly unusual in that this protein has been shown to colocalize with Brp in a wildtype axon. These data suggest that SRPK79D closely associates with Brp during axonal transport of the Brp protein to the presynaptic nerve terminal. It is possible that the distribution of the tagged-SRPK79D protein does not reflect the wild-type SRPK79D protein distri.