Ive larvae with anti-Brp and anti-Discs Large antibodies and examined both the peripheral nerves and the neuromuscular synapse for defects. P10036 is the only mutation identified to date PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19859661 that causes 
the observed accumulation of anti-Brp staining in peripheral axons. The P10036 transposon resides within an intron of the previously uncharacterized gene CG11489, which resides at chromosomal position 79D and is predicted to encode a member of the 
SRPK family. Due to the dramatic effect on Bruchpilot protein accumulation in peripheral axons, we named this mutant air traffic controller, and we refer to P10036 as srpk79Datc throughout this article. We next developed quantitative measures of the axonal Brp accumulations to further characterize and 946128-88-7 cost analyze the srpk79Datc mutant phenotype. In all cases, genetic controls were dissected, processed, stained, and imaged identically and in parallel with srpk79Datc mutants. We found a statistically significant increase in total nerve Brp fluorescence in srpk79Datc mutants compared to wild-type and heterozygous controls. We also found a highly significant increase in the average puncta fluorescence intensity compared to wild-type and heterozygous controls. Indeed, the entire distribution of puncta intensities was shifted toward larger values. Finally, we estimate that the frequency of these aberrant accumulations corresponds to 0.03 accumulations per micron of individual motor axon length. From these data, we conclude that Brp-positive puncta in srpk79Datc mutant axons represent larger, abnormal, protein aggregates compared to observations made in wild-type axons. Next, we assayed synaptic Brp staining intensity and NMJ morphology in the srpk79Datc mutant. We found that synaptic Brp staining intensity is significantly decreased compared to wild-type animals, assayed as both total Brp fluorescence and as the distribution of individual puncta intensities. This effect occurs at NMJ throughout the animal, and there is no evidence for a strong anteriorposterior gradient of this phenotype. Our data suggest that the accumulation of Brp aggregates in the axon of the srpk79Datc mutant depletes Brp protein from the presynaptic nerve terminal. Aglafoline site Consistent with this conclusion, we found that total Brp protein levels, assayed by western blot, are unaltered in the srpk79Datc mutant background despite the dramatic increase in nerve Brp. We also determined whether the decrease in total Brp fluorescence causes a decrease in total Brp puncta number, which would be indicative of a change in AZ number. We found, however, that Brp puncta density within srpk79Datc mutant NMJs is identical to wild type and that total bouton numbers are wild type in the srpk79Datc mutant background. Moreover, anti-Dlg, anti-Synaptotagmin 1, and anti-Cysteine String Protein staining at srpk79Datc mutant synapses are not different compared to wild type. Thus, synapse growth, morphology, and AZ number appear normal in the srpk79Datc mutant. Consistent with the observed lack of morphological change, we found no change in neurotransmitter release in the srpk79Datc mutant background. We assayed neurotransmission by recording SRPK-Dependent Control of T-Bar Assembly from the third-instar NMJ of homozygous sprk79Datc mutants, as well as homozygous srpk79Datc mutants lacking one copy of the brp gene . In all cases, evoked excitatory junctional potential amplitude and spontaneous miniature EJP amplitudes were wild type. There was also no difference in th.Ive larvae with anti-Brp and anti-Discs Large antibodies and examined both the peripheral nerves and the neuromuscular synapse for defects. P10036 is the only mutation identified to date PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19859661 that causes the observed accumulation of anti-Brp staining in peripheral axons. The P10036 transposon resides within an intron of the previously uncharacterized gene CG11489, which resides at chromosomal position 79D and is predicted to encode a member of the SRPK family. Due to the dramatic effect on Bruchpilot protein accumulation in peripheral axons, we named this mutant air traffic controller, and we refer to P10036 as srpk79Datc throughout this article. We next developed quantitative measures of the axonal Brp accumulations to further characterize and analyze the srpk79Datc mutant phenotype. In all cases, genetic controls were dissected, processed, stained, and imaged identically and in parallel with srpk79Datc mutants. We found a statistically significant increase in total nerve Brp fluorescence in srpk79Datc mutants compared to wild-type and heterozygous controls. We also found a highly significant increase in the average puncta fluorescence intensity compared to wild-type and heterozygous controls. Indeed, the entire distribution of puncta intensities was shifted toward larger values. Finally, we estimate that the frequency of these aberrant accumulations corresponds to 0.03 accumulations per micron of individual motor axon length. From these data, we conclude that Brp-positive puncta in srpk79Datc mutant axons represent larger, abnormal, protein aggregates compared to observations made in wild-type axons. Next, we assayed synaptic Brp staining intensity and NMJ morphology in the srpk79Datc mutant. We found that synaptic Brp staining intensity is significantly decreased compared to wild-type animals, assayed as both total Brp fluorescence and as the distribution of individual puncta intensities. This effect occurs at NMJ throughout the animal, and there is no evidence for a strong anteriorposterior gradient of this phenotype. Our data suggest that the accumulation of Brp aggregates in the axon of the srpk79Datc mutant depletes Brp protein from the presynaptic nerve terminal. Consistent with this conclusion, we found that total Brp protein levels, assayed by western blot, are unaltered in the srpk79Datc mutant background despite the dramatic increase in nerve Brp. We also determined whether the decrease in total Brp fluorescence causes a decrease in total Brp puncta number, which would be indicative of a change in AZ number. We found, however, that Brp puncta density within srpk79Datc mutant NMJs is identical to wild type and that total bouton numbers are wild type in the srpk79Datc mutant background. Moreover, anti-Dlg, anti-Synaptotagmin 1, and anti-Cysteine String Protein staining at srpk79Datc mutant synapses are not different compared to wild type. Thus, synapse growth, morphology, and AZ number appear normal in the srpk79Datc mutant. Consistent with the observed lack of morphological change, we found no change in neurotransmitter release in the srpk79Datc mutant background. We assayed neurotransmission by recording SRPK-Dependent Control of T-Bar Assembly from the third-instar NMJ of homozygous sprk79Datc mutants, as well as homozygous srpk79Datc mutants lacking one copy of the brp gene . In all cases, evoked excitatory junctional potential amplitude and spontaneous miniature EJP amplitudes were wild type. There was also no difference in th.
