Analysis of the Dstac Gene, a Novel Regulator of Neuronal Function and Behavior in Drosophila Melanogaster

Abstract

The stac genes encode a family of proteins with conserved CRD (cysteine-rich domain) and SH3 (Src Holomogy 3) domains found throughout the animal kingdom. stac1 and stac2 are expressed by subsets of neurons, and stac3 by skeletal muscles in vertebrates. One process regulated by a Stac protein is excitation-contraction (EC) in vertebrate skeletal muscles. EC coupling is the process that transduces changes in muscle membrane potential to increases in cytosolic Ca2+ that initiate muscle contraction. EC coupling is mediated by the interaction between the L-type voltage gated calcium channel (Cav channel), DHPR, in the transverse tubules (T tubules) and the ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR). Studies from the Kuwada lab demonstrated that Stac3 regulates EC coupling by regulating the stability, organization and voltage dependency of L-type Cav channel in vertebrate skeletal muscles. The goal of this dissertation is to understand the function of stac genes expressed by neurons, which was completely unknown. Towards this goal, we identified the stac gene in Drosophila melanogaster (Dstac) in order to take advantage of its unparalleled genetic toolbox and found that Dstac is expressed both by muscles and by specific classes of neurons. We first investigated the muscle function of Dstac. We found that Dmca1D, the sole Drosophila L-type Cav channel, and Dstac were expressed in stripes within muscles. Knocking down Dmca1D or Dstac selectively in larval Drosophila body-wall muscles reduced Ca2+ transients during locomotion. Furthermore, immunolabeling showed decreased Dmca1D levels in Dstac mutant muscles, showing that Dstac regulates L-type Cav channels as does Stac3 in vertebrate skeletal muscles. These results suggest that muscle Dstac regulates Dmca1D, which induces cytosolic Ca2+ increases for proper EC coupling in Drosophila body-wall muscles. In the Drosophila adult brain, we found that a set of clock neurons that releases the neuropeptide, pigment dispersing factor (PDF), to regulate circadian rhythm expresses Dstac as well as Dmca1D. Interestingly, selective knockdown of Dstac in PDF neurons disrupted circadian activity. This was the first neural function identified for a Stac protein. The results suggested the hypothesis that Dstac might regulate the Dmca1D L-type Cav channel and this in turn regulates the release of PDF for normal circadian rhythm. We found that Dstac is also expressed by a subset of neurons including motor neurons in the larval CNS. We examined whether Dstac might regulate Dmca1D and neuropeptide release at the larval neuromuscular junction to take advantage of the accessibility of motor boutons for cellular and physiological analysis. We found that Dstac, Dmca1D and the proctolin neuropeptide are expressed by motor boutons. Previously it was shown that proctolin enhances muscle contractions in various insects including Drosophila. By a combination of immunolabeling, Ca2+ imaging, electrophysiology, live imaging of neuropeptide release and behavioral analysis in genetically manipulated larvae we found that Dstac regulates the voltage response of Dmca1D channels and the release of neuropeptides from motor boutons to regulate locomotion by larvae. Since Dstac is expressed by other neuropeptide containing neurons, including the PDF+ clock neurons, in the CNS our results may be applicable to other neurons in the Drosophila CNS. Furthermore the expression of Stac1 and Stac2 in neurons in the vertebrate nervous system opens the intriguing possibility that these Stac proteins may also regulate the release of neuropeptides in at least some vertebrate neurons.