Nucleobindin-1 is an evolutionarily conserved, uniquely modular calcium (Ca2+)-binding protein, with a distinctive biological profile. It has been characterized as the major Golgi-associated Ca2+-binding protein, integrally involved in the establishment of the relatively understudied Golgi Ca2+ store, across wide-ranging vertebrate cell types. It has however never been satisfactorily analysed in any model genetic organism.

The present study was thus designed as a foundational effort for long-term in vivo analyses into diverse aspects of Nucleobindin-1 function, in Drosophila melanogaster (dmNUCB1). Three different resources were therefore combinatorially leveraged, as a function of their non-overlapping phenotypic / molecular identities.

Firstly, an adult-viable, fully fertile transposon insertion mutant (dmnucb1c01508) was analysed in terms of predicted truncation of the G protein α-subunit Binding and Activating motif. This homozygous allele did not manifest any obvious fitness-related, morphological / developmental or behavioural abnormalities by itself. dmnucb1c01508 however dominantly rescued pleiotropic mutant phenotypes arising from impaired IP3 receptor (ITPR) activity. It was additionally also shown to considerably reverse lifespan and mobility improvements effected by itpr mutants, in a Drosophila model of Amyotrophic Lateral Sclerosis. Homology modelling further predicted significant conformational conservation in Drosophila, of biochemically validated determinants that dictate the highly specific interaction of vertebrate NUCB1 with Gαi1/3.

Male infertility in a different adult-viable mutant (dmnucb1f04688) was refined to the level of disorganized sperm bundles, severely depleted seminal vesicles and smaller accessory glands. The associated transposon insertion was localized to the 3 end of the lone intron in dmnucb1. This allele also exhibited cardiac arrhythmia-like phenotypes in Optical Coherence Tomography assays. Both sets of defects were significantly rescued against the background of itpr mutants. We speculate that dmnucb1f04688 genetically likely represents a neomorph and phenotypically, a hypermorph, reflecting unregulated cytosolic Ca2+ levels (in contrast to the presumed, relatively nuanced, Gα-based regulation in dmnucb1c01508).

Independent experiments involving RNAi strategies and transposon insertion mutants alike, revealed multi-level pro-, as well as anti-apoptotic, involvements for dmNUCB1. These investigations implicate dmNUCB1 as a putative convergence point uniquely linking unconventional activation of a noncanonical Ca2+ store with ubiquitination pathways, in apoptosis. Targeted muscle-specific knockdown also uncovered hitherto unsuspected, largely Ca2+-independent roles for dmNUCB1 in sarcomere assembly. At the level of Transmission Electron Microscopy, a specific protein aggregate myopathy never reported to date, with respect to any Golgi-associated protein was further evidenced.

In conclusion, our nuanced reverse genetic approaches have yielded at least five non-overlapping functional involvements for dmNUCB1. The associated assay systems are likely to yield valuable insights with regard to meaningfully re-evaluating the Golgi Ca2+ store across diverse functional contexts, as also specific disease states.

Publications
1) Vidhya Balasubramanian and Bharath Srinivasan. Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate mediated intracellular calcium homeostasis Genome. 2020 Feb;63(2):61-90. doi: 10.1139/gen-2019-0113.

2) Aradhyam GK, Balivada LM, Kanuru M, Vadivel P, Vidhya B.S. Calnuc: Emerging roles in calcium signalling and human diseases IUBMB Rummy. 2010 Jun;62(6):436-46. doi: 10.1002/iub.341.