Genetic Analyses Uncover Pleiotropic Compensatory Roles for Drosophila Nucleobindin-1 in Inositol Trisphosphate- Mediated Intracellular Calcium Homeostasis
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Genome Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate- mediated intracellular calcium homeostasis Journal: Genome Manuscript ID gen-2019-0113.R2 Manuscript Type: Article Date Submitted by the 11-Sep-2019 Author: Complete List of Authors: Balasubramanian, Vidhya; Indian Institute of Technology Madras, Department of Biotechnology SRINIVASAN, BHARATH; Indian Institute of Technology Madras, DepartmentDraft of Biotechnology Intracellular calcium homeostasis, Golgi, IP<sub>3</sub> receptor, Keyword: <i>Drosophila melanogaster</i>, Nucleobindin-1 Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 86 Genome Page 1 of 49 1 Title Page 2 Genetic analyses uncover pleiotropic compensatory roles for Drosophila 3 Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium 4 homeostasis 5 6 Authors: Vidhya Balasubramanian1 and Bharath Srinivasan1* 7 1Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute 8 of Technology-Madras, Chennai, 600036, India. 9 Telephone: +9181222 48706 10 11 Email: [email protected] 12 *Corresponding Author 13 Draft 14 15 16 17 18 19 20 21 22 23 24 25 26 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 86 Page 2 of 49 27 Abstract: 28 Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly 29 localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative 30 involvements in intracellular calcium (Ca2+) homeostasis have however never been 31 phenotypically characterized in any model organism. We have analyzed an adult-viable mutant 32 that completely disrupts the G protein α-subunit Binding and Activating (GBA) motif of 33 Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious 34 fitness-related, morphological / developmental or behavioral abnormalities. A single copy of this 35 mutation or the knockdown of dmnucb1 in restricted sets of cells, however variously rescues 36 pleiotropic mutant phenotypes arising from impaired Inositol 1,4,5-trisphosphate receptor 37 (IP3R) activity (in turn depleting cytoplasmicDraft Ca2+ levels across diverse tissue types). 38 Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and 39 mobility improvements effected by IP3R mutants, in a Drosophila model of Amyotrophic 40 Lateral Sclerosis. Homology modeling-based analyses further predict a high degree of 41 conformational conservation in Drosophila, of biochemically validated structural determinants 42 in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of 43 NUCB1 with Gαi subunits. The broad implications of our findings are hypothetically discussed, 44 regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in 45 health and disease. 46 47 List of Keywords 48 Nucleobindin-1, Intracellular calcium homeostasis, Golgi, IP3 receptor, Drosophila 49 50 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 86 Genome Page 3 of 49 51 1. Introduction: 52 An elaborate ‘toolkit’ is well known to orchestrate Ca2+ signaling phenomena in diverse 53 vertebrate and invertebrate cell types with varied spatiotemporal dynamics, ranging from a brief 54 localized increase to repetitive spikes and waves spreading across wider regions. Ca2+ channels 55 on the plasma membrane and the IP3 receptor on the endoplasmic reticulum (ER) facilitate rapid 56 increases in cytosolic Ca2+. These are buffered by the action of various Ca2+-binding proteins 57 and also sequestered by Ca2+ pumps on the plasma membrane or intracellular membranes [such 58 as the Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA) or the Secretory Pathway 59 Ca2+/Mn2+-ATPase (SPCA), on the ER] and Ca2+ exchangers at both locations (Berridge et al. 60 2003; Chorna and Hasan 2012). The ER has traditionally been regarded as the primary store of 61 intracellular Ca2+ and its depletion is wellDraft established to activate the Store Operated Calcium 62 Entry (SOCE) pathway, to facilitate extracellular Ca2+ influx through the Orai plasma membrane 63 channel (Taylor and Machaca 2019). In recent years however, evidence has been accumulating 64 to suggest that other organelles such as the Golgi apparatus, mitochondria, peroxisomes and 65 endolysosomal compartments also store significant amount of ionic Ca2+, although the 66 functional implications of these stores are only beginning to be appreciated (Michelangeli et al. 67 2005). 68 69 Nucleobindin-1 (also known as NUCB1 or Calnuc) is a multi-domain EF-hand calcium-binding 70 protein, phylogenetically conserved from worms to humans (Aradhyam et al. 2010). It has been 71 identified as the major Golgi-associated calcium (Ca2+) binding protein and to be involved in the 72 establishment and maintenance of an agonist-mobilizable Golgi Ca2+ store, in a wide variety of 73 vertebrate cell and tissue types (Lin et al. 1998; Lin et al. 1999). Indeed, NUCB1 has also been https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 86 Page 4 of 49 74 identified as the second most abundant Golgi-associated protein in a quantitative proteomics 75 study evaluating fractions isolated from rat liver (Gilchrist et al. 2006). 76 77 Biochemical and cell culture-based approaches have made important contributions to the 78 understanding of select aspects of NUCB1 function (Garcia-Marcos et al. 2011; Kapoor et al. 79 2010; Weiss et al. 2001). Vertebrate NUCB1 has been shown through these approaches, to 80 harbor a motif of 15-25 amino acids (aa) that can directly bind to G protein α subunits and 81 stimulate their guanine nucleotide exchange activity, at the cytoplasmic surface of Golgi 82 membranes and without the involvement of a G protein coupled Receptor (GPCR) (Aznar et al. 83 2016; Garcia-Marcos et al. 2011). Among the small class of proteins currently known to contain 84 this G protein α-subunit Binding and ActivatingDraft (GBA) motif, vertebrate NUCB1 and its paralog 85 NUCB2 are the only members known to bind Ca2+ and the only ones localizing primarily to the 86 Golgi. Indeed, since the GBA motif in these proteins overlaps with one of their EF hands, 87 binding of Ca2+ has been shown to abolish their respective interactions with Gαi1/3 subunits. 88 Despite their relatively unique and highly interesting profiles, however, potential in vivo roles 89 for NUCB1 or NUCB2 have not been represented by informative mutant phenotypes in any 90 model genetic organism. 91 92 Drosophila melanogaster (henceforth referred to as, simply, Drosophila, except where it needs 93 to be distinguished from other Drosophila species) is among the most tractable experimental 94 organisms and routinely affords a wealth of resources for molecular genetic investigations, 95 especially in relation to Ca2+ signaling phenomena and the modeling of many human diseases 96 (Chorna and Hasan 2012; Ugur et al. 2016). NUCB1 is represented by a single copy gene in the 97 Drosophila genome, in turn encoding just a single isoform [as opposed to vertebrate genomes in https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 86 Genome Page 5 of 49 98 which the NUCB1 and NUCB2 / NEFA genes both encode multiple isoforms (Kawano et al. 99 2000; Aradhyam et al. 2010)]. Potential hurdles arising from functional redundancy with respect 100 to in vivo genetic approaches are thus easily circumvented in Drosophila. Most of the functional 101 domains predicted for vertebrate NUCB1 are also conserved in the Drosophila homolog, which 102 shares an overall sequence similarity of 58% with its human counterpart (Otte et al. 1999). 103 104 This study was undertaken to examine whether genetic disruption of the GBA motif in the 105 Drosophila NUCB1 ortholog could be characterized in terms of discrete mutant phenotypes. 106 Our efforts were also aimed at clarifying whether such impairment could be compared or 107 genetically linked with independently documented phenotypes known to reflect specific 108 molecular aspects of intracellular Ca2+Draft homeostasis. Mutants corresponding to the Drosophila 109 IP3 receptor were preferred in this regard, since it represents genetically, the best-studied core 110 component of intracellular Ca2+ homeostasis in Drosophila (Chorna and Hasan 2012). Wherever 111 possible, knockdown approaches in restricted cell, tissue or organ types were also attempted to 112 supplement these primary goals. Our approaches were intentionally broad-based in design, as 113 opposed to being focused on a specific cell / tissue / organ type or biological process. This 114 aspect of our work was based not only on the wide expression patterns of NUCB1 in vertebrates 115 and invertebrates, but also on the fact that neither NUCB1, nor any GBA motif harboring protein 116 has been genetically analyzed in any model organism, to date. 117 118 We report that a piggyBac transposon insertion in the GBA motif of Drosophila NUCB1 (that 119 presumably renders the motif functionally inactive) does not affect the overall fitness of flies, 120 even in the homozygous condition. Flies carrying two copies of this insertion are also 121 phenotypically normal with regard to many different behavioral or morphological assessments. https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 86 Page 6 of 49 122 A single copy of this insertion however variously rescues wing posture, cold sensitivity