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Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. We therefore expressed a low affinity Ca2+ indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau ∼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching ∼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions.SIGNIFICANCE STATEMENT Phototransduction in Drosophila is mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca2+ stores play any role. We therefore engineered flies expressing a genetically encoded Ca2+ indicator in the photoreceptor ER. Although NCX Na+/Ca2+ exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca2+ stores mediated by Na+/Ca2+ exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.02.041 Byline: Kristy Doyle, Justin Hogan, Meagan Lester, Simon Collier Keywords: Drosophila; Planar Cell Polarity; Wing membrane topography; Prickle; Frizzled Abstract: The Drosophila wing is a primary model system for studying the genetic control of epithelial Planar Cell Polarity (PCP). Each wing epithelial cell produces a distally pointing hair under the control of the Frizzled (Fz) PCP signaling pathway. Here, we show that Fz PCP signaling also controls the formation and orientation of ridges on the adult wing membrane. Ridge formation requires hexagonal cell packing, consistent with published data showing that Fz PCP signaling promotes hexagonal packing in developing wing epithelia. In contrast to hair polarity, ridge orientation differs across the wing and is primarily anteroposterior (A-P) in the anterior and proximodistal (P-D) in the posterior. We present evidence that A-P ridge specification is genetically distinct from P-D ridge organization and occurs later in wing development. We propose a two-phase model for PCP specification in the wing. P-D ridges are specified in an Early PCP Phase and both A-P ridges and distally pointing hairs in a Late PCP Phase. Our data suggest that isoforms of the Fz PCP pathway protein Prickle are differentially required for the two PCP Phases, with the Spiny-legs isoform primarily active in the Early PCP Phase and the Prickle isoform in the Late PCP Phase. Author Affiliation: Department of Biological Sciences, Marshall University, 1 John Marshall Drive, Huntington, WV 25755, USA Article History: Received 5 November 2007; Revised 31 January 2008; Accepted 21 February 2008

Much of our understanding of the genetic mechanisms that control planar cell polarity (PCP) in epithelia has derived from studies of the formation of polarized cell hairs during Drosophila wing development. The correct localization of an F-actin prehair to the distal vertex of the pupal wing cell has been shown to be dependent upon the polarized subcellular localization of Frizzled and other core PCP proteins. However, the core PCP proteins do not organize actin cytoskeletal polarity directly but require PCP effector proteins such as Fuzzy and Inturned to mediate this process. Here we describe the characterization of a new PCP effector gene, fritz, that encodes a novel but evolutionarily conserved coiled-coil WD40 protein. We show that the fritz gene product functions cell-autonomously downstream of the core PCP proteins to regulate both the location and the number of wing cell prehair initiation sites.

The Drosophila Prickle LIM domain protein is discussed. Balance between isoforms of the protein is critical for planar polarity in imaginal discs. The polarity gene prickle (pk) encodes a protein with a triple LIM domain and a novel domain that is seen in human, mouse and Caenorgabditis elegans homologs now designate PET.