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First published online January 16, 2009
Journal of Experimental Biology 212, 387-400 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.024513
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Organellar calcium signalling mechanisms in Drosophila epithelial function

Shireen A. Davies and Selim Terhzaz

Integrative and Systems Biology Group, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK


Figure 1
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  		Fig. 1. Aequorin action. Reversible binding of calcium to apoaequorin in the presence of coelenterazine and oxygen. Based on: http://biophysics.bumc.bu.edu/faculty/head/index.ht

 

Figure 2
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  		Fig. 2. Diagrammatic summary of protein-based calcium reporters. (A) As shown in Fig. 1, the apoprotein apoaequorin (rod-shaped structure) binds calcium leading to peroxidation (red circles indicate the peroxide) of the coenzyme, coelenterazine (blue), resulting in the release of blue light. (B) GFP-based probes (a) Cameleon, based on fusion of calmodulin (CaM) and the CaM-binding peptide M13 with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Binding of Ca2+ leads to FRET between CFP and YFP, with decreased CFP fluorescence and increased YFP fluorescence. (b) Camgaroo probe: Ca2+-induced conformational change in CaM leads to an increased YFP fluorescence. (c) Pericam: the Ca2+-induced interaction between CaM and M13 leads to changes in the fluorescence characteristics of circularly permuted (cp)YFP. The EGFP pericam is similar. Figure reproduced with permission from Rudolf et al. (Rudolf et al., 2003Go).

 

Figure 3
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  		Fig. 3. Spatial resolution of calcium signals in vivo. (A–E) shows schematic diagrams of GAL4 expression patterns (green) in the Malpighian tubule of adult Drosophila. (A) Ubiquitous expression via heat-shock GAL4. (B) c42 GAL4 driver, which drives expression in the principal cells in main segment, bar-shaped cells of initial and transitional segments, and lower tubule. (C) c724 GAL4 driver, which marks all stellate cells in main segment, and bar-shaped cells of initial and transitional segments (Sozen et al., 1997Go). (D) Line 649 marks bar-shaped cells. (E) Line 507 marks the lower tubule and ureter. (F) Representation of paired anterior tubules connected to the hindgut by the ureter (u), with initial (i), transitional (t), main (m) and lower (l) segments also indicated. (G–J) GAL4-driven GFP expression, confirming expression expected as shown in panels A–E. (K–P) Typical calcium responses (in nmol Ca2+) of the different GAL4/UAS-apoaequorin lines to 1 µmol CAP2b are shown. The inset in panel M shows the typical response of the c724/aequorin Malpighian tubule to 10–8 mol leucokinin, showing that lack of response to CAP2b is not due to a compromised tubule. Reproduced from Rosay et al. (Rosay et al., 1997Go).

 

Figure 4
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  		Fig. 4. Differential responses of principal and stellate cells to thapsigargin in intact Malpighian tubule. (A,B) Representative [Ca2+]cyt traces for (A) Type I cells (c42-aeq) and (B) Type II cells (c710-aeq) in normal medium, and stimulated with 1 mmol thapsigargin (arrow). Thapsigargin increases [Ca2+]cyt in both type I and type II cells. (C,D) Representative [Ca2+]cyt traces for (C) Type I cells (c42-aeq) and (D) Type II cells (c710-aeq) in Ca2+-free medium, and stimulated with 1 mmol thapsigargin (arrow). Thapsigargin increases [Ca2+]cyt only in type II cells. From Rosay et al. (Rosay et al., 1997Go).

 

Figure 5
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  		Fig. 5. Immunocytochemistry of ryanodine receptor (RyR) in Malpighian tubules. RyR expression (red staining) in principal cells in intact Malpighian tubules using a rabbit polyclonal antibody to Drosophila RyR verified in Pollock (Pollock, 2005Go). (A) Red staining is observed in only principal cells; note exclusion of star-shaped stellate cell from staining. (B) Higher magnification shows reticular pattern of RyR expression, in a principal cell containing a DAPI-stained nucleus. Data from S.T.

 

Figure 6
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  		Fig. 6. Drosophila sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in Malpighian tubules. (A) Immunocytochemical staining in tubule principal cells using a mouse monoclonal antibody (VE121G9) against SERCA1 ATPase from skeletal muscle SR (Knudson et al., 1989Go). (B) Green fluorescent protein (GFP) was C-terminally fused to the open reading frame of CG3725 and cloned into the UAS transformation vector pUAST. Transgenic UAS-CG3725 flies were generated, and crossed to the principal cell driver, c42 to target SERCA expression to principal cells. Intact tubules were dissected and imaged with confocal microscopy. (C) Transgenic lines bearing an RNAi contruct against the SERCA gene were generated from plasmid constructs as follows: an inverted repeat of a 685 base pair fragment of SERCA was cloned into the P-element vector pWIZ (Lee and Carthew, 2003Go). Primers (5'-GGGCTCTAGAGATGGAAGACGGTCACTCG-3' and 5'-GGGCTCTAGACAGGCCAGTGCCGATGACG-3') were used to generate the SERCA region containing AvrII restriction sites on both ends: the PCR product was subcloned into the pWIZ vector using AvrII and NheI sites as a tail–tail inverted repeat flanking the w (white) intron. The cloning of the SERCA fragment into pWIZ was analysed by restriction analysis and verified by sequencing. Transgenic lines were generated using standard methods for P-element-mediated germline transformation (BestGene Inc, USA) with independent insertions of pWIZ-SERCA. The transgenic line was crossed with a c42/aequorin line, which allows expression of aequorin and SERCA RNAi in Malpighian tubule principal cells (see Fig. 1). Calcium levels were measured via luminometry in intact Malpighian tubules, which were treated with 10–7 mol l–1 (final concentration) of capa-1 (Kean et al., 2002Go) to stimulate PLC-mediated IP3 and calcium signalling. A typical calcium trace from such an experiment is shown. (D) Pooled data from experiments shown in C, data are nmol [Ca2+] ± s.e.m., N=8. `Primary' refers to the fast (ms) rise in Ca2+ (panel C) whereas `secondary' refers to the slower, sustained Ca2+ rise (panel C). Significance assessed by Student's t-test, where P<0.05 (*) or P<0.001 (***). Data from S.T.

 

Figure 7
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  		Fig. 7. Intracellular localisation of SPoCk isoforms in vivo in principal cells of the Malpighian tubule. (A) Golgi apparatus localisation of SPoCk A, shown by staining of c-myc tagged-SPoCk A with Texas Red secondary antibody, in principal cells of the tubule (B–D) Colocalisation (D) of SPoCk A [c-myc tagged, FITC secondary antibody, green (B)] with affinity-purified rabbit anti-SPoCk A (red; C). (E) ER-localisation of SPoCk B. The nucleus is labeled blue with 4',6-diamidino-2-phenylindole (DAPI). (F–H) Colocalization (H) of SPoCk B [green fluorescent protein (GFP)-tagged, green; F] with blue ER tracker dye (blue; G). (I) Peroxisomal localisation of SPoCk C. Endogenous SPoCk C in the tubule was detected with a SPoCk C-specific antibody; nuclei are labelled blue with DAPI. (J–L) Colocalisation (L) of SPoCk C [yellow fluorescent protein (YFP)-tagged, green; J] with catalase (red; K). All scale bars, 10 µm. Reproduced with permission from Southall et al. (Southall et al., 2006Go). The specificity of all antibodies, including anti-catalase and anti-COPII antibodies was verified by Western blot analysis (Southall et al., 2006Go).

 

Figure 8
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  		Fig. 8. SPoCk A modulates calcium signalling and fluid transport. (A) UAS-SPoCk A was crossed with a c42/aequorin line, which allows expression of aequorin and SPoCk A in Malpighian tubule principal cells (see Fig. 1). Calcium levels were measured via luminometry in intact tubules, which were treated with 10–7 mol (final concentration) of capa-1 (Kean et al., 2002Go). A typical calcium trace from such an experiment is shown, comparing SPoCk A over-expressors (aeq/+; SPoCk A/+; c42, red) with parental c42/aequorin tubules (black). Cytosolic calcium levels are expressed as nmol [Ca2+]cyt (y-axis). (B) Pooled data from experiments shown in A, including data from SPoCk B and SPoCk C over-expressors. Data are nmol [Ca2+] ± s.e.m., N=8. `Primary' refers to the fast (ms) rise in Ca2+ (panel A) whereas `secondary' refers to the slower, sustained Ca2+ rise (panel A). (C) Treatment of intact SPoCk A/+; c42 tubules with Brefeldin A (panel `BFA') shows disruption of Golgi and loss of Golgi-associated SPoCk A (c-myc epitope-tagged, green). (D) Brefeldin A (BFA) treatment results in significantly lower capa-1-stimulated primary calcium rises in aeq/+; SPoCk-A/+; c42 compared with parental c42/aequorin Malpighian tubules. Data are nmol [Ca2+] ± s.e.m., N=8. (E) Over-expression of SPoCk A in tubule principal cells (red) is sufficient to confer increased basal rates of fluid transport, as well as increased capa-1-stimulated fluid transport, compared with parental lines (black, blue). Data are expressed as mean fluid transport rates over time (nl min–1), ± s.e.m., N=10. Significance assessed by Student's t-test, where P<0.05 (*), P<0.01 (**) or P<0.001 (*). From Southall et al. (Southall et al., 2006Go).

 

Figure 9
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  		Fig. 9. Calcium and mitochondrial activity in capa-1 stimulated Malpighian tubules. Left panel: confocal section through mitycam-expressing tubule, showing packing of mitochondria into apical (luminal) microvilli). Right three panels: schematic of selective action of capa-1 on apical microvilli, under resting conditions, within a second of capa-1 stimulation, and during the sustained phase 1–10 min later. Reproduced with permission from Terhzaz et al. (Terhzaz et al., 2006Go).

 

Figure 10
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  		Fig. 10. Functional correlates of SPoCk C. (A) Sexually dimorphic SPoCk C expression in Malpighian tubules: mRNA expression of SPoCk C in female anterior tubules was significantly higher than in males and was increased compared with female posterior (***P<0.0001), male anterior (**P=0.0015) and male posterior tubules (**P=0.001). Data are shown as means±s.e.m., N=4. SPoCk C protein expression was significantly increased in female anterior tubules by ~32% [band intensity determined using ImageJ sotftware (http://rsb.info.nih.gov/ij/)] compared with male anterior tubules. (B–E) Peroxisome-specific expression of SPoCk C isoform. (B) Bright-field image of initial segment of the anterior tubule; (C) confocal image of the initial segment of the anterior tubule, stained with anti-SPoCk C antibody; (D) overlay of B and C. Note that the lumen of the tubule is packed with SPoCk C-positive concretion bodies. Nuclei are stained blue with DAPI; (E) high-power view of the tubule initial segment showing the annular nature of anti-SPoCk C staining. Peroxisomal SPoCk C is associated with Ca2+ storage excretion. (F–G) Ca2+ storage (F) and transport (G) of 45Ca2+ in SPoCk C-overexpressing tubules. Tubules were incubated in radioactively labeled Ca2+, and the `normalised storage ratio' was calculated as the ratio of specific activities of the tubules to bathing fluid. Over-expression of SPoCk C resulted in a significantly increased Ca2+ storage compared with the parental UAS-SPoCk C (*P<0.014) and c42 (*P<0.033) parental lines. Data are means±s.e.m., N≥4 tubules. The `transport ratio' was calculated as the ratio of specific activities of the secreted fluid to that of bathing fluid. Over-expression of SPoCk C resulted in a significantly increased Ca2+ transport compared with parental UAS-SPoCk C (*P<0.014) and c42 GAL4 (*P<0.04) lines. Data are means±s.e.m., N=8 tubules. From Southall et al. (Southall et al., 2006Go).

 

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© The Company of Biologists Ltd 2009