Apical localization of inositol 1,4,5-trisphosphate receptors is independent of extended synaptotagmins in hepatocytes.

Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca2+) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca2+ signaling is unknown. Ca2+ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca2+ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca2+ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity

The plasmodium receptor for activated C kinase protein inhibits Ca(2+) signaling in mammalian cells

Plasmodium falciparum, the most lethal malarial parasite, expresses an ortholog for the protein kinase C (PKC) activator RACK1. However, PKC has not been identified in this parasite, and the mammalian RACK1 can interact with the inositol 1,4,5-trisphosphate receptor (InsP3R). Therefore we investigated whether the Plasmodium ortholog PfRACK also can affect InsP3R-mediated Ca(2+) signaling in mammalian cells. GFP-tagged PfRACK and endogenous RACK1 were expressed in a similar distribution within cells. PfRACK inhibited agonist-induced Ca(2+) signals in cells expressing each isoform of the InsP3R, and this effect persisted when expression of endogenous RACK1 was reduced by siRNA. PfRACK also inhibited Ca(2+) signals induced by photorelease of caged InsP3. These findings provide evidence that PfRACK directly inhibits InsP3-mediated Ca(2+) signaling in mammalian cells. Interference with host cell signaling pathways to subvert the host intracellular milieu may be an important mechanism for parasite survival. (C) 2009 Elsevier Inc. All rights reserved.NIH[DK45710]U.S. National Institutes of Health (NIH)U.S. National Institutes of Health (NIH)NIH[DK57751]U.S. National Institutes of Health (NIH)NIH[DK34989]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPES

Apical organic anion secretion is not affected by E-Syt knockdown.

<p>(<b>A</b>) Representative differential interference contrast (DIC) (left), and confocal fluorescence time-lapse images at baseline (middle) and after 4 min (right) of CMFDA secretion in control non-treated rat hepatocytes in collagen sandwich culture. The cell-permeant fluorescent Mrp2 substrate was added to cells, and its secretion into the canalicular spaces (green) was monitored every second for 4 minutes by confocal microscopy. (<b>B</b>) and (<b>C</b>) Quantification of CMFDA canalicular accumulation under control (n = 55 canaliculi), scrambled (n = 49 canaliculi), E-Syt1 (n = 52 canaliculi), E-Syt2 (n = 65 canaliculi) and E-Syt1+2 (n = 52 canaliculi) siRNA conditions. Pre-treatment with the cytosolic Ca²⁺ buffer BAPTA-AM (50 µM) (n = 48 canaliculi) was used as a negative control (*p<0.05; n = 3 experiments). (<b>D</b>) and (<b>E</b>) Quantification of CMFDA canalicular accumulation under control (n = 163 canaliculi), scrambled siRNA (n = 73 canaliculi), BAPTA-AM (n = 111 canaliculi), scrambled siRNA+BAPTA-AM (n = 81 canaliculi), E-Syt1 siRNA+BAPTA-AM (n = 163 canaliculi), E-Syt2 siRNA+BAPTA-AM (n = 146 canaliculi) and E-Syt1+2 siRNA+BAPTA-AM (n = 164 canaliculi) conditions. (***p<0.0001; n = 3 experiments). Values are mean ± SD. Data were analyzed by one-way ANOVA, followed by Bonferroni's post-tests.</p

Rat hepatocytes express E-Syt1 and E-Syt2 but not E-Syt3.

<p>Relative mRNA expression of E-Syt isoforms was measured by Real Time quantitative PCR in whole liver extracts (A) and in fully polarized rat hepatocytes (after 96 hrs in sandwich culture) (B). E-Syt 1 and 2 were detected, and E-Syt3 was absent from both samples. E-Syt1 was the most abundantly expressed isoform in both samples. (**p<0.001; ***p<0.0001; n = 3 experiments). Values are mean ± SD. Data were analyzed by one-way ANOVA, followed by Bonferroni's post-tests.</p

Average InsP3R-II cluster size is not altered by E-Syt knockdown.

<p>Confocal immunofluorescence (A, B, D) and STED super-resolution (C) images of a control rat hepatocyte canaliculus. InsP3R-II (green) was co-labelled with Mrp2 (blue). Scale bar = 3.5µm. Box in (A) represents the area depicted in (C) and (D). (<b>C</b>) Individual clusters (arrows) were observed in STED image, whereas they could not be resolved as well in confocal image (D). (<b>E</b>) Average InsP3R-II cluster area was determined based on STED images (control: n = 118 clusters; scrambled siRNA: n = 129 clusters; E-Syt1 siRNA: n = 118 clusters; E-Syt2 siRNA: n = 109 clusters; E-Syt1+2 siRNA: n = 103 clusters). Data were analyzed by one-way ANOVA, followed by Bonferroni's post-tests.</p

E-Syt1, E-Syt2 and InsP3RII expression is maintained during rat hepatocyte collagen sandwich culture.

<p>Relative E-Syt1(A) and E-Syt2 (B) mRNA expression was assessed by Real Time PCR in rat hepatocytes at the indicated times in collagen sandwich culture. (<b>C</b>) Representative immunoblottings of E-Syt1, E-Syt2 and InsP3R-II in rat hepatocytes at the indicated time points of collagen sandwich culture. GAPDH was used as loading control. HeLa cell lysate was used as a positive control for E-Syts and whole liver lysate was used as a positive control for InsP3R-II. Correct molecular weight is indicated by the arrows. (<b>D</b>) Densitometric analysis of E-Syt1 blots in (C). (n = 4 experiments). (<b>E</b>) Densitometric analysis of E-Syt2 blots in (C). (n = 4 experiments). (<b>F</b>) Densitomteric analysis of InsP3R-II blots in (C). (n = 4 experiments). Values are mean ± SD. Data were analyzed by one-way ANOVA, followed by Bonferroni's post-tests. Expression was compared among time points ranging from 0 to 96 hrs in collagen sandwich culture.</p

InsP3-induced Ca²⁺ signals are unaltered by E-Syt knockdown.

<p>(<b>A</b>) Representative DIC (left panels) and confocal fluorescence time-lapse images (right panels) of R-GECO-transfected, fully polarized rat hepatocytes under non-treated, scrambled, E-Syt1, E-Syt2 and E-Syt1+2 siRNA conditions and stimulated with 20µM ATP. Images were pseudocolored according to the scale shown at the bottom. Outlined structures represent the canalicular area of each cell, where Ca²⁺ waves begin. Scale bar = 20µm (<b>B</b>) Representative tracings from cells under each experimental condition are shown. Based on these tracings, peak signal amplitude (C), rise time (D) and Ca²⁺ wave speed (E) were measured. (n = 3 experiments, n = 20 cells per condition). Values are mean ± SD. Data were analyzed by one-way ANOVA, followed by Bonferroni's post-tests.</p

Apical localization of InsP3Rs is independent of E-Syt1 and E-Syt2.

<p>Confocal immunofluorescence images of InsP3R-II under control, non-treated (A), scrambled (B), E-Syt1 (C), E-Syt2 (D) and E-Syt1+2 (E) siRNA conditions. InsP3R-II (green) was co-labelled with Rhodamine phalloidin (red), the apical membrane marker Mrp2 (blue) and the nuclear stain DAPI (magenta). Scale bar = 10µm. (<b>F</b>) Distribution of InsP3R-II was quantified by its normalized fluorescence intensity along a 6µm line perpendicular to the canalicular membrane. (n = 3 experiments; control: n = 63 canaliculi; scrambled siRNA: n = 48 canaliculi; E-Syt1 siRNA: n = 48 canaliculi; E-Syt2 siRNA: n = 51 canaliculi; E-Syt1+2 siRNA: n = 30 canaliculi). Values are mean ± SEM. (<b>G</b>) Total InsP3R-II fluorescence intensity per canaliculus. (<b>H</b>) Relative InsP3R-II fluorescence intensity, calculated as total InsP3R-II fluorescence normalized by canalicular area. (n = 3 experiments; control: n = 234 canaliculi; scrambled siRNA: n = 134 canaliculi; E-Syt1 siRNA: n = 163 canaliculi; E-Syt2 siRNA: n = 299 canaliculi; E-Syt1+2 siRNA: n = 220 canaliculi). (<b>I</b>) Canalicular diameter is shown as an indicator of canalicular morphology. (n = 3 experiments; control: n = 63 canaliculi; scrambled siRNA: n = 48 canaliculi; E-Syt1 siRNA: n = 48 canaliculi; E-Syt2 siRNA: n = 51 canaliculi; E-Syt1+2 siRNA: n = 30 canaliculi). Values are mean ± SD. Data were analyzed by one-way ANOVA, followed by Bonferroni's post-tests.</p

Specific siRNAs against E-Syt1 and E-Syt2 efficiently reduce E-Syt1 and E-Syt2 protein expression but do not affect InsP3R-II expression.

<p>Representative immunoblotings of E-Syt1 (A) and E-Syt2 (B) in rat hepatocytes after treatment with 25 nM of scrambled, E-Syt1 (A), E-Syt2 (B) or E-Syt1+2 siRNAs (A and B) for 96 hrs in sandwich culture. (<b>C</b>) Densitometric analysis of blots in (A). (***p<0.0001; n = 3 experiments) (<b>D</b>) Densitometric analysis of blots in (B) (*p<0.05; n = 3 experiments). (<b>E</b>) Representative immunoblots of InsP3R-II after individual or simultaneous treatment with E-Syt1 and E-Syt2 siRNAs. (<b>F</b>) Densitometric analysis of blots in (E) (n = 3 experiments). GAPDH was used as loading control. Values are mean ± SD. Data were analyzed by one-way ANOVA, followed by Bonferroni's post-tests.</p