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Analysis of Ca²⁺ uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber

Monica Hagedorn and Andreas Ziegler^*

Zentrale Einrichtung Elektronenmikroskopie, Universität Ulm, 89069 Ulm, Germany

View larger version (20K): [in a new window]   Fig. 1. (A) Schematic diagram of the perfusion chamber with anterior (ASE) and posterior (PSE) sternal epithelia mounted on grids. Solutions can easily be exchanged at the side of the chamber without disturbing the specimen (large arrows). (B) Side view of the perfusion chamber at the level indicated in A by the small arrow and a dotted line.

View larger version (112K): [in a new window]   Fig. 2. Light micrographs, (A) phase-contrast and (B) polarisation microscopy image, of the anterior sternal epithelium of Porcellio scaber after a calcium oxalate loading experiment. The birefringent calcium oxalate crystals appear as bright signals (arrows). Scale bar, 20 µm. g, grid bar.

View larger version (17K): [in a new window]   Fig. 3. Calcium oxalate loading experiments in the anterior sternal epithelium of Porcellio scaber during late premoult. (A) Intensity signal in the loading medium at a Ca2+ activity (aCa) of 0.68 µmol l-1. The intensity units given in A—C represent the difference between the mean grey-scale value and that in the first image. Each point represents the mean ± S.E.M. of nine animals. (B) Effect of ATP on calcium oxalate loading. After removal of Na2ATP, calcium oxalate loading is immediately and reversibly blocked. (C) Effect of the specific smooth endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) on calcium oxalate loading at a aCa of 0.38 µmol l-1. After addition of 1 µmol l-1 CPA, the increase in the intensity signal is immediately blocked. (D) Effect of aCa on calcium oxalate loading in the anterior sternal epithelium from late premoult animals. Each data point gives the mean intensity value (± S.E.M.) of 4-6 loading experiments. The rates were normalised to the highest rate at a aCa of 2.05 µmol l-1 and plotted against aCa. The line was drawn using GraphPad Prism 3.0 software.

View larger version (168K): [in a new window]   Fig. 4. Electron micrographs of unstained sections of the anterior sternal epithelium during late premoult after calcium oxalate loading experiments. (A) Example with larger and (B) with smaller calcium oxalate precipitates. (C,D) Details of calcium oxalate precipitates within smooth membranous cisternae (arrows). Scale bars, 1 µm in A and B; 0.5 µm in C and D. n, nucleus; cl, cuticular layer; m, mitochondria.

View larger version (62K): [in a new window]   Fig. 5. Electron energy-loss imaging (ESI) and electron energy-loss spectral (EELS) analysis of 20 nm thick sections of cryofixed, freeze-substituted anterior sternal epithelium after loading the smooth endoplasmic reticulum with calcium oxalate. Some of the calcium oxalate precipitates are indicated by arrows. cl, cuticular layer. Scale bars, 0.5 µm. (A) ESI image at E=320±5 eV. (B) ESI image at E=360±5 eV; the calcium oxalate precipitates transmit a bright signal. (C) EELS of the precipitate within smooth membrane cisterns; CaL2,3 indicates the absorption edges for calcium.

View larger version (22K): [in a new window]   Fig. 6. Comparison of Ca2+ uptake rates in sternal epithelial cells at different stages of the moulting cycle (means + S.E.M.). The number of samples is given in parentheses. Asterisks indicate significant differences from the mid premoult stage; ***P<0.001, **P<0.01. ASE, anterior sternal epithelium; PSE, posterior sternal epithelium; EPRE, early premoult; MPRE, mid premoult; LPRE, late premoult; INTRA, intramoult.