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First published online 17 July 2006
doi: 10.1242/jcs.03058

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Dynamic and reversible restructuring of the ER induced by PDMP in cultured cells

Teresa Sprocati¹, Paolo Ronchi¹, Andrea Raimondi^1,*, Maura Francolini¹ and Nica Borgese^1,2,

¹ Consiglio Nazionale delle Ricerche Institute of Neuroscience and Department of Medical Pharmacology, via Vanvitelli 32, University of Milano, 20129 Milano, Italy
² Department of Pharmacobiological Science, `Magna Graecia' University of Catanzaro, 88100 Catanzaro, Italy

View larger version (69K): [in a new window]   Fig. 1. Illustration of GFP-b(5)tail and characterisation of HeLa TetOff cells expressing the construct. (A) Schematic representation of GFP-b(5)tail. The construct is composed of GFP fused at its C-terminus to a linker (zig-zagged line) consisting of the myc epitope followed by a repeated Gly-Ser sequence. This is attached to the entire tail region of rat cytochrome b(5), which contains the transmembrane domain flanked upstream and downstream by polar sequences. The orientation of the construct, with the N-terminal GFP in the cytosol and the C-terminus in the ER lumen is indicated. (B) Western blot analysis of GFP-b(5)tail expression in stably transfected HeLa TetOff cells. Cells were harvested at the indicated times after removal of doxycycline from the culture medium. Each lane contained 55 µg of protein. (C) Confocal analysis of HeLa TetOff cells expressing GFP-b(5)tail. Cells were fixed 3 days after exposure to doxycycline-free medium. Bar, 10 µm. (D) G-6-Pase EM cytochemistry of HeLa TetOff cells, induced (right) or not induced (left) to express GFP-b(5)tail. The product of the cytochemical reaction is found within the nuclear envelope and in ER cisternae. Note the higher density of ER cisternae in the induced cells. N, nucleus. Bar, 1 µm.

View larger version (101K): [in a new window]   Fig. 2. Effect of PDMP on the ER of HeLa cells expressing GFP-b(5)tail. Cells grown without doxycycline for 3 days were treated with 100 µM PDMP for 3 hours, then fixed and examined by confocal microscopy, either without further manipulation (A,D,E), or after immunostaining for calnexin (B) or giantin (C), with the use of Texas-Red-conjugated secondary antibodies. Note the presence of calnexin in the PDMP-induced structures (B), and the normal appearance of the Golgi complex (C), which is similar in GFP-expressing and non-expressing cells (the latter indicated by an arrow in the middle and lower images of C). (D) PDMP-induced ER patches at higher magnification. (E) Vertical section of a cell with PDMP-induced ER patches (lower panel) and a horizontal section of the same cell (upper panel). The red line indicates the y position along which the x-z scan was performed. Bars, 10 µm (A-C); 2 µm (D-E).

View larger version (28K): [in a new window]   Fig. 3. FRAP analysis of PDMP-induced SER patches. The area delimited by the rectangle was bleached with unattenuated laser power and the cells were then imaged at low laser power for 3 minutes following the bleach. The numbers in the lower left corner of the panels indicate the time (in seconds) after returning to low laser power. Note that three SER patches within the boxed area recovered fluorescence at the expense of another SER patch indicated by the arrow. This effect was not due to bleaching, because the fluorescence of two other cells in the field remained constant during the imaging. Bar, 10 µm. The movie of this experiment is shown in supplementary material Movie 2.

View larger version (163K): [in a new window]   Fig. 4. Ultrastructure of PDMP-induced ER structures in induced HeLa TetOff cells. Conventional EM analysis at low (A) and high (B) magnification. (A) An SER patch is indicated by the arrow. Abundant free ribosomes are visible in the surrounding cytosol. The asterisk indicates the intercellular space. N, nucleus; M, mitochondrion; ER, elongated ER cisterna partly covered with ribosomes and containing a coated protrusion, which probably corresponds to an ER exit site (arrowhead). (B) A portion of a particularly convoluted SER patch consisting in a dense network of frequently branching tubules of small calibre. (C) G-6-Pase EM cytochemistry of a PDMP-treated cell. The arrow indicates an area of packed G-6-Pase-positive tubules that presumably corresponds to an SER patch. N, nucleus. Bars, 0.5 µm (A); 0.1 µm (B); 1 µm (C).

View larger version (116K): [in a new window]   Fig. 5. SER patches are dynamic and are rapidly reabsorbed into the ER polygonal meshwork upon removal of PDMP. (A) Live imaging by wide-field microscopy of PDMP-treated HeLa TetOff cells. Each row shows a different field of cells, imaged in the presence of PDMP. The arrow in the top row images indicates an SER patch that has fused with a smaller one at 30 seconds. After 6 minutes the patch has become less elongated and has changed position relative to the nuclear envelope. The arrow in the bottom row indicates a large patch, which appears to be undergoing fission at the end of the imaging period. See Movie 1 in supplementary material for an illustration of the genesis and dynamics of SER patches. (B) PDMP-treated cells were placed in PDMP-free medium and immediately subjected to time-lapse imaging by wide-field microscopy. A movie of this sequence is available in supplementary material Movie 3). In all panels, the numbers on the left indicate the time (in seconds) from the start of the imaging experiment. Bars, 10 µm.

View larger version (85K): [in a new window]   Fig. 6. Distribution of ER proteins between SER patches and polygonal meshwork ER. (Top row) HeLa TetOff cells expressing GFP-b(5)tail and microinjected with a plasmid coding for G2A b5R were treated with PDMP and immunostained for b5R. Three bottom rows: PDMP-treated cells were immunostained for Sec61ß (second row), CLIMP-63 (third row) or calreticulin (bottom row), with the use of Alexa Fluor 568-conjugated secondary antibodies. The cells were then analysed by confocal microscopy. The arrows in the Sec61ß and CLIMP-63 panels indicate evident SER patches with low content of the two RER markers. Note the tendency of CLIMP-63 to encircle the SER patches. Bars, 10 µm.

View larger version (115K): [in a new window]   Fig. 7. Distribution of lipid dyes between SER patches and polygonal meshwork ER. Induced HeLa TetOff were microinjected with DiIC16 (3) (two upper rows) or with FAST-DiI (two lower rows) dissolved in soybean oil. One set of cells was then exposed to PDMP whereas another set was exposed to the vector, as indicated to the left of the figure. Live cells were imaged by wide-field microscopy 3 hours later. The asterisks are placed at the centre of the microinjected oil droplets. Note in the bottom row (inset) the prevalence of the GFP fluorescence in the PDMP-induced patches compared with FAST-DiI. The boxes in the right panel of the second and fourth rows indicate areas that are enlarged (threefold) in the insets. The arrows in the bottom row indicate uncharacterised structures in which FAST-DiI is particularly concentrated. Bar, 10 µm.

View larger version (21K): [in a new window]   Fig. 8. Sucrose density gradient analysis of PDMP-treated and untreated cells. PNS obtained from total homogenates of induced untreated (A) or PDMP-treated (B) HeLa TetOff cells were centrifuged on sucrose density gradients. An equal aliquot of each fraction was analysed by western blotting for Sec61ß and GFP-b(5)tail, as indicated. The grey line in both panels indicates the percentage of sucrose in each fraction, determined gravimetrically. The arrow and arrowhead below the abscissa indicate the average sucrose concentration at which elements containing GFP-b(5)tail (arrow) and Sec61ß (arrowhead) equilibrate.

View larger version (76K): [in a new window]   Fig. 9. Intracellular Ca2+ chelation partially reverses the effect of PDMP on ER architecture. Induced HeLa TetOff cells were treated with 100 µM PDMP for 3 hours. During the last hour of incubation the medium was supplemented with 50 µM BAPTA-AM (right panel) or vector (left panel). After fixation, the cells were analysed by confocal microscopy. The percentage of cells showing SER patches was determined on eight randomly selected fields, such as those illustrated in the figure. The difference between the percentages of responding cells under the two conditions (indicated under the panels ± s.e.m.) was highly significant by Student's t-test (P=1.7x10-5).

View larger version (11K): [in a new window]   Fig. 10. Model of SER patch formation. (A) In HeLa cells not expressing GFP-b(5) tail, ribosome-free and ribosome-covered areas are interspersed along the same tubules. A protein or protein complex (red squares) is responsible for maintaining relatively straight tubules and for regulating the branching frequency. (B) GFP-b(5)tail expression induces an increase in ER surface area without a concomitant increase in the expression of ER membrane proteins. This results in dilution of the protein complex that regulates tubule geometry. (C) Minor perturbations induced by PDMP cause the regulatory protein(s) to cluster with ribosomes, leaving large areas of ER free to collapse into a random tubular network. See text for further explanation.

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