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First published online 13 March 2007
doi: 10.1242/jcs.03402

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Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function

Galina V. Beznoussenko^1,*, Viacheslav V. Dolgikh^2,*, Elena V. Seliverstova³, Petr B. Semenov², Yuri S. Tokarev², Alvar Trucco¹, Massimo Micaroni¹, Daniele Di Giandomenico¹, Peter Auinger⁴, Igor V. Senderskiy², Sergei O. Skarlato⁵, Ekaterina S. Snigirevskaya⁵, Yan Yu. Komissarchik⁵, Margit Pavelka⁴, Maria A. De Matteis¹, Alberto Luini¹, Yuliya Ya. Sokolova^5, and Alexander A. Mironov^1,,

¹ Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale, 66030 Santa Maria Imbaro (Chieti), Italy
² Laboratory of Microbiological Control, All-Russian Institute for Plant Protection, Russian Academy of Agricultural Sciences, Shosse Podbelskogo, 3, 189620, St. Petersburg-Pushkin, Russia
³ Laboratory of Renal Physiology, Sechenov Institute of Evolutionary Physiology and Biochemistry, 44 Moris Torez Prospekt, St. Petersburg, Russia
⁴ Institute of Histology and Embryology, University of Vienna, Schwarzspanierstrafle 17, A-1090 Vienna, Austria
⁵ Institute of Cytology, Russian Academy of Sciences, Tikhoretsky Av., 4, 194064, St. Petersburg, Russia

View larger version (98K): [in this window] [in a new window]   Fig. 1. Ultrastructure of the Golgi in microsporidia P. grylli. Cells from cricket fat bodies were infected with P. grylli, fixed and prepared for routine EM. (A-E) Clusters (300-700 nm in diameter, thin arrows) of elongated round profiles (TNs) in meronts, which are often connected with the ER (B,C, thick arrows) and sometimes to the plasma membrane (D,E, thick arrows). (F-I) In sporonts, the number of ER cisternae (F, thick arrows) and the size of the TNs (G-I, TN) are increased. TNs are mostly composed of round profiles (G,H, thick arrows) and often connected with the ER (H,I, thin arrows) and with the plasma membrane (I, thick arrow). Bar, 1400 nm (A); 130 nm (B); 160 nm (C); 270 nm (D,G,I); 1000 nm (F); 200 nm (E,H).

View larger version (65K): [in this window] [in a new window]   Fig. 2. Three-dimensional reconstructions of tubular networks in P. grylli. Cells from cricket fat bodies were infected with P. grylli and fixed chemically (A,C-F) or prepared by high pressure freezing and cryosubstitution (B). 200-nm-thick sections of microsporidia were subjected to EM tomography. (A) General overview of the meront TN surrounded by the ER (green), which contains tubular (brown) and varicose (yellow) parts. Arrow indicates connection between TN and ER. (B-D) TNs in meronts pretreated without (B) and with (C,D) NEM for 15 minutes. (E,F) In the sporont, the varicose part is larger, with the smooth tubular part still connected (E,F, arrows) to the ER (green). (B-D) TNs, red (B) or orange (C,D); ER, green. Bar, 80 nm (A-D); 100 nm (E,F).

View larger version (94K): [in this window] [in a new window]   Fig. 3. The round profiles within the tubular networks are not vesicles, but varicose tubules. Cells from cricket fat bodies were infected with P. grylli and fixed and prepared for EM tomography (A-D), routine EM (F-J) or for EM by high pressure freezing and cryosubstitution (E). (A-D) 200-nm-thick sections of a meront as extracted virtual sections of part of a TN (with three perpendicular views of the same single voxel indicated by yellow crosses). Enlargements of the white boxes are indicated by white arrows. Thick arrows indicate connections between round profiles and other structures. (E-J) Samples were pretreated without (E) and with NEM for 15 minutes (F,G) or AlF4 for 20 minutes (H-J). Thin arrows in F,G indicate TNs, thick arrows in G indicate microsporidia plasmalemma. Asterisks in G,H,J indicate the vesiculated Golgi of the host cell. Normal TNs are seen in microsporidia treated with both NEM (F,G) and AlF4 (I,J). Bar, 120 nm (A-D, original sections); 35 nm (A-D, enlargements); 150 nm (E,F,H,I); 500 nm (G,J).

View larger version (128K): [in this window] [in a new window]   Fig. 4. Localization of cis-Golgi markers in the tubular networks. Cells from cricket fat bodies were infected with P. grylli and treated with an aqueous 1% solution of OsO4 for 24 hours (A) or prepared for cryo-immuno EM (B-H). (A) Arrow shows TN stained with osmium black. (B) Immuno-gold EM staining of cryosections for GNA lectin in the sporoblast. (C,D) Immuno-EM localization of COP in the meront (C) and sporont (D). (E,F) Immunolocalization of Sec13 in meronts (E,F, arrow) and in the sporont (F, right side). The plasmalemma of the sporont is indicated with a dashed line. (G,H) Immunolocalization of giantin-like (G, thick arrow over round profiles) and GM130-like (H, thick arrow) proteins in meronts; thin arrows in G indicate microsporidia plasma membrane. Bar, 250 nm (A); 100 nm (B-F); 180 nm (G); 120 nm (H).

View larger version (62K): [in this window] [in a new window]   Fig. 5. Glycosylation of p40 and PTP A in microsporidia. Cells from cricket fat bodies were infected with P. grylli and prepared for PAGE. (A) SDS-PAGE analysis followed by Coomassie Brilliant Blue (left) and Schiff reagent (right) staining. Lane 1, MW markers; lane 2, intact spores broken up in phosphate-buffered saline and boiled for 10 minutes in SDS-PAGE sample buffer; lane 3, intact spores boiled as for lane 2; lane 4, intact P. grylli spores in alkaline saline solution (10 mM KOH, 170 mM KCl; 30 minutes) selectively solubilizes the major 40-kDa protein; lane 5, spore extrusion followed by washing in 3% SDS and PTPs solubilized with 50% 2-mercaptoethanol; lane 6, soluble; and lane 7, membrane, proteins of the host (cricket Gryllus bimaculatus) fat body. (B) Precipitation of p40 by GNA-agarose. mw, molecular weight markers; lanes 1, 3, 5, supernatant after p40 solubilization in 10 mM KOH, 170 mM KCl; lanes 2, 4, 6, proteins precipitated by GNA-agarose and eluted by boiling in SDS-PAGE sample buffer; lanes 1, 2, pretreatment with reaction buffer for mannosidase (control); lanes 3, 4, treatment with -mannosidase; lanes 5, 6, treatment with -mannosidase.

View larger version (130K): [in this window] [in a new window]   Fig. 6. Transport of p40 through the Golgi in microsporidia. Cells from cricket fat bodies were infected with P. grylli and prepared for cryo-immuno EM. (A-F) Cryosections of meronts (A-C) and sporoblasts (D-F) with labeling for p40. The TNs (C, thin arrows) are labeled for GM130 (15 nm; C) and p40 (10 nm; A-C). p40 can be seen over the forming spore wall (D,F, thick arrows) and in the peripheral parts of the enlarged TN (F, thin arrow), but not over the forming polar tube (E, thin arrows). Thin arrows in D indicate polar tubes. Bars, 80 nm (A-D,F); 90 nm (E).

View larger version (146K): [in this window] [in a new window]   Fig. 7. Transport of PTP A through the Golgi in microsporidia. Cells from cricket fat bodies were infected with P. grylli and prepared for cryo-immuno EM. Cryosections of a sporoblast (A) and maturing spores (B-D) with labeling for PTP A. (C) represents the enlarged area from the black box in B. (A) PTP A is concentrated over the large TN (thin arrow, forming spore). (B-D), PTP A is visible over polar tubes (thick arrows), but not over the ER (C, thin arrows). Polar tubes are connected with the TN (D, thin arrow). Bars, 100 nm (A,C); 300 nm (B); 70 nm (D).

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