JCS03402 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Representation of the stages in microsporidia development drawn from EM data. (A) The spore. (B) Infection by spore germination. (C) The meront. (D) Transition from meront to sporont. (E) The sporoblast. ER, endoplasmic reticulum; TN, tubular network; N, nucleus; PT, polar tube; PV, posterior vacuole; SW, spore wall.

• Supplemental Figure 2 -

  Fig. S2. Stages in the development of P. locustae. Cells from cricket fat bodies were infected with P. locustae and fixed and prepared for routine EM. (A) Meront. (B) Early sporont. (C) Sporont. (D) Early sporoblast. (E) Sporoblast. (F, G) Early and late spores. Bar, 500 nm (A-D); 800 nm (E-G).

• Supplemental Figure 3 -

  Fig. S3. Ultrastructure of tubular networks in P. locustae, showing structural identity between P. grylli (see Figs. 1, 2) and P. locustae. Cells from cricket fat bodies were infected with P. locustae and fixed chemically and prepared for routine EM (A, E-H) or prepared by high pressure freezing and cryosubstitution (B-D) that was also combined with electron tomography (B). (A) A TN in a meront, composed of elongated and round profiles. (B) Three-dimensional reconstruction of a TN in meront. (C) The TN of a meront connected to the ER (arrow). (B, D, H) Samples were pretreated with NEM for 15 minutes and then prepared for EM. No vesiculation was observed. (E, F) In early sporonts, the TNs are larger (arrows) and composed of tubular and varicose parts. (G) Samples were pretreated with AlF₄ for 20 minutes and then prepared for EM. Arrows in A, D, F, G show TNs. Arrow in B shows a bud (blue) on a TN (red). Arrow and asterisk in H show the vesiculated Golgi in a host cell. NE in C indicates the nuclear envelope. Bar, 120 nm (A,B); 300 nm (C); 80 nm (D, G); 250 nm (E, H); 100 nm (F).

• Supplemental Figure 4 -

  Fig. S4. Cytochemistry of the Golgi and glycosylation of p40 and PTP A in P. locustae. Cells from cricket fat bodies were infected with P. locustae and fixed and prepared for routine EM (A) or cryo-immuno EM labeling (B-H). (A) Prolonged (48 hour) treatment of meronts with an aqueous 1% solution of OsO₄ stained the TN (as indicated) with osmium black. (B-H) Cryosections of meronts (D, G), sporonts (B, C) and sporoblasts (E, F, H) with labeling for GM130 (B), giantin (C) as Golgi markers, GNA (D, E) as a glycosylation indicator; PTP A (F) and p40 (G, H) as transport markers. (B, C) Concentration of GM130 (B) and giantin (C) over TNs. (D, E) Decoration of terminal α-mannose residues in TNs (D), polar tubes and, much less, over the spore wall (E) with Galanthus nivalis agglutinin. (F) PTP A is visible over tubular networks. (G, H) Transport of p40 is seen in the TN (G) and over the spore wall (H). Bars: 200 nm (A, B); 100 nm (C, D, G); 150 nm (E, F, H).

• Supplemental Figure 5 -

  Fig. S5. Immunolocalization of COP-I and COP-II proteins in tubular networks of P. glylli and P. locustae. Cells from cricket fat bodies were infected with P. grylli and P. locustae, and prepared for western blotting (A, B, J), or fixed and prepared for cryo-immuno EM (C-I). (A) Western blotting of protein extracts from P. grylli with the Ab against Sec13 of N. locustae. (B) Western blotting of P. grylli (1) and P. locustae (2) protein extracts with Abs against γCOP. (C-E) Immunolabelling for γCOP of vesicular-tubular clusters (VTCs) in host cells (C,D) and in the TN of a sporoblast (E) of P. grylli. (F-I) Immuno-EM labelling for Sec13 of the TNs in a meront (F), early sporonts (G, I) and a sporoblast (H). (J) Immunoblotting of P. grylli spore proteins with purified anti-PTP A (lane 1) and anti-p40 (lane 2) Abs. Bars, 400 nm (C, D); 100 nm (E, I); 150 nm (F, G, H).

• Supplemental Figure 6 -

  Fig. S6. Immunocytochemistry of the tubular networks in P. grylli. Cells from cricket fat bodies were infected with P. grylli and prepared for western blotting (A, B) or fixed and prepared for immunofluorescence (C-D) and cryo immuno EM (F,G). (A,B) P. grylli protein extracts were prepared for western blotting with Abs against giantin (A; 1:2000) and GM130 (B; 1:1000). Lane 1, uninfected cricket fat-body proteins; lane 2, P. grylli intracellular stages; lane 3, P. grylli spores. (A) The anti-giantin Ab recognized a single band of ∼190 kDa in the intracellular stages, but not in the spores or the uninfected host tissue. (B) The anti-GM130 Ab recognized a single band of ∼90 kDa in the intracellular stages and the microsporidia spores, but not in the uninfected host tissue. (C-E) Immunofluorescence for giantin (C,D) and GM130 (E) in meronts (C,E) and an early sporont (D). (F,G) Immuno-gold EM staining of cryosections for giantin in a sporont (F), and for GM130 in a sporoblast (G). Arrows indicate TNs. Bars, 1 μm (C-E); 100 nm (F, G).