Inhibition of Contractile Vacuole Function by ... - Universität zu Köln

More documents

Recommendations

Info

204 vacuole at late diastole stages was never observed even when serial sections were used to investigate a larger surface area of the CV (data not shown). Effect of BFA on the contractile vacuole Upon addition of BFA to S. dubia cells, the two CVs failed to expel liquid and swelled (Fig. 2B, 5 min of BFA treatment, video 1 of the Supplementary material) which is accompanied by an increase in total cell volume indicating the uptake of water by the cell (see video 1 of the Supplementary material). About 10 min after BFA addition, the cells lost their flagella. Finally, >90% of the cells developed an apparent single LCV (Fig. 2C, video 1 of the Supplementary material). Within 30 min, the LCV reached an average diameter of 6.7 ± 1.3 µm (n = 20). A few cells (maximum 10% of the cell population) remained motile and completely unaffected by BFA treatment as judged by LM. The formation of an LCV was completely reversible (tested for up to 1 h of BFA treatment at 15°C, data not shown). BFA-induced formation of an LCV was temperature dependent. At 0°C (cells incubated with BFA on ice), no LCVs were The contractile vacuole of S. dubia Fig. 3 Electron microscopic analysis of the BFAinduced formation of large central vacuoles (LCVs). Flagellar regeneration was induced by experimental amputation and the cells allowed to regenerate flagella for 20 min before BFA was added. Cells were treated for the indicated time with BFA and processed for standard TEM. (A and B) After 5 min of BFA treatment. (A) Longitudinal section through a cell and (B) a cross-section of a cell: the CV was enlarged, showed an irregular appearance and coated pits (arrows) are associated with the enlarged CV. (C) After 10 min of BFA treatment: a round vacuole is formed. (D) After 15 min of BFA treatment. (E) After 30 min of BFA treatment. bb, basal body; c, chloroplast; cv, contractile vacuole; g, Golgi stack; lcv, large central vacuole; n, nucleus; v, putative polyphosphate-containing vacuole. Bar = 0.5 µm. formed at all (Fig. 2D) and in cells incubated with BFA at 15°C the formation of LCVs was significantly slower when compared with cells treated with BFA at 25°C (Fig. 2D). Ultrastructure of the CV in BFA-treated S. dubia cells Cells were deflagellated and incubated for 20 min at 20°C in culture medium before BFA was added. During the incubation time (without BFA), the cells started to regenerate new flagella and synthesized new flagellar scales (see Fig. 4A). At 20 min after deflagellation, all Golgi cisternae contain scales except for the two to three cis-most cisternae (see below for details). Cells were chemically fixed and processed for EM prior to addition of BFA and 5, 10, 15, 20 and 30 min after adding BFA. As a control, methanol alone was added 20 min after deflagellation and cells were fixed for EM after 15 and 30 min incubation time, respectively. No effect on the ultrastructure of S. dubia cells was observed in the control samples (not shown). Cells fixed at 5 min after addition of BFA contained enlarged CVs of an irregular geometry (Fig. 3A, B). In contrast to control cells, numerous coated pits are associated with the CV of BFA-treated cells (Fig. 3C, arrows). At 10 min after
addition of BFA, the CVs were larger and had a more round geometry (Fig. 3D). Only a few coated pits associated with the CVs were observed. EM of later stages showed either a single LCV (Fig. 3E, 15 min), which is in agreement with the LM observations, or two LCVs (Fig. 3F, 30 min). Most probably, cells always contained two LCVs which could not be resolved by LM (see also below). In cells treated with BFA for >15 min, only remnants of the SR were observed, indicating that the SR was mainly adsorbed into the LCV. The number of pentagonal scales per µm 2 of the LCV membrane decreased with BFA The contractile vacuole of S. dubia 205 Table 2 Effect of BFA on the structure of the Golgi complex in S. dubia Fig. 4 BFA affects the structure of the Golgi complex in S. dubia. Flagellar regeneration of S. dubia cells was induced by experimental amputation and the cells allowed to regenerate flagella for 20 min before BFA was added. Cells were treated for the indicated time with BFA and processed for standard TEM. (A) Cells were fixed 20 min after deflagellation. A longitudinal section is shown. The Golgi stack is filled with scales (arrowheads). At the rim of the stack, numerous transition vesicles can be seen (small arrows). All types of scales are transported within the same vesicles to the flagellar surface (arrow). (B) Cells were treated with BFA for 5 min before they were chemically fixed and processed for EM. The number of Golgi cisternae is reduced and only few transition vesicles can be seen. The arrow points to a vesicular tubular element which seems to leave the stack. (C) After 10 min of BFA treatment. The number of cisternae has decreased further and all cisternae contain scales (arrowheads). Numerous vesicles of 150 nm in diameter accumulate at the cisface of the stack (arrows). bb, basal body; c, chloroplast; g, Golgi stack; f, flagellum; lcv, large central vacuole; m, mitochondrion; n, nucleus; sr, scale reticulum. Bar = 1 µm. incubation time (Table 1), indicating that the membrane source for LCV growth had a lower scale content than the CV and ‘diluted’ the number of scales per µm 2 of membrane in the LCV. Similar results were obtained when interphase cells (no flagellar regeneration) were treated with BFA. Ultrastructure of the Golgi complex in BFA-treated cells Beside affecting the structure of the CV, BFA also induced changes in the structure of the Golgi complex of S. dubia. In flagellar regenerating cells of S. dubia, the two Golgi stacks Control cells 5 min BFA 10 min BFA No. of stacks analysed 24 17 15 No. of cisternae a 14.3 ± 0.8 11.6 ± 1.1 8.3 ± 1.2 No. of trans-cisternae containing scales a 11.3 ± 0.7 10.5 ± 1.1 8.0 ± 1.3 No. of transition vesicles/stack a 10.6 ± 3.3 0.7 ± 1.1 0 Cells were deflagellated and allowed to regenerate for 20 min before BFA was added. Cells were fixed and processed for EM prior to and 5 or 10 min after addition of BFA. a Only clear cross-sections of Golgi stacks were analysed.
Page 1 and 2: Plant Cell Physiol. 46(1): 201-212
Page 3: Fig. 2 BFA interferes with the func
Page 7 and 8: Fig. 7 The CVs and the LCVs do not
Page 9 and 10: information regarding the presence
Page 11 and 12: Inhibitor treatments A stock soluti

Inhibition of Contractile Vacuole Function by ... - Universität zu Köln

Create successful ePaper yourself

Delete template?

Save as template?