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First published online 13 May 2003
doi: 10.1242/jcs.00468

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Pollen tubes exhibit regular periodic membrane trafficking events in the absence of apical extension

Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JU, UK

View larger version (36K): [in a new window]   Fig. 9. Periodic growth fluctuations for different pollen species determined from bright field image sequences. Distance grown and interval growth rate (between consecutive images) are plotted against time.

View larger version (93K): [in a new window]   Fig. 1. BFA effects the organisation of living pollen tube tips. Confocal images of FM4-64 distribution in L. longiflorum (A,B) and N. plumbaginifolia (C,D) before and 30-60 minutes after 3.6 µM BFA treatment. (E-F) Confocal images of mitofluor staining before and after BFA treatment in L. longiflorum. (G-H) Schematic of cytoplasmic movements in (G) growing and (H) BFA-treated pollen tube, based upon image sequences. (I) Projected time series of an FM4-64-stained, BFA-treated tube showing tracks of movement. Bars, 15 µm (A,B,E,F) or 10 µm (C,D); corresponding bright field images shown at one-third size.

View larger version (68K): [in a new window]   Fig. 2. Movement of material into and through the BIA of L. longiflorum pollen tubes followed by confocal FRAP of FM4-64. (A) Bleaching within the BIA; (B) bleaching of the apical region. Sites of photobleaching indicated on bright field images. Time is in seconds after bleaching. (A') Fluorescence intensity at the original location of the bleach site and `following' the displaced region of bleached fluorescence (see insert diagrams). (B') Fluorescence intensity at an apical bleach site and within the BIA. Bars, 15 µm.

View larger version (79K): [in a new window]   Fig. 3. A functional actin cytoskeleton is required to maintain the BIA and associated movements. Effects of (A) 5.0 µM cytochalasin D and (B) 2.0 µM Jasplakinolide on BFA (3.6 µM)-treated L. longiflorum pollen tubes labeled with FM4-64. Times are in seconds. Bars, 15 µm.

View larger version (34K): [in a new window]   Fig. 4. Consecutive confocal images (interval 1.5 seconds) of a BFA (3.6 µM)-treated tube expressing cytoplasmic targeted GFP. Bar, 15 µm.

View larger version (134K): [in a new window]   Fig. 5. ER and Golgi localisation in growing L. longiflorum pollen tubes shown by targeted fluorescent protein expression. (A-B) mGFP5-ER in (A) growing tube, sequential images (image/3 seconds) showing the dynamic nature of ER; (B) onion epidermis, showing the typical reticulate ER structure. (C-E') GT-EYFP: (C) Sequential images (image/2 seconds) of a growing tube revealing movement of targeted structures; (D) high expression levels; (E) in onion epidermis; (E') effects of BFA on labeling in onion epidermis. (F-G) GONST1-YFP expressed in growing L. longiflorum pollen tubes: (F) Sequential images (as for C); (G) high expression levels showing labelling of the apical vesicle accumulation. Inserts show bright field images at one-third size. Bars, 15 µm (A,C,D,F,G); 100 µm (B,E).

View larger version (125K): [in a new window]   Fig. 6. (A-C) Differences in ER and Golgi involvement in the BIA and associated aggregates revealed by fluorescein protein expression in living L. longiflorum pollen tubes treated with 3.6 µM BFA. (A) mGFP5-ER redistribution (times in minutes). Arrowheads indicate fine structures projecting into the apex in the main picture and contrast adjusted insert. (B-C) Redistribution of Golgi markers GT-EYFP and GONST1-YFP (times after applying BFA in minutes). In all cases, before the 15-minute time-point tubes were still extending so images have been aligned from the tips. Images for time-points 40 minutes and longer are contrast adjusted to increase brightness relative to earlier time points. Arrowheads in C indicate the BIA. Inserts show bright field images at one-third size. Bars, 15 µm.

View larger version (104K): [in a new window]   Fig. 7. GT-EYFP distribution after 40 minutes BFA (3.6 µM) treatment shows similarity to BFA effects on mGFP5-ER. The zoomed region shows labeled `projections' extending from the site of the BIA towards the apex (arrowheads). Bar, 15 µm.

View larger version (61K): [in a new window]   Fig. 8. Periodic `trafficking events' at the tip of BFA-treated (1 hour, 3.6 µM) L. longiflorum pollen tubes. (A-A') DIC bright field image sequence and corresponding plot recording movement of material along the pollen tube axis as pixel intensity changes in a `sampling window' (highlighted in A). A single trafficking event is indicated in red. (B) As in A but with FM4-64 labelling. (B') Periodic movements of FM4-64-stained material along the pollen tube axis plotted as pixel intensity changes (gray circles) in a `sampling window' highlighted in the insert (white rectangle) over time, each movement event is marked with a vertical bar. The insert image is false-coloured to reveal the pattern of relative staining intensity: red, high fluorescence; blue, low fluorescence intensity. FM4-64 signal intensity within the BIA (black rectangle in insert image) is also plotted (black squares). The two plots were aligned by a -2.5 second shift in the BIA intensity plot. Bars, 15 µm.

View larger version (43K): [in a new window]   Fig. 10. Temperature alters periodic behaviour in L. longiflorum pollen tubes. (A) A 10°C rise shortens the period of growth pulses and (B) increases tube diameter. (C) A 10°C rise shortens the period of trafficking events associated with the BIA. Bar, 20 µm.

View larger version (56K): [in a new window]   Fig. 11. Calcium ratio imaging of non-growing, BFA-treated (A) and growing (B) L. longiflorum pollen tubes. (A) Plots show the typical movement of material into the subapical membrane aggregation (closed circles; c.f. Fig. 2) and the apical intracellular calcium concentration (open squares: ratio OG 488/TR). Images are (top to bottom): a Texas Red fluorescence image (the BIA and associated aggregates appears dark); the corresponding projected TR image time-series displayed as a single `negative' image to show the path of movement of material; the corresponding ratio image. The areas sampled to produce the plots (rectangles) and location of the BIA (circled) and associated aggregates (arrowhead) are indicated. (B) Untreated pollen tube: plots show the typical oscillatory fluctuation in apical intracellular calcium concentration during growth (open squares: ratio OG 488/TR; the area sampled is shown on the insert image) and corresponding fluctuation in growth rate (closed circles). Insert images show OG 488/TR ratios corresponding to a consecutive minima and maxima in the tip-focused cytosolic calcium gradient. Bars, 10 µm.