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First published online 16 October 2007
doi: 10.1242/jcs.012138

Journal of Cell Science 120, 3804-3819 (2007)
Published by The Company of Biologists 2007
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Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold

Alessandra Moscatelli1,*, Fabrizio Ciampolini2, Simona Rodighiero3, Elisabetta Onelli1, Mauro Cresti2, Nadia Santo4 and Aurora Idilli1

1 Dipartimento di Biologia L. Gorini, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy
2 Dipartimento di Scienze Ambientali G. Sarfatti, Università degli Studi di Siena, Via P. A. Mattioli 4, 53100 Siena, Italy
3 CIMAINA, Università degli Studi di Milano, Via Celoria 16, 20133 Milan, Italy
4 CIMA, Università degli Studi di Milano, Via Celoria 16, 20133 Milan, Italy


Figure 1
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  		Fig. 1. (a,b) Tube lengths with and without probes were compared. Probes do not significantly influence tube length. x axis shows time in minutes (m). (c) Percentage of stained vesicles in the tips of pollen tubes incubated with negatively or positively charged nanogold. (d) Pollen tube growth in the presence of Ika (yellow bars) was compared with tubes grown in control medium (dark-purple bars) or in medium containing the Ika solvent (DMSO) (light-purple bars). Error bars indicate 95% confidence intervals (n=100-140).

 

Figure 2
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  		Fig. 2. Internalization of positively charged nanogold for 30 minutes. (a) After 30 minutes, positively charged nanogold (black dots) was regularly distributed on the PM and was trapped in invaginating vesicles (black arrowhead) and in vesicles immediately beneath the PM (asterisks). Vesicles of the TGN were stained (yellow arrows) as well as vesicles associated with the cis and medial rims of Golgi cisternae (G, blue arrowhead). Black arrows indicate labelled cytoplasmic vesicles. (b) A Golgi apparatus showing labelled vesicles associated with cis and medial cisternae (arrowheads). (c) Gold particles were internalized in vesicles containing cell wall components (arrowheads). (d) MVB-like compartments were labelled (arrows) as well as Golgi body vesicles (blue arrowhead). (e) A tip region showing a few stained vesicles (asterisks). (f,g) Regions far from the tip showing gold particles (black) regularly spaced on the PM surface. Bars, 0.5 µm (a,b,d-g), 1 µm (c).

 

Figure 3
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  		Fig. 3. Internalization of positively charged nanogold for 1-2 hours. (a,b) After 1 hour, vacuoles were labelled (arrows). (c) After 2 hours, gold uptake occurred in subapical regions of the tube (black arrow). (d) Gold particles in vesicles and cisternae of the cis, medial and trans faces of Golgi bodies. (e) A large number of stained vesicles were observed in the tip region. Gold particles were observed in tubules of smooth ER (arrows). Bars, 0.5 µm.

 

Figure 4
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  		Fig. 4. Pulse chase using charged nanogold. (a,b) Positively charged nanogold (+) accumulated in vacuoles (arrow), but was not observed in other organelles. (c,d) Negatively charged nanogold (–) was found in vesicles in the tip region (c) and in vacuoles (d, arrow). Bars, 0.5 µm.

 

Figure 5
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  		Fig. 5. Internalization of negatively charged nanogold for 15-30 minutes. (a,b) After 15 minutes, negatively charged nanogold was seen in vesicles in the TGN region (a, arrows) and in vacuole-like compartments (a, arrowheads and b, arrows). (c) After 30 minutes, invaginating vesicles were seen in the tip (arrowhead), whereas few vesicles were labelled in the apex. (d) No internalization was observed in the subapical region of the tube and Golgi (G) bodies were not labelled. Arrowhead indicates gold particles on the PM in the organelle-rich zone. (e) Organelle-rich zone showing unlabelled Golgi apparatus (G). Bars, 0.5 µm (a,b,e), 1 µm (c,d).

 

Figure 6
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  		Fig. 6. Internalization of negatively charged nanogold for 1 hour. (a) Accumulation of probe was not observed in the tip region, in which a limited number of vesicles was labelled (arrows). (b-e) Internalization was not observed in the subapical region (b-d) or in the region far from the tip (e). (b,d,f) Negatively charged nanogold was never observed within membranous organelles such as the ER or Golgi bodies (G). Bars, 0.5 µm (a-e), 1 µm (f).

 

Figure 7
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  		Fig. 7. Internalization of negatively charged nanogold for 2 hours. (a) Gold particles were seen associated with the PM (arrows) far from the tip. (b) In the tip region, only a few vesicles were stained. (c) Golgi bodies were not labelled. (d) Gold particles were observed in vacuoles after 2 hours of incubation (arrows). Bars, 0.5 µm.

 

Figure 8
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  		Fig. 8. Time-lapse experiments. (A) Control tubes. (Aa-Aa'') green ROI indicates the area measured in the tip region. Orange and violet ROI indicate the areas measured on the flanks of tubes. (Ab) Graph showing the mean fluorescence at the tip and in the subapical regions for 300 seconds. Arrow indicates the time of FM4-64 addition. (B) Ika-treated tubes. (Ba-Ba'') Coloured areas indicate regions in which fluorescence was measured (see above). (Bb) Graph showing mean fluorescence at the tip and in subapical regions for 400 seconds. Arrow indicates time of FM4-64 addition. (Bc-Be') Medial plane images of the pollen tube 15-60 minutes after FM4-64 loading. Bars, 10 µm.

 

Figure 9
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  		Fig. 9. Statistical analysis of quantitative FM4-64 uptake in the tip with respect to subapical regions of pollen tubes grown in control medium and with Ika. (a) Internalization of FM4-64 in flank regions was almost double that at the apex. (b) Quantitative analysis of FM4-64 internalization in two regions of the tube, in control medium and in the presence of Ika. (a,b) Error bars indicate 95% confidence intervals (n=6). t-test analysis of FM4-64 uptake in the presence and absence of Ika gave P values in control tubes and tubes incubated with Ika that indicated that inhibition of FM4-64 uptake was significant in the apical and subapical regions (control, P=0.01401; Ika, P=0.05294). Apical (Ika) versus apical (control): P=0.01149. Subapical (Ika) versus subapical (control): P=0.00142. For quantization, the last ten frames after reaching the steady state were considered in six independent experiments.

 

Figure 10
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  		Fig. 10. Pulse-chase experiments using FM4-64, LysoSensor Blue and BODIPY TR-Ceramide. (A) Pulse-chase experiments using FM4-64. (Aa,Aa') Internalized PM is accumulated in the tip region 15 minutes after the pulse. (Aa'') Five hours after the pulse, the staining in the apex was faint or absent. (Ab-Ab'') Pollen tube 24 hours after the pulse with FM4-64 (Ab); colocalization experiments using LysoSensor Blue (Ab') showed that the probes overlap in the vacuolar membranes (Ab''). (Ab''') Citofluogram that visualizes the frequency distribution of intensity in a 2D scatter plot. (B) Colocalization of FM4-64 with BODIPY-labelled membranes. (Ba,Bb) BODIPY TR-Ceramide-stained spots are distributed in the cytoplasm and are more concentrated in the tip region to form a collar-like structure (arrowheads) just behind the extreme tip at 5 (a) and 30 (b) minutes after the addition of FM4-64. (Ba',Bb') FM4-64 staining pattern after 5 (Ba') and 30 (Bb') minutes. (Ba'',Bb'') Colocalization analysis showing that probe staining overlaps in the collar-like structure and in the whole tip (white spots) after 5 and 30 minutes, respectively, after the addition of FM4-64. (Bc,Bd) Citofluograms that allow the distribution of intensity of green and red channels to be visualized in a 2D scatter plot referred to time-course analysis 5 and 30 minutes after FM4-64 addition, respectively. The ROIs select colocalized pixels. Bars, 10 µm.

 

Figure 11
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  		Fig. 11. Effect of Ika on positively and negatively charged nanogold internalization. (a,b) After 30 minutes incubation with Ika, positively charged nanogold was found in vacuoles (arrows and arrowheads), whereas the Golgi body (b, G) was not labelled. (c) Gold particles in vesicles containing fibrillar cell wall material after 30 minutes of incubation. (d) After 1 hour, positively charged nanogold was seen in small vesicles (black arrowheads). Large vesicles/endosomes were also labelled (asterisks). Golgi bodies in the picture only showed two gold particles (blue arrowheads), whereas one vesicle of the TGN was labelled (yellow arrowhead). (e-g) Negatively charged nanogold was not seen in vacuoles and was only sometimes observed in vesicles (f, arrowhead). Bars, 0.5 µm.

 

Figure 12
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  		Fig. 12. Effect of Ika on the distribution of CHC. (A) The specificity of the 4A8 monoclonal antibody (Mab) was tested by SDS-PAGE and western blot of pollen tube crude extracts (CE). 4A8 antibody specifically recognized a 167 kDa polypeptide (lane 4A8, arrow). In the control (lane C) assay, the primary antibody was omitted. St, molecular-mass markers. (B) Immunostaining to reveal the effect of Ika on CHC distribution with 4A8 Mab. (Ba-Bb') Medial sections of tubes (1 µm) treated with 3 µM Ika for 15 or 45 minutes showed increased association of CHC with the PM in the apical and subapical regions. (Bc,Bc') In control (Co) experiments, 4A8 antibody gave punctate staining on the PM (arrowheads) and in the cytoplasm. Bars, 20 µm. (C) Cytoplasmic dissection with 3 µM Ika suggested that CHC was found in the microsomal fraction (P2) in preference to the soluble fraction (S2) (row 4A8). In the control microsome preparation, CHC appeared to be more equally distributed between P2 and S2 (row 4A8). Staining of membrane used for CHC detection with an anti-{alpha} tubulin antibody is also reported. (D) Densitometric analysis of anti-CHC western blot with Ika showed an integrated optical density (IOD) of CHC in P2 samples that was almost sixfold greater than that in S2, whereas, in the control experiments, it was only 1.6-fold greater than S2. Error bars indicate standard errors (n=3). 15 µg of proteins were loaded in each lane.

 

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© The Company of Biologists Ltd 2007