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Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells

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Summary.

The vacuole is a characteristic organelle of plant cells and fulfills several important functions related to metabolism and growth of the cell. To shed light on the details of vacuolar structural changes in plant cells, we explored the three-dimensional organization and dynamics of living Nicotiana tabacum L. cv. Bright Yellow 2 cell vacuoles by real-time confocal time-lapse imaging. For imaging, the cells were pulse-labeled with the amphipathic styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), which is delivered to the plant vacuole by endocytic uptake and then incubated overnight. Imaging of the membrane-labeled vacuole revealed a complex vacuole morphology underlaid by constant remodeling. The vacuole is traversed by multiple transvacuolar strands which move along each other and fuse in multiple manners. New strands were created by fission of large membrane sheets. Endocytic vesicle trafficking was followed within the dynamic transvacuolar strands. The movement occurred in a stop-and-go fashion with an average vesicle velocity of 0.46 μm/s and a peak velocity of 0.82 μm/s. Transvacuolar-strand reduction and creation is a characteristic event observed during mitosis. Here we propose a mechanistic model for the alteration of the number of transvacuolar strands, on the basis of their fusion and fission.

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Authors and Affiliations

  1. Cellome Research Group, Institute for Molecular Biotechnology, Aachen University, Aachen

    N. Ruthardt, H. Spiegel, R. Fischer & N. Emans

  2. Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg-Grafschaft

    R. Fischer

  3. Visitron Systems, Pucheim

    N. Gulde

Authors
  1. N. Ruthardt
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  2. N. Gulde
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  3. H. Spiegel
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  4. R. Fischer
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  5. N. Emans
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Correspondence and reprints: Cellome Research Group, Institute for Molecular Biotechnology, Rheinisch-Westfälische Technische Hochschule Aachen, Worringerweg 1, 52074 Aachen, Federal Republic of Germany.

Electronic supplementary material

Fig. S1.

Plant vacuoles labeled with FM 1-43 are complex in three dimensions and traversed by transvacuolar strands. Three-dimensional reconstructions of a confocal 20 μm deep Z-section of the central three cells from a BY-2 suspension cell chain labeled with FM 1-43. The QuickTime movie shows a 3-D reconstruction rotating 360 degrees around the Z-axis

Fig. S2A.

Transvacuolar strand dynamics in living plant cell vacuoles. 3-D time-lapse movie of an 8 μm deep Z-section of a forked transvacuolar strand showing membrane bolus movement across the upper fork of the strand. The time-lapse was acquired at 15 s intervals. The QuickTime movie shows a sequence playing at 225-fold real time

Fig. S2B.

Transvacuolar strand dynamics in living plant cell vacuoles. 3-D time-lapse movie of a 20 μm deep Z-section showing strand-to-strand movement followed by a bolus migrating through one strand. The QuickTime movie shows a sequence playing at 150-fold real time

Fig. S3A.

Transvacuolar strands in tobacco suspension cells undergo fusion within the vacuolar lumen. 3-D time-lapse movie of a 10 μm deep confocal Z-section of a suspension cell showing two strands that undergo fusion. The QuickTime movie shows a sequence playing at 160-fold real time

Fig. S3B.

Transvacuolar strands in tobacco suspension cells undergo fusion within the vacuolar lumen. Time-lapse movie of 3-D confocal images of a 12 μm deep Z-section acquired at 15 s intervals, showing multiple transvacuolar strand fusion. The QuickTime movie shows the sequence playing at 180-fold real time

Fig. S3C.

Transvacuolar strands in tobacco suspension cells undergo fusion within the vacuolar lumen. Transvacuolar strand fusion on the whole-cell level in a 16 μm deep Z-section over time from four different perspectives. The image of the fusing strands rotates around the Z-axis as the movie progresses in time. For each segment, the projection angle increases by one degree every 15 s. The upper left shows panel projection angles from 1 to 60 degrees, the upper right from 61 to 120 degrees, lower left from 121 to 180 degrees, lower right from 181 to 240 degrees

Fig. S4.

Transvacuolar strands evolve from splitting membrane sheets. 3-D confocal time-lapse movie of a 16 μm deep Z-section acquired at 15 s intervals, showing the formation of new strands. The movie shows the sequence playing at 125-fold real time

Fig. S5.

Endosome trafficking occurs within mobile trans-vacuolar strands. 3-D confocal time-lapse movie of a 4 μm deep Z-section showing a suspension cell labeled by a 2 h pulse of 20 μM FM 1-43 and overnight chase, followed by plasma membrane labeling with 20 μM FM 4-64 prior to imaging of FM 4-64 internalization. The QuickTime movie shows the overlay sequence of endosome movement playing at 720-fold real time

Fig. S6.

Endosome tracking in living plant cells. Endosomes were tracked in 3-D time-lapse movies taken of suspension cells that had internalized FM 1-43 for 50 min. The movie shows the tracking of two endosomes (red overlay) in a 1.5 μm deep confocal section of a transvacuolar strand and plays at 10-fold real time

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Ruthardt, N., Gulde, N., Spiegel, H. et al. Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells. Protoplasma 225, 205–215 (2005). https://doi.org/10.1007/s00709-005-0093-7

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