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Fluorescent phosphocholine—a specific marker for the endoplasmic reticulum and for lipid droplets in Chara internodal cells

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Abstract

The staining pattern of 1,2-bis(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (Bodipy PC) was investigated in internodal cells of the green alga Chara corallina. Ten minutes after dye addition, Bodipy-PC-derived fluorescence appeared in lipid droplets and after 1 h in the cortical endoplasmic reticulum (ER) and in the inner ER tubes. Staining of the ER required energy but was independent of an intact actin or microtubule cytoskeleton and independent of vesicular endocytosis. The size of the lipid droplets varied between 0.25 µm in elongating cells and 3.2 µm in senescent internodes. They moved together with or along the cortical ER cisternae in a cytoskeleton-independent manner or remained immobile up to several minutes. Detachment of lipid droplets from the cortical ER or fusion of lipid droplets was never observed. The results of this study suggest that Bodipy PC is a valuable, less toxic alternative to 3,3′-dihexyloxacarbocyanine iodide (DiOC6) staining of the ER in Chara. They confirm an earlier report about microtubule-dependent cortical ER morphology and dynamics in elongating internodes and offer new perspectives for the study of organelle interactions.

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Abbreviations

Bodipy PC:

Bis-BODIPY© FL C11-PC = 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine

CD:

Cytochalasin D

CLSM:

Confocal laser scanning microscope/microscopy

DIC:

Differential interference contrast

DiOC6 :

3,3′-Dihexyloxacarbocyanine iodide

DMSO:

Dimethyl sulfoxide

DNP:

2,4-Dinitrophenole

EDTA:

Ethylenediaminetetraacetic acid

EGTA:

Ethylene glycol tetraacetic acid

IPC:

Isopropyl N-(3-chlorophenyl)-carbamate

fps:

Frames per second

NBD PC:

1-Acyl-2-(N-4-nitrobenz∼2-oxa-l,3-diazole)aminoacyl phosphatidyl choline

PIPES:

Piperazine-N,N′-bis (2-ethanesulfonic acid)

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Acknowledgements

I am grateful to Patric Schmölzer and Christian Pritz for valuable discussion.

Conflict of interest

The author declares that she has no conflict of interest.

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Authors and Affiliations

  1. Department of Cell Biology, Division of Plant Physiology, University of Salzburg, Hellbrunnerstrasse 34, 5020, Salzburg, Austria

    Ilse Foissner

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  1. Ilse Foissner
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Correspondence to Ilse Foissner.

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Suppl. Fig. 1

Bodipy-PC-stained cortical ER in an internodal cell of C. corallina before (a) and after 1 h treatment with 50 µM IPC (b). Bar is 8 µm (JPEG 618 kb)

Suppl. Movie 1

Mature C. corallina internodal cell stained with Bodipy PC (green) for 2 h. During this time series, the focus was manually changed from the cortical to the inner cytoplasm which are separated from each other by the stationary layer of autofluorescent chloroplasts (red). Bright green spots visible at the upper and lower side of images 1–10 are Bodipy-PC-stained epiphytes on the outer surface of the cell wall. The horizontal axis of the images corresponds to the long axis of the internode. Note striking contrast in the dynamics of the cortical and subcortical ER. Images were collected at 600-ms interval and played back at 7 fps. Total elapsed time is 27 s (AVI 8645 kb)

Suppl. Movie 2

Bodipy-PC-stained cortical ER and lipid droplets from a mature internodal cell. Time-lapse images were collected at 6.5-s intervals and played back at 5 fps. Total elapsed time is 248 s (AVI 30424 kb)

Suppl. Movie 3

Bodipy-PC-stained cortical ER and lipid droplets from a rapidly elongating internodal cell (compare Fig. 5l–p). Time-lapse images are positioned with horizontal sides parallel to the long axis of the internode. They were collected at 8.8-s intervals and played back at 5 fps. Total elapsed time is 106 s (AVI 3617 kb)

Bodipy-PC-stained cortical ER and lipid droplets from an internodal cell that had recently stopped elongation (compare Fig. 5e–k). Time-lapse images were positioned with horizontal sides slightly oblique to the long axis of the internode, were collected at 7.7-s intervals and played back at 5 fps. Total elapsed time is 453 s (AVI 34722 kb)

Suppl. Movie 5

Bodipy-PC-stained cortical ER and lipid droplets from a mature internodal cell treated with 5 µM CD for 30 min. Time-lapse images were collected at 4.6-s intervals and played back at 5 fps. Total elapsed time is 109 s (AVI 19504 kb)

Suppl. Movie 6

Z-series through cytoplasmic droplet of unstained cell squeezed into Bodipy-PC-containing perfusion solution. Note reticulate meshwork and intense labelling of the nuclear (N) surface. Bodipy fluorescence is absent from chloroplasts (not seen but their positions are indicated by C). Optical sections were taken at 1.03-µm intervals, total stack size is 8.2 µm (AVI 6916 kb)

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Foissner, I. Fluorescent phosphocholine—a specific marker for the endoplasmic reticulum and for lipid droplets in Chara internodal cells. Protoplasma 238, 47–58 (2009). https://doi.org/10.1007/s00709-009-0072-5

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