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Localized endocytosis in tobacco pollen tubes: visualisation and dynamics of membrane retrieval by a fluorescent phospholipid

  • Cell Biology and Morphogenesis
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Abstract

Two modes of endocytosis are known to occur in eucaryotic cells: fluid phase and receptor-mediated endocytosis. Fluid-phase endocytosis in plant cells resembles the retrieval of excess plasma membrane material previously incorporated by exocytosis. Pollen tubes need to carry out strong membrane retrieval due to their fast polar tip growth. Plasma membrane labelling of pollen tubes, grown in suspension, was achieved by the incorporation of a fluorescently modified phospholipid, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (20 μM) and measured with a confocal laser-scanning microscope. Time course experiments revealed a highly localised and relatively fast plasma membrane retrieval below the tip within the first 5 min after phospholipid application. The retrieved fluorescent plasma membrane was quickly re-integrated into parts of the endomembrane pool and then redistributed to the pollen tube base and very tip of the apex, with the exception of the cortical endoplasmic reticulum (ER) and the mitochondria even after 1-h incubation period. Low temperature (10°C) and the actin filament depolymerizing cytochalasin D (2 μM) completely abolished plasma membrane retrieval, whereas the microtubule destabilizing herbicide oryzalin (1 μM) had no effect. Our results provide strong support for a highly localised endocytotic pathway in tobacco pollen tubes. Passive uptake of bis-Bodipy FL C11-phosphocholine by mere penetration can be excluded. It is a valuable alternative to the styryl dyes often used in endocytotic studies, and may also be used to follow lipid turnover because membrane flow of labelled membranes occurs apparently not in a default manner as ascertained by its fast distribution.

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Abbreviations

bis-Bodipy FL C11-phosphocholine:

(1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine

β-Bodipy 581/591C5 HPC:

2-(4,4-difluoro-5-(4-phenyl 1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine

DiOC6(3):

3,3-dihexyloxacarbocyanine iodine

FM4-64:

N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridium dibromide

mitotracker RedCMXRos:

8-(4′-chloromethyl)phenyl-2,3,5,6,11, 12, 14,15-octahydro-1H, 4H, 10H13H-diquinolizino-8H-xanthylium chloride

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Acknowledgments

This work was supported by the Bundesministerium für Forschung (Grant 50WB0010 to G.S. and H.Q.). The gift of the clathrin heavy chain antibody by Prof. D.G. Robinson and the technical assistance of Mrs. E. Woelken is gratefully acknowledged.

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Authors and Affiliations

  1. Biozentrum Klein Flottbek, University of Hamburg, Ohnhorst-Str. 18, 22609, Hamburg, Germany

    S. Lisboa & H. Quader

  2. Institute of Zierpflanzenbau, University of Hannover, Hannover, Germany

    G. E. F. Scherer

Authors
  1. S. Lisboa
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  2. G. E. F. Scherer
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  3. H. Quader
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Correspondence to H. Quader.

Additional information

Communicated by R. Reski.

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Fig8

S1: Endocytotic plasma membrane retrieval: distribution of the fluorescent marker phospholipid into endomembranes . Staining of endocytotic pathway with the phospholipid bis-Bodipy FL C11-phosphocholine (excitation: blue, emission: green; (1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine) and the mitochondria with the mitotracker RedCMXRos (excitation: green, emission: red; 8-(4′-chloromethyl)phenyl-2,3,5,6,11, 12, 14,15-octahydro-1H,4H,10H13H-diquinolizino-8H-xanthylium chloride) for 30 min. Time sequence of 15 imgages taken with a confocal scanning laser microscope (Leica, TCS 4D) without moving the microscopical stage neither in z- nor in xy-direction throughout the acquisition time (3 min). Note the changes in the distribution of brightly green fluorescent large vesicle like compartments which could represent endosomes or lipid bodies, but we do not think that it concerns dictyosomes. Besides these structures the endoplasmic reticulum became labelled for the most part by the fluorescent marker phospholipid. In the very tip of the pollen tube the dynamic formation of a sheet like ER domain is recognizable. Mitochondria are visible as small orange/red fast moving compartments.

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Lisboa, S., Scherer, G.E.F. & Quader, H. Localized endocytosis in tobacco pollen tubes: visualisation and dynamics of membrane retrieval by a fluorescent phospholipid. Plant Cell Rep 27, 21–28 (2008). https://doi.org/10.1007/s00299-007-0437-1

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