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JXB Advance Access published online on July 4, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern154
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Clathrin-dependent and independent endocytic pathways in tobacco protoplasts revealed by labelling with charged nanogold

Elisabetta Onelli1, Cristina Prescianotto-Baschong2, Marco Caccianiga1 and Alessandra Moscatelli1,*

1Department of Biology, University of Milan, Milan, Italy
2Department of Biochemistry, Biozentrum, University of Basel, Basel, Switzerland

* To whom correspondence should be addressed. E-mail: alessandra.moscatelli{at}unimi.it

Positively charged nanogold was used as a probe to trace the internalization of plasma membrane (PM) domains carrying negatively charged residues at an ultrastructural level. The probe revealed distinct endocytic pathways within tobacco protoplasts and allowed the morphology of the organelles involved in endocytosis to be characterized in great detail. Putative early endosomes with a tubulo-vesicular structure, similar to that observed in animal cells, are described and a new compartment, characterized by interconnected vesicles, was identified as a late endosome using the Arabidopsis anti-syntaxin family Syp-21 antibody. Endocytosis dissection using Brefeldin A (BFA), pulse chase, temperature- and energy-dependent experiments combined with quantitative analysis of nanogold particles in different compartments, suggested that recycling to the PM predominated with respect to degradation. Further experiments using ikarugamycin (IKA), an inhibitor of clathrin-dependent endocytosis, and negatively charged nanogold confirmed that distinct endocytic pathways coexist in tobacco protoplasts.

Key words: Charged nanogold, endocytosis, membrane recycling, Nicotiana tabacum, protoplasts, vesicle trafficking

Received 28 February 2008; Revised 3 April 2008 Accepted 8 May 2008


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