A New Probe for Super-Resolution Imaging of Membranes Elucidates Trafficking Pathways

October 17, 2014

The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.

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Revelo NH, Kamin D, Truckenbrodt S, Wong AB, Reuter-Jessen K, Reisinger E, Moser T, and Rizzoli SO:
A New Probe for Super-Resolution Imaging of Membranes Elucidates Trafficking Pathways

JCB 205 (4): 591–606 (2014); doi: 10.1083/jcb.201402066

In the past two decades, super-resolution microscopy has been one of the fastest evolving fields through many technical improvements. However, the development of new labeling tools, probes and their biological application, is mostly lagging behind the technical capabilities. Most recently, Prof. Silvio O. Rizzoli from the Cluster of Excellence and DFGResearch Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) has developed together with his team a new technique that expands the benefit of superresolution microscopy to study biological questions. This method contributes to understand on how cells renew, distribute and transport their molecular and subcellular components. 

All cells rely on the recycling of membranes via various pathways (secretion, uptake, andmembrane turnover). Several types of cellular organelles such as the plasma membrane, theendoplasmic reticulum, the Golgi apparatus, endosomes and vesicles are involved in theseprocesses. However, it was difficult to identify the protein composition of the involvedorganelles since both, the membranes and the proteins of the same organelle need to bemarked simultaneously. Here the main difficulty comes with the membrane probe, as almostall dyes that work excellent in live cell experiments are only poorly fixable and get “lost”during the antibody staining procedure.

The research team with first author Natalia Revelo therefore developed a membrane probethat overcomes this problem. The probe mCLING (membrane-binding fluorophore-Cysteine-Lysine-Palmitoyl Group) is a composition of a short polypeptide coupled to a membraneanchor and a fluorophore.

The study, published in the Journal of Cell Biology, shows that mCLING can be used to label the plasma membrane, and also to faithfully track specific organelles, which can be done in conjunction with fixation and immunostaining, in both cellculture and in tissue. The utility of the mCLING probe could be characterized for variousimportant biological model systems and already enabled the authors to answer long-lasting question in the field of membrane recycling. Moreover, mCLING imaging could also beextended to different processes. For example, the structure and molecular organization ofisolated organelles in vitro, or the arrangement of proteins on the membranes of varioustypes of cells, can be easily tackled with mCLING. These efforts will be aided by the fact that mCLING can be optimized for any available super-resolution technique.

From left to right: Prof. Dr. Silvio O. Rizzoli, Natalia Revelo, Dr. Dirk Kamin, Sven Truckenbrodt. Foto: AG Rizzoli.

More information:
CNMPB: http://www.cnmpb.de

Prof. Dr. Silvio O. Rizzoli
University Medical Center Göttingen
Department Neuro- & Sensory Physiology
c/o European Neuroscience Institute (ENI)
Grisebachstraße 5, 37077 Göttingen
Phone +49 (0) 551 / 39-33630
srizzol@gwdg.de

CNMPB – Center for Nanoscale Microscopy and Molecular Physiology of the Brain
Cluster of Excellence 171 – DFG Research Center 103
Dr. Heike Conrad
Scientific Coordination, Press & Public Relations
Humboldtallee 23, 37073 Göttingen
Phone +49 (0) 551 / 39-7065
heike.conrad@med.uni-goettingen.de

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