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J Cell Biol. 2014 May 26;205(4):591-606. doi: 10.1083/jcb.201402066.

A new probe for super-resolution imaging of membranes elucidates trafficking pathways.

Author information

1
Department of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, GermanyDepartment of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, Germany International Max Planck Research School for Neurosciences, 37077 Göttingen, Germany Collaborative Research Center 889 and Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany.
2
Department of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, GermanyDepartment of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, Germany srizzol@gwdg.de dirk.kamin@mpibpc.mpg.de.
3
Department of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, GermanyDepartment of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, Germany International Max Planck Research School for Molecular Biology, 37077 Göttingen, Germany.
4
Department of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, Germany International Max Planck Research School for Neurosciences, 37077 Göttingen, Germany Collaborative Research Center 889 and Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany.
5
Department of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, Germany Collaborative Research Center 889 and Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany.
6
Department of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, Germany Collaborative Research Center 889 and Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany Collaborative Research Center 889 and Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany.
7
Department of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, GermanyDepartment of Neuro- and Sensory Physiology; European Neuroscience Institute; and InnerEarLab and Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology; University Medical Center Göttingen, 37099 Göttingen, Germany Collaborative Research Center 889 and Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany Collaborative Research Center 889 and Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, 37099 Göttingen, Germany srizzol@gwdg.de dirk.kamin@mpibpc.mpg.de.

Abstract

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.

PMID:
24862576
PMCID:
PMC4033769
DOI:
10.1083/jcb.201402066
[Indexed for MEDLINE]
Free PMC Article

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