RRC ID 75844
Author West MC, McDermott BM Jr.
Title Ribeye a-mCherry fusion protein: a novel tool for labeling synaptic ribbons of the hair cell.
Journal J Neurosci Methods
Abstract Synaptic ribbons are presynaptic cytomatrices that are required for efficient transfer of auditory information from hair cells to the central nervous system. In the hair cell, each electron-dense ribbon tethers numerous synaptic vesicles by fine filaments. The ribbon generally resides juxtaposed to the active zone plasma membrane. A dearth of appropriate tools to visualize the ribbon synapse has limited our knowledge of its development. Here we present the design and implementation of a method to visualize synaptic ribbons in hair cells. This scheme uses a tagged version of the protein Ribeye a, which is specific to ribbons. We generate the DNA construct Tg(pvalb3b:ribeye a-mCherry) to transgenically express the fusion protein Ribeye a-mCherry in zebrafish hair cells. The fusion protein localizes to the basolateral surface of the hair cell with a pattern similar to that of a hair cell labeled with an antiserum that recognizes ribeye proteins. Moreover, using this antiserum to label transgenics that express Ribeye a-mCherry, we demonstrate that the fusion protein and antibody-associated fluorescent signals overlap. In addition, ribbons labeled with the fusion protein are proximal to afferent nerve endings. Finally, the fusion protein labels hair-cell ribbons of zebrafish at different developmental time points. These findings indicate that the fusion protein is an effective tool to label ribbons in live and fixed hair cells, which will make it useful in the study of ribbon synapse development and to characterize zebrafish mutants with defects in synapse formation.
Volume 197(2)
Pages 274-8
Published 2011-4-30
DOI 10.1016/j.jneumeth.2010.11.011
PII S0165-0270(10)00645-X
PMID 21334379
MeSH Animals Animals, Genetically Modified Biomarkers / metabolism Hair Cells, Auditory / metabolism* Hair Cells, Auditory / ultrastructure Luminescent Proteins / genetics* Luminescent Proteins / metabolism Microscopy, Confocal / methods Recombinant Fusion Proteins / genetics* Recombinant Fusion Proteins / metabolism Staining and Labeling / methods Synapses / metabolism* Synapses / ultrastructure Zebrafish Zebrafish Proteins / genetics*
IF 2.214
Zebrafish HGn39D