PUBLICATION

Ribeye a-mCherry fusion protein: A novel tool for labeling synaptic ribbons of the hair cell

Authors
West, M.C., and McDermott, B.M. Jr.
ID
ZDB-PUB-110316-6
Date
2011
Source
Journal of Neuroscience Methods   197(2): 274-8 (Journal)
Registered Authors
McDermott Jr., Brian M., West, Megan
Keywords
Balance, Hair cell, Hearing, Transgenic, Zebrafish
MeSH Terms
  • 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*
PubMed
21334379 Full text @ 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.
Genes / Markers
Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes