Characterization of the three zebrafish orthologs of the mitochondrial GTPase Miro/Rhot.
Mitochondria exhibit dynamic locomotion and spatial rearrangement. This movement is necessary for a cell to maintain basic metabolic functions, and disruption of motility often results in cell death. Miro is a mitochondrial outer membrane Rho GTPase essential for mitochondrial movement and distribution in diverse systems, including yeast, animals, and plants. We sought to study the previously uncharacterized Miro protein family in zebrafish. We confirmed that, like human Miro, the zebrafish Miro proteins (Rhot1a, Rhot1b, and Rhot2) localize to mitochondria in mammalian tissue culture cells by both biochemical fractionation and immunofluorescent colocalization. In addition, using whole mount in situ hybridization, we observed ubiquitous expression of all three mRNAs throughout development. By microinjecting three antisense morpholino oligonucleotides targeted to each of the rhot genes, we knocked down all three proteins simultaneously in developing zebrafish embryos. The triple morphants demonstrated a dose-dependent defect in posterior body-axis elongation, while a single knockdown of each protein at the same dose produced no effect. This phenotype could be rescued with human Miro1 mRNA and is most likely due to increased cell death. Taken altogether, this research demonstrates the importance of the Rhot proteins during vertebrate development.
Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN 37232-2358, USA.
Brittany M
BM
Hollister
Department of Cell Biology, Duke University, Durham, NC 27710, USA.
Kelsey A
KA
Oonk
Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
Douglas C
DC
Weiser
Department of Biology, Rollins College, Winter Park, FL 32789, USA. Electronic address: sjwalsh@rollins.edu.
Susan
S
Walsh
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D006801:Humans; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D020741:rho GTP-Binding Proteins
Danio rerio; Miro; Mitochondria; Rhot; Trak; Zebrafish