In vitro expression and characterization of MYH9 mutant alleles linked to hereditary hearing loss
This article was presented at the 2009 AAO–HNSF Annual Meeting & OTO EXPO, San Diego, CA, October 4-7, 2009.
Received 9 September 2009; received in revised form 20 November 2009; accepted 10 December 2009. published online 25 March 2010.
Abstract
Objective
To assess whether MYH9 mutant alleles linked to hereditary hearing loss induce disruption of cellular functions and associated phenotype following transient expression within cultured human cell lines.
Study Design
Dominantly inherited MYH9 mutant alleles, MYH9R702C and MYH9R705H, were integrated within eukaryotic expression vector and then transfected into cultured human cell lines for transient expression and analysis. The transfected cells were assessed for transgene-induced alterations of the cellular phenotype, including NMHC-IIA-dependent cell shape, actin cytoskeleton integrity, and inhibition of cytokinesis.
Setting
Laboratories of Molecular Otology and Molecular Genetics at the New York University School of Medicine.
Subjects and Methods
HeLa and MDA-MB-231 cultured cell lines were transiently transfected with an expression vector carrying a wild type or mutant MYH9 alleles, linked to nonsyndromic and syndromic hearing loss. Expression of exogenous transgene product was detected with antibodies directed toward its N-terminal HA tag, and transfection efficiency was greater than 95 percent. Host cells were characterized for cell shape, integrity of actin-myosin cytoskeleton, and nuclear status before and after transfections via immunofluorescence.
Results
MDA-MB-231 cells transfected with MYH9R705H but not MYH9R702C were found to have a greater than two-fold increase in cells with filopodia and a ten-fold increase in proportion of cells with multiple nuclei, indicating inhibition of cytokinesis, relative to the control cells transfected with wild type MYH9. Actin cytoskeleton configuration within MDA-MB-231 cells was unaffected by expression of MYH9R702C or MYH9R705H. Unlike MDA-MB-231 cells, HeLa cells were refractory to MYH9R705H and MYH9R702C.
Conclusions
MYH9R705H-induced altered phenotype of the MDA-MB-231 cell line supports the pathogenicity of the mutation and represents a suitable assay system for identification and characterization of its dysfunction.
aLaboratory of Molecular Otology, Department of Otolaryngology, New York University School of Medicine, New York, NY
bLaboratory of Molecular Genetics, Department of Otolaryngology, New York University School of Medicine, New York, NY
cDepartment of Physiology and Neuroscience, New York University School of Medicine, New York, NY
dDepartment of Pediatrics, New York University School of Medicine, New York, NY
Corresponding author: Anand N. Mhatre, PhD, Assistant Professor, Department of Otolaryngology, New York University School of Medicine, 560 First Ave., TCH 513, New York, NY 10016
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
ABSTRACT