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Blood, 1 January 2005, Vol. 105, No. 1, pp. 161-169.
Prepublished online as a Blood First Edition Paper on August 31, 2004; DOI 10.1182/blood-2004-06-2067.
 Previous Article | Next Article 
Submitted June 2, 2004
Accepted August 12, 2004
Rod mutations associated with MYH9-related disorders disrupt non-muscle myosin-IIA assembly
Josef D Franke, Fan Dong, Wayne L Rickoll, Michael J Kelley, and Daniel P Kiehart*
Department of Cell Biology, Duke University Medical Center, Durham, NC, USA
Department of Medicine, Duke University Medical Center, Durham, NC, USA; Hematology/Oncology, Durham Veterans Affairs Medical Center, Durham, NC, USA
Department of Biology, University of Puget Sound, Tacoma, WA, USA
Department of Biology, DCMB Group, Duke University Medical Center, Durham, NC, USA; Department of Cell Biology, Duke University Medical Center, Durham, NC, USA
* Corresponding author; email: dkiehart{at}duke.edu.
MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human non-muscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt non-muscle myosin-IIA function, we examined the in vitro behavior of four common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared to wild-type. We used negative stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild-type. Using circular dichroism we find that two mutants affect the  -helical coiled-coil structure of individual molecules, and two mutants disrupt the lateral associations between individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in non-muscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease.
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