TY - JOUR
T1 - A Novel Method of Determining the Functional Effects of a Minor Genetic Modification of a Protein
AU - Nagwekar, Janhavi
AU - Duggal, Divya
AU - Midde, Krishna
AU - Rich, Ryan
AU - Liang, Jingsheng
AU - Kazmierczak, Katarzyna
AU - Huang, Wenrui
AU - Fudala, Rafal
AU - Gryczynski, Ignacy
AU - Gryczynski, Zygmunt
AU - Szczesna-Cordary, Danuta
AU - Borejdo, Julian
N1 - Funding Information:
This work was supported by the National Institutes of Health (NIH) grants R01AR048622 (JB), R01HL108343 and HL123255 (DSC), R01HL090786 (JB and DSC), and R01EB12003 (ZG).
Publisher Copyright:
© Copyright © 2015 Nagwekar, Duggal, Midde, Rich, Liang, Kazmierczak, Huang, Fudala, Gryczynski, Gryczynski, Szczesna-Cordary and Borejdo.
PY - 2015/11/18
Y1 - 2015/11/18
N2 - Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being “kicked” by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either “Standard” (Std) (decaying monotonically in time) or “Non-standard” (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.
AB - Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being “kicked” by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either “Standard” (Std) (decaying monotonically in time) or “Non-standard” (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.
KW - actin
KW - autocorrelation function
KW - fluorescence polarization
KW - low expressing mutations
KW - myosin light chain
KW - phalloidin-actin
UR - http://www.scopus.com/inward/record.url?scp=85074514422&partnerID=8YFLogxK
U2 - 10.3389/fcvm.2015.00035
DO - 10.3389/fcvm.2015.00035
M3 - Article
AN - SCOPUS:85074514422
SN - 2297-055X
VL - 2
JO - Frontiers in Cardiovascular Medicine
JF - Frontiers in Cardiovascular Medicine
M1 - 35
ER -