Biomedical subjects

Melissa McKane

Publications and source records attributed to Melissa McKane.

3 recordsLinked to original sources

PubMed2006

Effect of the substitution of muscle actin-specific subdomain 1 and 2 residues in yeast actin on actin function.

Muscle and yeast actins display distinct behavioral characteristics. To better understand the allosteric interactions that regulate actin function, we created a muscle/yeast hybrid actin containing a muscle-specific outer domain (subdomains 1 and 2) and a yeast inner domain (subdomains 3 and 4). Actin with muscle subdomain 1 and the two yeast N-terminal negative charges supported viability. The four negative charge muscle N terminus in a muscle subdomain 1 background caused death, but in the same background actin with three N-terminal acidic residues (3Ac/Sub1) led to sick but viable cells. Addition of three muscle subdomain 2 residues (3Ac/Sub12) produced no further deleterious effects. These hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, and mitochondrial and endocytosis defects. 3Ac/Sub1 G-actin exchanged bound epsilonATP more slowly than wild type actin, and the exchange rate for 3Ac/Sub12 was even slower, similar to that for muscle actin. The mutant actins polymerized faster and produced less stable and shorter filaments than yeast actin, the opposite of that expected for muscle actin. Unlike wild type actin, in the absence of unbound ATP, polymerization led to ADP-F-actin, which rapidly depolymerized. Like yeast actin, the hybrid actins activated muscle myosin S1 ATPase activity only about one-eighth as well as muscle actin, despite having essentially a muscle actin-specific myosin-binding site. Finally, the hybrid actins behaved abnormally in a yeast Arp2/3-dependent polymerization assay. Our results demonstrate a unique sensitivity of yeast to actin N-terminal negative charge density. They also provide insight into the role of each domain in the control of the various functions of actin.

Actins↗

PubMed2005

A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype.

To determine the reason for the inviability of Saccharomyces cerevisiae with skeletal muscle actin, we introduced into yeast actin the first variant muscle residue from the C-terminal end, H372R. Arg is also found at this position in non-yeast nonmuscle actins. The substitution caused retarded growth on glucose and an inability to use glycerol as a sole carbon source. The mitochondria were clumped and had lost their DNA, the vacuole appeared hypervesiculated, and the actin cytoskeleton became somewhat depolarized. Introduction of the second muscle actin-specific substitution, S365A, rescued these defects. Suppression was also achieved by introducing the four acidic N-terminal residues of muscle actin in place of the two found in yeast actin. The H372R substitution results in an increase in polymerization-dependent fluorescence of Cys-374 pyrene-labeled actin. H372R actin polymerizes slightly faster than wild-type (WT) actin. Yeast actin-related proteins 2 and 3 (Arp2/3) accelerates the polymerization of H372R actin to a much greater extent than WT actin. The two suppressors did not affect the rate of H372R actin polymerization in the absence of an Arp2/3 complex. In contrast, the S365A substitution dampened the rate of Arp2/3 complex-stimulated H372R actin polymerization, and the addition of the four acidic N-terminal residues caused this rate to decrease below that observed with WT actin in the presence of Arp2/3. Structural analysis of the mutations suggests the presence of stringent steric and ionic requirements for the bottom of actin subdomain 1 and also suggests that there is allosteric communication through subdomain 1 within the actin monomer between the N and C termini.

Actins↗

PubMed2004

Early myocardial function affects endocardial cushion development in zebrafish.

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih(-/-)) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk(-/-)) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk(-/-) embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfk(s11) variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.

Actins↗