scholarly journals Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities.

1996 ◽  
Vol 134 (3) ◽  
pp. 675-687 ◽  
Author(s):  
C A Kelley ◽  
J R Sellers ◽  
D L Gard ◽  
D Bui ◽  
R S Adelstein ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Heavy Chain Isoforms ◽  
Enzymatic Activities ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Nonmuscle Myosin ◽  
In Vitro Motility Assay ◽  
Interphase Cells

There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. We compared the enzymatic activities as well as the subcellular localizations of these isoforms in Xenopus cells. MHC-A and MHC-B were purified from cells by immunoprecipitation with isoform-specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A was 3.3-fold faster than that by MHC-B. Likewise, the Vmax of the actin-activated Mg(2+)-ATPase activity of MHC-A was 2.6-fold greater than that of MHC-B. Immunofluorescence microscopy demonstrated distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium was dramatically enriched for MHC-B suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A was absent from the cell periphery and was arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also had distinct localizations throughout mitosis. During prophase, the MHC-B redistributed to the nuclear membrane, and then resumed its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localized to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalized to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions.

Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia

2001 ◽  
Vol 90 (6) ◽  
pp. 2508-2513 ◽  
Author(s):  
Thomas L. Clanton ◽  
Valerie P. Wright ◽  
Peter J. Reiser ◽  
Paul F. Klawitter ◽  
Nanduri R. Prabhakar
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Intermittent Hypoxia ◽  
Contractile Function ◽  
Myosin Heavy Chain Isoforms ◽  
Adaptive Responses ◽  
Low Frequencies ◽  
Myosin Heavy Chain Isoform ◽  
Obstructive Sleep

Intermittent hypoxia (IH), associated with obstructive sleep apnea, initiates adaptive physiological responses in a variety of organs. Little is known about its influence on diaphragm. IH was simulated by exposing rats to alternating 15-s cycles of 5% O2 and 21% O2 for 5 min, 9 sets/h, 8 h/day, for 10 days. Controls did not experience IH. Diaphragms were excised 20–36 h after IH. Diaphragm bundles were studied in vitro or analyzed for myosin heavy chain isoform composition. No differences in maximum tetanic stress were observed between groups. However, peak twitch stress ( P < 0.005), twitch half-relaxation time ( P < 0.02), and tetanic stress at 20 or 30 Hz ( P < 0.05) were elevated in IH. No differences in expression of myosin heavy chain isoforms or susceptibility to fatigue were seen. Contractile function after 30 min of anoxia (95% N2-5% CO2) was markedly preserved at all stimulation frequencies during IH and at low frequencies after 15 min of reoxygenation. Anoxia-induced increases in passive muscle force were eliminated in the IH animals ( P < 0.01). These results demonstrate that IH induces adaptive responses in the diaphragm that preserve its function in anoxia.

Sliding velocity of isolated rabbit cardiac myosin correlates with isozyme distribution

1992 ◽  
Vol 263 (2) ◽  
pp. H464-H472 ◽  
Author(s):  
H. Yamashita ◽  
S. Sugiura ◽  
T. Serizawa ◽  
T. Sugimoto ◽  
M. Iizuka ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Motility Assay ◽  
Cardiac Myosin ◽  
Sliding Velocity ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Actin Cables

To investigate the relationship between the mechanical and biochemical properties of cardiac myosin, the sliding velocity of isolated cardiac myosin obtained from both euthyroid and hyperthyroid rabbits on actin cables was measured with an in vitro motility assay system. Ten rabbits (T) were treated with L-thyroxine to induce hyperthyroidism, and eight nontreated animals (N) were used as controls. Myosin was purified from the left ventricles of anesthetized animals. Myosin isozyme content was analyzed by the pyrophosphate gel electrophoresis method, and myosin adenosinetriphosphatase (ATPase) activity was determined on the same sample. Long well-organized actin cables of green algae, Nitellopsis, were used in the in vitro motility assay. Small latex beads were coated with purified cardiac myosin and introduced onto the Nitellopsis actin cables. Active unidirectional movement of the beads on the actin cables was observed under a photomicroscope, and the velocity was measured. The velocity was dependent on ATP concentrations, and the optimal pH for bead movement was approximately 7.0-7.5. The mean velocity was higher in T than in N (0.66 +/- 0.12 vs. 0.32 +/- 0.09 micron/s, P less than 0.01). Both Ca(2+)-activated ATPase activity and the percentage of alpha-myosin heavy chain were also higher in T than in N (0.691 +/- 0.072 vs. 0.335 +/- 0.072 microM Pi.mg-1.min-1, P less than 0.01, and 79 +/- 12 vs. 26 +/- 7%, P less than 0.01, respectively). The velocity of myosin closely correlated with both Ca(+2)-activated myosin ATPase activity (r = 0.87, P less than 0.01) and the percentage of alpha-myosin heavy chain (r = 0.87, P less than 0.01).

scholarly journals A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair

2016 ◽  
Vol 213 (6) ◽  
pp. 631-640 ◽  
Author(s):  
Marisa Encarnação ◽  
Lília Espada ◽  
Cristina Escrevente ◽  
Denisa Mateus ◽  
José Ramalho ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cell Periphery ◽  
Perinuclear Region ◽  
Nonmuscle Myosin ◽  
Membrane Repair ◽  
Rab Family ◽  
Plasma Membrane Repair ◽  
Molecular Machinery

Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.

scholarly journals Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries

2012 ◽  
Vol 303 (10) ◽  
pp. C1090-C1103 ◽  
Author(s):  
Margaret C. Hubbell ◽  
Andrew J. Semotiuk ◽  
Richard B. Thorpe ◽  
Olayemi O. Adeoye ◽  
Stacy M. Butler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Chronic Hypoxia ◽  
Myosin Heavy Chain Isoforms ◽  
Wall Structure ◽  
Vegf Receptor ◽  
Age Dependent ◽  
Endothelial Growth Factor

Chronic hypoxia increases vascular endothelial growth factor (VEGF) and thereby promotes angiogenesis. The present study explores the hypothesis that hypoxic increases in VEGF also remodel artery wall structure and contractility through phenotypic transformation of smooth muscle. Pregnant and nonpregnant ewes were maintained at sea level (normoxia) or 3,820 m (hypoxia) for the final 110 days of gestation. Common carotid arteries harvested from term fetal lambs and nonpregnant adults were denuded of endothelium and studied in vitro. Stretch-dependent contractile stresses were 32 and 77% of normoxic values in hypoxic fetal and adult arteries. Hypoxic hypocontractility was coupled with increased abundance of nonmuscle myosin heavy chain (NM-MHC) in fetal (+37%) and adult (+119%) arteries. Conversely, hypoxia decreased smooth muscle MHC (SM-MHC) abundance by 40% in fetal arteries but increased it 123% in adult arteries. Hypoxia decreased colocalization of NM-MHC with smooth muscle α-actin (SM-αA) in fetal arteries and decreased colocalization of SM-MHC with SM-αA in adult arteries. Organ culture with physiological concentrations (3 ng/ml) of VEGF-A165 similarly depressed stretch-dependent stresses to 37 and 49% of control fetal and adult values. The VEGF receptor antagonist vatalanib ablated VEGF's effects in adult but not fetal arteries, suggesting age-dependent VEGF receptor signaling. VEGF replicated hypoxic decreases in colocalization of NM-MHC with SM-αA in fetal arteries and decreases in colocalization of SM-MHC with SM-αA in adult arteries. These results suggest that hypoxic increases in VEGF not only promote angiogenesis but may also help mediate hypoxic arterial remodeling through age-dependent changes in smooth muscle phenotype and contractility.

Molecular and phenotypic effects of heterozygous, homozygous, and compound heterozygote myosin heavy-chain mutations

2005 ◽  
Vol 288 (3) ◽  
pp. H1097-H1102 ◽  
Author(s):  
Norman R. Alpert ◽  
Saidi A. Mohiddin ◽  
Dorothy Tripodi ◽  
Jacqueline Jacobson-Hatzell ◽  
Kelly Vaughn-Whitley ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mechanical Performance ◽  
Left Ventricular ◽  
Familial Hypertrophic Cardiomyopathy ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Common Causes ◽  
Disease Penetrance

Autosomal dominant familial hypertrophic cardiomyopathy (FHC) has variable penetrance and phenotype. Heterozygous mutations in MYH7 encoding β-myosin heavy chain are the most common causes of FHC, and we proposed that “enhanced” mutant actin-myosin function is the causative molecular abnormality. We have studied individuals from families in which members have two, one, or no mutant MYH7 alleles to examine for dose effects. In one family, a member homozygous for Lys207Gln had cardiomyopathy complicated by left ventricular dilatation, systolic impairment, atrial fibrillation, and defibrillator interventions. Only one of five heterozygous relatives had FHC. Leu908Val and Asp906Gly mutations were detected in a second family in which penetrance for Leu908Val heterozygotes was 46% (21/46) and 25% (3/12) for Asp906Gly. Despite the low penetrance, hypertrophy was severe in several heterozygotes. Two individuals with both mutations developed severe FHC. The velocities of actin translocation ( Vactin) by mutant and wild-type (WT) myosins were compared in the in vitro motility assay. Compared with WT/WT, Vactin was 34% faster for WT/D906G and 21% for WT/L908V. Surprisingly Vactin for Leu908Val/Asp906Gly and Lys207Gln/Lys207Gln mutants were similar to WT. The apparent enhancement of mechanical performance with mutant/WT myosin was not observed for mutant/mutant myosin. This suggests that Vactin may be a poor predictor of disease penetrance or severity and that power production may be more appropriate, or that the limited availability of double mutant patients prohibits any definitive conclusions. Finally, severe FHC in heterozygous individuals can occur despite very low penetrance, suggesting these mutations alone are insufficient to cause FHC and that uncharacterized modifying mechanisms exert powerful influences.

scholarly journals Prdm1 (Blimp-1) and the Expression of Fast and Slow Myosin Heavy Chain Isoforms during Avian Myogenesis In Vitro

PLoS ONE ◽  
2010 ◽  
Vol 5 (4) ◽  
pp. e9951 ◽  
Author(s):  
Mary Lou Beermann ◽  
Magdalena Ardelt ◽  
Mahasweta Girgenrath ◽  
Jeffrey Boone Miller
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Heavy Chain Isoforms ◽  
Slow Myosin ◽  
Slow Myosin Heavy Chain

Characterization of isoform diversity in smooth muscle myosin heavy chains

1994 ◽  
Vol 72 (11) ◽  
pp. 1351-1360 ◽  
Author(s):  
Christine A. Kelley ◽  
Robert S. Adelstein
Keyword(s):  
Smooth Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Smooth Muscle Myosin ◽  
Motility Assay ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Amino Terminal ◽  
Muscle Myosin

In this paper we review some of our recent work on the structural and biochemical characterization of isoforms of the heavy chain of vertebrate smooth muscle myosin II. There exist both amino-terminal and carboxyl-terminal alternatively spliced isoforms of the smooth muscle myosin heavy chain (MHC). mRNA splicing at the 3′ end generates two MHCs, which differ in length and amino acid sequence in the carboxyl terminus. We will refer to the longer, 204-kDa isoform as MHC204 and the shorter, 200-kDa isoform as MHC200. We found that MHC204, but not MHC200, can be phosphorylated by casein kinase II on a serine near the carboxyl terminus, suggesting that these isoforms may be differentially regulated. The physiological significance of this phosphorylation is not known. However, as demonstrated in this paper, phosphorylation does not appear to affect filament formation, velocity of movement of actin filaments by myosin in an in vitro motility assay, actin-activated Mg2+ ATPase activity, or myosin conformation. Our results also show that MHC204 and MHC200 form homodimers, but not heterodimers. Purified MHC204 and MHC200 homodimers are not enzymatically different, at least as measured using an in vitro motility assay. The amino-terminal spliced MHC204 and MHC200 isoforms are the result of the specific insertion or deletion of seven amino acids near the ATP-binding region in the myosin head. We refer to these isoforms as inserted (MHC204-I; MHC200-I) or noninserted (MHC204; MHC200), respectively. In contrast to the carboxyl-terminal spliced isoforms, the amino-terminal spliced inserted and noninserted myosin heavy chain isoforms are enzymatically different. The inserted isoform, which is expressed in intestinal, phasic-type smooth muscle, has a higher actin-activated Mg ATPase activity and moves actin filaments at a greater velocity in an in vitro motility assay than the noninserted MHC isoform, which is expressed in tonic-type vascular smooth muscle. The results presented in this review suggest that the alternative splicing of smooth muscle mRNA results in at least four different isoforms of the myosin heavy chain molecule. The potential relevance of these molecular isoforms to smooth muscle function is discussed.Key words: myosin, heavy chain isoforms.

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