Aberrant pattern formation in myosin heavy chain mutants of Dictyostelium

Development ◽  
1994 ◽  
Vol 120 (3) ◽  
pp. 591-601 ◽  
Author(s):  
D. Traynor ◽  
M. Tasaka ◽  
T. Takeuchi ◽  
J. Williams
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cell Types ◽  
Culture Conditions ◽  
Wild Type ◽  
Multicellular Aggregate ◽  
A Cells ◽  
Air Water Interface ◽  
Shape Changes ◽  
Roller Tube Culture

In mutant Dictyostelium strains that fail to accumulate the myosin heavy chain (MHC A), development is relatively normal up to the tight aggregate stage but is arrested prior to formation of the apical tip (DeLozanne and Spudich 1987, Knecht and Loomis, 1987). We show that in aggregates formed by such MHC A deficient (MHC A-) strains the proportions of pstA and pstB cells, the two prestalk cell types, and of prespore cells are similar to those found during normal development but their distribution is radically different. During the initial stages of normal slug formation, pstA cells move to the tip, pstB cells accumulate in the base and prespore cells occupy the remainder of the aggregate. In the aggregates initially formed by MHC A- mutants pstA cells are present in a central core, pstB cells are present in the cortex and prespore cells lie sandwiched between them. Eventually, cells within the cortex differentiate into mature stalk cells but spores are never formed. Mixing experiments, in which MHC A- cells are allowed to co-aggregate with an excess of normal cells, show that MHC A- prestalk cells enter the aggregate relatively normally but are unable to enter the slug tip or to migrate into the stalk at culmination and that MHC A- prespore cells accumulate in the lower part of the spore head during culmination. Thus MHC A- cells appear to be able to move within the multicellular aggregate but are incapable of participating in normal morphogenesis. The structures formed by MHC A- cells are very similar to those of the agglomerates that form when wild-type cells are developed in roller-tube culture, conditions that result in loss of the polarity imparted by the presence of an air-water interface. We propose formation of such a structure by MHC A- cells to be a default response, caused by their inability to undertake the shape changes and intercalatory cell movements that are necessary to form and extend the tip.

scholarly journals Novel Myosin Heavy Chain Kinase Involved in Disassembly of Myosin II Filaments and Efficient Cleavage in MitoticDictyostelium Cells

2002 ◽  
Vol 13 (12) ◽  
pp. 4333-4342 ◽  
Author(s):  
Akira Nagasaki ◽  
Go Itoh ◽  
Shigehiko Yumura ◽  
Taro Q.P. Uyeda
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Ii ◽  
Kinase Domain ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Daughter Cells ◽  
Cleavage Process ◽  
Interphase Cells ◽  
Myosin Heavy Chain Kinase

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, themhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.

scholarly journals A novel positive selection for identifying cold-sensitive myosin II mutants in Dictyostelium.

Genetics ◽  
1995 ◽  
Vol 140 (2) ◽  
pp. 505-515 ◽  
Author(s):  
B Patterson ◽  
J A Spudich
Keyword(s):  
Positive Selection ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Scale ◽  
Myosin Ii ◽  
Rapid Onset ◽  
Chemical Mutagenesis ◽  
Wild Type ◽  
Cold Sensitive ◽  
Selection For

Abstract We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.

scholarly journals Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster.

1988 ◽  
Vol 107 (6) ◽  
pp. 2613-2621 ◽  
Author(s):  
M Chun ◽  
S Falkenthal
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Flight Muscle ◽  
Microscopic Analysis ◽  
Thick Filament ◽  
Indirect Flight Muscle ◽  
Chain Gene ◽  
Wild Type ◽  
Electron Microscopic ◽  
Sarcomere Assembly

Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.

scholarly journals Point Mutations in Human β Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly

1997 ◽  
Vol 137 (1) ◽  
pp. 131-140 ◽  
Author(s):  
K. David Becker ◽  
Kim R. Gottshall ◽  
Reed Hickey ◽  
Jean-Claude Perriard ◽  
Kenneth R. Chien
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Subcellular Localization ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Point Mutations ◽  
Neonatal Rat ◽  
Atp Binding ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Thick Filaments

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased βMHC function.

Divergence in species and regulatory role of β-myosin heavy chain proximal promoter muscle-CAT elements

2002 ◽  
Vol 283 (6) ◽  
pp. C1761-C1775 ◽  
Author(s):  
Richard W. Tsika ◽  
John McCarthy ◽  
Natalia Karasseva ◽  
Yangsi Ou ◽  
Gretchen L. Tsika
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transgene Expression ◽  
Weight Bearing ◽  
Nuclear Extract ◽  
Proximal Promoter ◽  
Wild Type ◽  
Mobility Shift

We examined the functional role of distinct muscle-CAT (MCAT) elements during non-weight-bearing (NWB) regulation of a wild-type 293-base pair β-myosin heavy chain (βMyHC) transgene. Electrophoretic mobility shift assays (EMSA) revealed decreased NTEF-1, poly(ADP-ribose) polymerase, and Max binding at the human distal MCAT element when using NWB soleus vs. control soleus nuclear extract. Compared with the wild-type transgene, expression assays revealed that distal MCAT element mutation decreased basal transgene expression, which was decreased further in response to NWB. EMSA analysis of the human proximal MCAT (pMCAT) element revealed low levels of NTEF-1 binding that did not differ between control and NWB extract, whereas the rat pMCAT element displayed robust NTEF-1 binding that decreased when using NWB soleus extracts. Differences in binding between human and rat pMCAT elements were consistent whether using rat or mouse nuclear extract or in vitro synthesized human TEF-1 proteins. Our results provide the first evidence that 1) different binding properties and likely regulatory functions are served by the human and rat pMCAT elements, and 2) previously unrecognized βMyHC proximal promoter elements contribute to NWB regulation.

Tempol prevents cardiac oxidative damage and left ventricular dysfunction in the PPAR-α KO mouse

2013 ◽  
Vol 304 (11) ◽  
pp. H1505-H1512 ◽  
Author(s):  
Aziz Guellich ◽  
Thibaud Damy ◽  
Marc Conti ◽  
Victor Claes ◽  
Jane-Lise Samuel ◽  
...  
Keyword(s):  
Glutathione Peroxidase ◽  
Glutathione Reductase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Papillary Muscle ◽  
Ex Vivo ◽  
Left Ventricular ◽  
Lv Function ◽  
Wild Type ◽  
Contractile Parameters

Peroxisome proliferator-activated receptor (PPAR)-α deletion induces a profound decrease in MnSOD activity, leading to oxidative stress and left ventricular (LV) dysfunction. We tested the hypothesis that treatment of PPAR-α knockout (KO) mice with the SOD mimetic tempol prevents the heart from pathological remodelling and preserves LV function. Twenty PPAR-α KO mice and 20 age-matched wild-type mice were randomly treated for 8 wk with vehicle or tempol in the drinking water. LV contractile parameters were determined both in vivo using echocardiography and ex vivo using papillary muscle mechanics. Translational and posttranslational modifications of myosin heavy chain protein as well as the expression and activity of major antioxidant enzymes were measured. Tempol treatment did not affect LV function in wild-type mice; however, in PPAR-α KO mice, tempol prevented the decrease in LV ejection fraction and restored the contractile parameters of papillary muscle, including maximum shortening velocity, maximum extent of shortening, and total tension. Moreover, compared with untreated PPAR-α KO mice, myosin heavy chain tyrosine nitration and anion superoxide production were markedly reduced in PPAR-α KO mice after treatment. Tempol also significantly increased glutathione peroxidase and glutathione reductase activities (∼ 50%) in PPAR-α KO mice. In conclusion, these findings demonstrate that treatment with the SOD mimetic tempol can prevent cardiac dysfunction in PPAR-α KO mice by reducing the oxidation of contractile proteins. In addition, we show that the beneficial effects of tempol in PPAR-α KO mice involve activation of the glutathione peroxidase/glutathione reductase system.

scholarly journals Structure and function of the cytoskeleton of a Dictyostelium myosin-defective mutant.

1990 ◽  
Vol 110 (2) ◽  
pp. 367-378 ◽  
Author(s):  
Y Fukui ◽  
A De Lozanne ◽  
J A Spudich
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Insertional Mutagenesis ◽  
Chain Gene ◽  
Mitotic Spindles ◽  
Wild Type ◽  
Surface Receptors ◽  
Dictyostelium Myosin ◽  
Thick Filaments ◽  
Defective Mutant

To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A. Spudich. 1987. Science (Wash. DC). 236: 1086-1091). Western blot analysis using different mAbs confirms that hmm cells express a truncated myosin fragment of 140 kD (HMM-140 protein) instead of the normal 243-kD myosin heavy chain (MHC). Spontaneous revertants appear at a frequency less than 4 x 10(-5), which synthesize normal myosin and are capable of forming thick filaments. In hmm cells, the HMM-140 protein is diffusely distributed in the cytoplasm, indicating that it cannot assemble into thick filaments. The actin distribution in these mutant cells appears similar to that of wild-type cells. However, there is a significant abnormality in the organization of cytoplasmic microtubules, which penetrate into lamellipodial regions. The microtubule networks consist of approximately 13 microtubules on average and their pattern is abnormal. Although hmm cells can form mitotic spindles, mitosis is not coordinated with normal furrow formation. The hmm cells are clearly defective in the contractile events that lead to normal cytokinesis. The retraction of different regions of the cell can result in the occasional pinching off of part of the cell. This process is not coupled with formation of mitotic spindles. There is no specific accumulation of HMM-140 in such constrictions, whereas 73% of such cells show actin concentrated in these regions. The mutant hmm cells are also deficient in capping of Con-A-bound surface receptors, but instead internalize this complex into the cytoplasm. The hmm cells display active phagocytosis of bacteria. Whereas actin is concentrated in the phagocytic cups, HMM-140 protein is not localized in these regions. cAMP, a chemoattractant that induces drastic rounding up and formation of surface blebs in wild type cells, does not induce rounding up in the hmm cells. A Triton-permeabilized cell model of the wild-type amebae contracts on reactivation with Mg-ATP, whereas a model of the hmm cell shows no detectable contraction. Our data demonstrate that the conventional myosin participates in the significant cortical motile activities of Dictyostelium cells, which include rounding up, constriction of cleavage furrows, capping surface receptors, and establishing cell polarity.

scholarly journals Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 121 (3) ◽  
pp. 463-476 ◽  
Author(s):  
A Bender ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Types ◽  
Haploid Cell ◽  
Wild Type ◽  
Cell Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
A Cells ◽  
Alpha Factor ◽  
A Cell ◽  
Specific Proteins

Abstract Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.

Abstract 200: Beta-Myosin Heavy Chain Induction after Pressure Overload Is in a Minor Sub-population of Myocytes and Requires the Alpha-1A-Adrenergic Receptor

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Javier E Lopez ◽  
Bat-Erdene Myagmar ◽  
Philip Swigart ◽  
Marty Bigos ◽  
Manoj Rodrigo ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Adrenergic Receptor ◽  
Pressure Overload ◽  
Transverse Aortic Constriction ◽  
Wild Type ◽  
Rt Pcr ◽  
Aortic Constriction ◽  
Double Knockout ◽  
A Minor

Background: Induction of the fetal hypertrophic gene beta-myosin heavy chain (MHC) is a signature feature of pressure overload, and is thought to occur in most hypertrophied myocytes. Beta-MHC mRNA is not induced after transverse aortic constriction (TAC) in a double knockout (KO) model of the alpha-1A and alpha-1B-adrenergic receptor (AR) subtypes, but it is unknown whether the A or B or both are required. Hypothesis: We tested the hypothesis that native beta-MHC protein induction is in a sub-population of myocytes and requires only a single alpha-1 subtype. Methods: TAC was done in wild type (WT) and alpha-1 KO male mice ages 12–14 weeks. Beta-MHC protein was measured in isolated adult myocytes by a novel flow cytometry approach, with antibodies validated for beta-MHC and total sarcomeric MHC. Results: In WT mice, the fraction of myocytes expressing beta-MHC was 3% in shams (SE =1, n =4 hearts), and increased to 26% of myocytes at 1–3 weeks after TAC (SE =4, n =8, p< 0.01 vs. sham). Myocytes expressing beta-MHC were predominantly large cells (see Figure ). In alpha-1A KO mice (AKO), beta-MHC was induced in only 6% of myocytes (SE =2, n =3, p<0.05 vs. WT TAC, p =NS vs. sham), or in only 15% as many myocytes as in WT hearts. Western blotting and quantitative RT-PCR confirmed reduced beta-MHC induction in alpha-1A KO myocytes. Conclusion: Beta-MHC induction after pressure overload is in only a minor sub-population of cardiac myocytes, contrary to common models of fetal hypertrophic gene induction. Furthermore, beta-MHC induction requires a single alpha-1-AR subtype, the alpha-1A, which cannot be compensated by other hypertrophic receptors.

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