Identification of a cytoskeletal protein localized in the myoplasm of ascidian eggs: localization is modified during anural development

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 425-436 ◽  
Author(s):  
B.J. Swalla ◽  
M.R. Badgett ◽  
W.R. Jeffery
Keyword(s):  
Embryonic Development ◽  
Muscle Cells ◽  
Insoluble Fraction ◽  
Cytoskeletal Protein ◽  
Western Blots ◽  
One Dimensional ◽  
Cytoskeletal Network ◽  
Ascidian Species ◽  
Triton X ◽  
Tail Muscle

The myoplasm of ascidian eggs is a localized cytoskeletal domain that is segregated to presumptive larval tail muscle cells during embryonic development. We have identified a cytoskeletal protein recognized by a vertebrate neurofilament monoclonal antibody (NN18) which is concentrated in the myoplasm in eggs and embryos of a variety of ascidian species. The NN18 antigen is localized in the periphery of unfertilized eggs, segregates with the myoplasm after fertilization, and enters the larval tail muscle cells during embryonic development. Western blots of one-dimensional and two-dimensional gels showed that the major component recognized by NN18 antibody is a 58 × 10(3) Mr protein (p58), which exists in at least three different isoforms. The enrichment of p58 in the Triton X-100-insoluble fraction of eggs and its reticular staining pattern in eggs and embryos suggests that it is a cytoskeletal protein. In subsequent experiments, p58 was used as a marker to determine whether changes in the myoplasm occur in eggs of anural ascidian species, i.e. those exhibiting a life cycle lacking tadpole larvae with differentiated muscle cells. Although p58 was localized in the myoplasm in eggs of four urodele ascidian species that develop into swimming tadpole larvae, this protein was distributed uniformly in eggs of three anural ascidian species. The eggs of two of these anural species contained the actin lamina, another component of the myoplasm, whereas the third anural species lacked the actin lamina. There was no detectible localization of p58 after fertilization or segregation into muscle lineage cells during cleavage of anural eggs. NN18 antigen was uniformly distributed in pre-vitellogenic oocytes and then localized in the perinuclear zone during vitellogenesis of urodele and anural ascidians. Subsequently, NN18 antigen was concentrated in the peripheral cytoplasm of post-vitellogenic oocytes and mature eggs of urodele, but not anural, ascidians. It is concluded that the myoplasm of ascidian eggs contains an intermediate filament-like cytoskeletal network which is missing in anural species that have modified or eliminated the tadpole larva.

NUMERICAL SIMULATION OF THE ROLE OF CO-TRANSMISSION BY ACETYLCHOLINE AND SEROTONIN ON MOTILITY OF THE GUT

2006 ◽  
Vol 06 (04) ◽  
pp. 399-428
Author(s):  
R. MIFTAHOF
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Transit Time ◽  
Receptor Agonist ◽  
Muscle Cells ◽  
Receptor Antagonists ◽  
One Dimensional ◽  
Circular Smooth Muscle ◽  
Spatio Temporal ◽  
Stimulation Of

Electrophysiological mechanisms of co-transmission by serotonin (5-HT) and acetylcholine (ACh), co-expression of their receptor types, i.e., 5-HT type 3 and 4, nicotinic cholinerginc (nACh) and muscarinic cholinergic (μACh), and effects of selective and non-selective 5-HT3 and 5-HT4 receptor agonists/antagonists, on electromechanical activity of the gut were studied numerically. Two series of numerical experiments were performed. First, the dynamics of the generation and propagation of electrical signals interconnected with the primary sensory (AH) neurons, motor (S) neurons and smooth muscle cells were studied in a one-dimensional model. Simulations showed that stimulation of the 5-HT3 receptors reduced the threshold of activation of the mechanoreceptors by 17.6%. Conjoint excitation of the 5-HT3 and 5-HT4 receptors by endogenous serotonin converted the regular firing pattern of electrical discharges of the AH and S neurons to a beating mode. Activation confined to 5-HT3 receptors, located on the somas of the adjacent AH and S type neurons, could not sustain normal signal transduction between them. It required ACh as a co-transmitter and co-activation of the nACh receptors. Application of selective 5-HT3 receptor antagonists inhibited dose-dependently the production of action potentials at the level of mechanoreceptors and the soma of the primary sensory neuron and increased the threshold activation of the mechanoreceptors. Normal mechanical contractile activity depended on co-stimulation of the 5-HT4 and μACh receptors on the membrane of smooth muscle cells. In the second series of simulations, which involved a spatio-temporal model of the functional unit, effects of co-transmission by ACh and 5-HT on the electromechanical response in a segment of the gut were analyzed. Results indicated that propagation of the wave of excitation between the AH and S neurons within the myenteric nervous plexus in the presence of 5-HT3 receptor antagonists was supported by co-release of ACh. Co-stimulation of 5-HT3, nACh and μACh receptors impaired propulsive activity of the gut. The bolus showed uncoordinated movements. In an ACh-free environment Lotronex (GlaxoSmithKline), a 5-HT3 receptor antagonist, significantly increased the transit time of the pellet along the gut. In the presence of ACh, Lotronex produced intensive tonic-type contractions in the longitudinal and circular smooth muscle layers and eliminated propulsive activity. The 5HT4 receptor agonist, Zelnorm (Novartis), preserved the reciprocal electromechanical relationships between the longitudinal and circular smooth muscle layers. The drug changed the normal propulsive pattern of activity to an expulsive (non-mixing) type. Treatment of the gut with selective 5HT4 receptor antagonists increased the transit time by disrupting the migrating myoelectrical complex. Cisapride (Janssen), a mixed 5HT3 and 5HT4 receptor agonist, increased excitability of the AH and S neurons and the frequency of slow waves. Longitudinal and circular smooth muscle syncytia responded with the generation of long-lasting tonic contractions, resulting in a "squeezing" type of pellet movement. Comparison of the theoretical results obtained on one-dimensional and spatio-temporal models to in vivo and in vitro experimental data indicated satisfactory qualitative, and where available, quantitative agreement.

scholarly journals Biosynthesis of titin in cultured skeletal muscle cells.

1989 ◽  
Vol 109 (5) ◽  
pp. 2189-2195 ◽  
Author(s):  
W B Isaacs ◽  
I S Kim ◽  
A Struve ◽  
A B Fulton
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Significant Progress ◽  
Large Protein ◽  
Time Period ◽  
And Function ◽  
Triton X ◽  
Developing Muscle ◽  
Synchronized Cultures

Although significant progress has been made regarding the structure and function of titin, little data exist on the biosynthesis of this large protein in developing muscle. Using pulse-labeling with [35S]methionine and immunoprecipitation with an anti-titin mAb, we have examined the biosynthesis of titin in synchronized cultures of skeletal muscle cells derived from day 12 chicken embryos. We find that: (a) titin synthesis increases greater than 4-fold during the first week in culture and during this same time period, synthesis of muscle-specific myosin heavy chain increases greater than 12-fold; (b) newly synthesized titin has a t1/2 of approximately 70 h; (c) titin is resistant to extraction with Triton X-100 both during and immediately after its synthesis. These observations suggest that newly synthesized titin molecules are stable proteins that rapidly associate with the cytoskeleton of developing myotubes.

Regulation of Na+ pump expression by vascular smooth muscle cells

2001 ◽  
Vol 280 (4) ◽  
pp. H1869-H1874 ◽  
Author(s):  
Aslihan Aydemir-Koksoy ◽  
Julius C. Allen
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Mammalian Cells ◽  
Muscle Cells ◽  
Transport Processes ◽  
Western Blots ◽  
Cytoplasmic Transport ◽  
Low K

The Na+ pump and its regulation is important for maintaining membrane potential and transmembrane Na+gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K+ (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [3H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either α1- or β1-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K+ media, the pump site upregulation was inhibited. These data suggest that the low-K+-induced upregulation of Na+ pump number can occur by translocation of preformed pumps from intracellular stores.

scholarly journals Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle

2010 ◽  
Vol 299 (3) ◽  
pp. G742-G750 ◽  
Author(s):  
P. Cong ◽  
V. Pricolo ◽  
P. Biancani ◽  
J. Behar
Keyword(s):  
Small Interfering Rna ◽  
Muscle Cells ◽  
High Cholesterol ◽  
Western Blots ◽  
Human Gallbladder ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Δ Opioid Receptor ◽  
Interfering Rna ◽  
Bulk Plasma

The contraction of gallbladders (GBs) with cholesterol stones is impaired due to high cholesterol concentrations in caveolae compared with GBs with pigment stones. The reduced contraction is caused by a lower cholecystokinin (CCK)-8 binding to CCK-1 receptors (CCK-1R) due to caveolar sequestration of receptors. We aimed to examine the mechanism of cholesterol-induced sequestration of receptors. Muscle cells from human and guinea pig GBs were studied. Antibodies were used to examine CCK-1R, antigens of early and recycling endosomes, and total (CAV-3) and phosphorylated caveolar-3 protein (pCAV-3) by Western blots. Contraction was measured in muscle cells transfected with CAV3 mRNA or clathrin heavy-chain small-interfering RNA (siRNA). CCK-1R returned back to the bulk plasma membrane (PM) 30 min after CCK-8 recycled by endosomes, peaking at 5 min in early endosomes and at 20 min in recycling endosomes. Pretreatment with cholesterol-rich liposomes inhibited the transfer of CCK-1R and of CAV-3 in the endosomes by blocking CAV-3 phosphorylation. 4-Amino-5-(4-chloro-phenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (inhibitor of tyrosine kinase) reproduced these effects by blocking pCAV-3 formation, increasing CAV-3 and CCK-1R sequestration in the caveolae and impairing CCK-8-induced contraction. CAV-3 siRNA reduced CAV-3 protein expression, decreased CCK-8-induced contraction, and accumulated CCK-1R in the caveolae. Abnormal concentrations of caveolar cholesterol had no effect on met-enkephalin that stimulates a δ-opioid receptor that internalizes through clathrin. We found that impaired muscle contraction in GBs with cholesterol stones is due to high caveolar levels of cholesterol that inhibits pCAV-3 generation. Caveolar cholesterol increases the caveolar sequestration of CAV-3 and CCK-1R caused by their reduced recycling to the PM.

The snail repressor establishes a muscle/notochord boundary in the Ciona embryo

Development ◽  
1998 ◽  
Vol 125 (13) ◽  
pp. 2511-2520 ◽  
Author(s):  
S. Fujiwara ◽  
J.C. Corbo ◽  
M. Levine
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Sites ◽  
Promoter Region ◽  
Muscle Cells ◽  
Specific Pattern ◽  
Binding Assays ◽  
Heterologous Promoter ◽  
Present Evidence ◽  
Tail Muscle

Previous studies have identified a minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra), which is sufficient to direct a notochord-specific pattern of gene expression. Here we present evidence that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages.

Activation of MAP kinases in airway smooth muscle

1997 ◽  
Vol 272 (2) ◽  
pp. L244-L252 ◽  
Author(s):  
W. T. Gerthoffer ◽  
I. A. Yamboliev ◽  
J. Pohl ◽  
R. Haynes ◽  
S. Dang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Map Kinase ◽  
Airway Smooth Muscle ◽  
Map Kinases ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
Mitogen Activated Protein ◽  
Intact Muscle ◽  
Triton X

To test the hypothesis that mitogen-activated protein (MAP) kinases are activated by contractile agonists in intact nonproliferating airway smooth muscle, kinase activities were compared in resting and stimulated canine tracheal smooth muscle. Kinase activities in sodium dodecyl sulfate extracts were assayed by a gel renaturation method. Myelin basic protein kinase activities corresponding to ERK1 and ERK2 immunoreactive proteins were activated twofold above the basal level within 5 min by 1 microM carbachol. MAP kinase activity assayed in crude homogenates using a synthetic peptide substrate (APRTPGGRR) also increased twofold above basal in muscles stimulated with 1 microM carbachol. Two protein kinases separated by Mono-Q chromatography were identified on Western blots as ERK1 and ERK2 MAP kinases. Carbachol stimulation increased caldesmon phosphorylation in intact muscle, and purified caldesmon was a substrate for activated murine ERK2 MAP kinase. Activated ERK2 MAP kinase added to Triton X-100-permeabilized fibers potentiated Ca2+-induced contraction. The results show that ERK MAP kinases are activated after stimulation of muscarinic receptors in airway smooth muscle, which is consistent with coupling of MAP kinases to phosphorylation of caldesmon in vivo.

scholarly journals Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture.

1982 ◽  
Vol 92 (1) ◽  
pp. 231-236 ◽  
Author(s):  
J Prives ◽  
A B Fulton ◽  
S Penman ◽  
M P Daniels ◽  
C N Christian
Keyword(s):  
Cell Surface ◽  
Internal Structure ◽  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Cells In Culture ◽  
Detergent Extraction ◽  
Embryonic Muscle ◽  
Alpha Bungarotoxin ◽  
Triton X

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent alpha-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction.

scholarly journals Zinc Modulates Several Transcription-Factor Regulated Pathways in Mouse Skeletal Muscle Cells

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 5098
Author(s):  
Parisa Vahidi Ferdowsi ◽  
Rachel Ng ◽  
John Adulcikas ◽  
Sukhwinder Singh Sohal ◽  
Stephen Myers
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Metal Ion ◽  
Insulin Signalling ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Western Blots ◽  
Insulin Signalling Pathway ◽  
C2c12 Skeletal Muscle Cells

Zinc is an essential metal ion involved in many biological processes. Studies have shown that zinc can activate several molecules in the insulin signalling pathway and the concomitant uptake of glucose in skeletal muscle cells. However, there is limited information on other potential pathways that zinc can activate in skeletal muscle. Accordingly, this study aimed to identify other zinc-activating pathways in skeletal muscle cells to further delineate the role of this metal ion in cellular processes. Mouse C2C12 skeletal muscle cells were treated with insulin (10 nM), zinc (20 µM), and the zinc chelator TPEN (various concentrations) over 60 min. Western blots were performed for the zinc-activation of pAkt, pErk, and pCreb. A Cignal 45-Reporter Array that targets 45 signalling pathways was utilised to test the ability of zinc to activate pathways that have not yet been described. Zinc and insulin activated pAkt over 60 min as expected. Moreover, the treatment of C2C12 skeletal muscle cells with TPEN reduced the ability of zinc to activate pAkt and pErk. Zinc also activated several associated novel transcription factor pathways including Nrf1/Nrf2, ATF6, CREB, EGR1, STAT1, AP-1, PPAR, and TCF/LEF, and pCREB protein over 120 min of zinc treatment. These studies have shown that zinc’s activity extends beyond that of insulin signalling and plays a role in modulating novel transcription factor activated pathways. Further studies to determine the exact role of zinc in the activation of transcription factor pathways will provide novel insights into this metal ion actions.

scholarly journals An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

1998 ◽  
Vol 9 (3) ◽  
pp. 599-609 ◽  
Author(s):  
Hans de Vries ◽  
Cobi Schrage ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Insoluble Fraction ◽  
Membrane Domains ◽  
Myelin Membrane ◽  
Influenza Virus Hemagglutinin ◽  
Polarized Cells ◽  
Vesicular Stomatitis ◽  
Triton X

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.

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