Myosin filaments isolated from skinned amphibian smooth muscle cells are side-polar

1989 ◽  
Vol 10 (3) ◽  
pp. 206-220 ◽  
Author(s):  
Peter H. Cooke ◽  
Fredric S. Fay ◽  
Roger Craig
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Myosin Filaments

scholarly journals Myosin filaments in smooth muscle cells do not have a constant length

2013 ◽  
Vol 591 (23) ◽  
pp. 5867-5878 ◽  
Author(s):  
Jeffrey C.-Y. Liu ◽  
Jörg Rottler ◽  
Lu Wang ◽  
Jenny Zhang ◽  
Chris D. Pascoe ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Constant Length ◽  
Myosin Filaments

scholarly journals Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

2002 ◽  
Vol 156 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Kyoungtae Kim ◽  
Thomas C.S. Keller
Keyword(s):  
Smooth Muscle ◽  
Ionic Strength ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Smooth Muscle Myosin ◽  
Globular Domain ◽  
Contractile Apparatus ◽  
Myosin Filaments ◽  
Muscle Myosin

Smooth muscle cells use an actin–myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

scholarly journals Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells.

1983 ◽  
Vol 97 (4) ◽  
pp. 1062-1071 ◽  
Author(s):  
W Z Cande ◽  
P J Tooth ◽  
J Kendrick-Jones
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Spatial Information ◽  
Muscle Cells ◽  
Thick Filament ◽  
Length Change ◽  
Intact Cells ◽  
Myosin Filaments ◽  
Skeletal Myosin

Isolated smooth muscle cells and cell fragments prepared by glycerination and subsequent homogenization will contract to one-third their normal length, provided Ca++ and ATP are present. Ca++-independent contraction was obtained by preincubation in Ca++ and ATP gamma S, or by addition of trypsin-treated myosin light chain kinase (MLCK) that no longer requires Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of this myosin is phosphorylated. The extent of myosin loss was ATP- and pH-dependent and occurred under conditions similar to those previously reported for the in vitro disassembly of gizzard myosin filaments. Ca++-dependent contraction was restored to extracted cells by addition of gizzard myosin under rigor conditions (i.e., no ATP), followed by addition of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by adding all these proteins in relaxing conditions (i.e., in EGTA and ATP) and then initiating contraction by Ca++ addition. Incubation with skeletal myosin will restore contraction, but this was not Ca++-dependent unless the cells were first incubated in troponin and tropomyosin. These results strengthen the idea that contraction in glycerinated cells and presumably also in intact cells is primarily thick filament regulated via MLCK, that the myosin filaments are unstable in relaxing conditions, and that the spatial information required for cell length change is present in the thin filament-intermediate filament organization.

Studies on isolated smooth muscle cells: The contractile apparatus

1977 ◽  
Vol 24 (1) ◽  
pp. 327-349
Author(s):  
J.V. Small
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Maximum Velocity ◽  
Muscle Cells ◽  
Myosin Filament ◽  
Contractile Apparatus ◽  
Myosin Filaments ◽  
Intact Muscle ◽  
10 Nm Filaments ◽  
Triton X

Smooth muscle cells may be isolated from the taenia coli muscle of the guinea pig which, when made permeable by treatment with Triton X-100 (0-05%) show a sensitivity to Ca for contraction with MgATP. The rate of contraction, about 10 micron s-1, corresponds closely to the maximum velocity of shortening of the intact muscle. Electron microscopy of such partially demembranated muscle cells shows that myosin filaments of about 16-nm diameter are present in both the rigor and the relaxes states. In addition, the actin and myosin filaments are commonly seen to be associated in groups corresponding approximately in size to the fibrils recognizable in cells in rigor in the light microscope. The dense bodies and the 10-nm filaments are found located between the actin-myosin filament groups. The thick myosin filaments may be isolated by fragmentation of the cells under relaxing conditions. These native filaments range up to about 8 micron in length and show the same structural organization as filaments aseembled from purified smooth muscle myosin: there is no central bare zone and bare edges, about 0-2 micrin long, occur at the filament ends. The lack of bipolarity of the native smooth muscle muosin filaments and the absence, in the contractile apparatus, of actin-associated structures equivalent to Z-lines suggests that the amount of shearing that can occur between the actin and myosin filaments is considerably greater than in skeletal muscle.

scholarly journals Physiological and structural properties of saponin-skinned single smooth muscle cells.

1987 ◽  
Vol 90 (1) ◽  
pp. 49-73 ◽  
Author(s):  
G J Kargacin ◽  
F S Fay
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Cells ◽  
Muscle Cells ◽  
Fibrillar Structure ◽  
High K ◽  
Myosin Filaments ◽  
Rigor Solution ◽  
Shortening Rate ◽  
Short Time

The study of the fundamental events underlying the generation and regulation of force in smooth muscle would be greatly facilitated if the permeability of the cell membrane were increased so that the intracellular environment of the contractile apparatus could be manipulated experimentally. To initiate such an analysis, we developed a saponin permeabilization procedure that was used to "skin" isolated smooth muscle cells from the stomach of the toad, Bufo marinus. Suspensions of single cells isolated enzymatically were resuspended in high-K+ rigor solution (0 ATP, 5 mM EGTA) and exposed for 5 min to 25 micrograms/ml saponin. Virtually all the cells in a suspension were made permeable by this procedure and shortened to less than one-third their initial length when ATP and Ca++ were added; they re-extended when free Ca++ was removed. Analysis of the protein content of the skinned cells revealed that, although their total protein was reduced by approximately 30%, they retained most of their myosin and actin. Skinning was accompanied by a rearrangement of actin and myosin filaments within the cells such that a fine fibrillar structure became visible under the light microscope and a tight clustering of acting filaments around myosin filaments was revealed by the electron microscope. Face-on views of saponin-treated cell membranes revealed the presence of 70-80-A-wide pits or holes. The shortening rate of skinned cells was sensitive to [Ca++] between pCa 7 and pCa 5 and was half-maximal at approximately pCa 6.2. Shortening was also dependent on [ATP] but could be increased at low [ATP] by pretreatment with adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), which suggests that myosin phosphorylation was more sensitive to low substrate concentrations than was cross-bridge cycling. To determine whether a significant limitation to free diffusion existed in the skinned cells, a computer model of the cell and the unstirred layer surrounding it was developed. Simulations revealed that the membrane, even in skinned cells, could, for short time intervals, significantly inhibit the movement of substances into and out of cells.

Immunogold localization of HMB-45 antigen in pulmonary lymphangiomyomatosis

1995 ◽  
Vol 53 ◽  
pp. 998-999
Author(s):  
J.M. Minda ◽  
E. Dessy ◽  
G. G. Pietra
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Muscle Cells ◽  
Ultrastructural Localization ◽  
Reproductive Age ◽  
Thin Sections ◽  
Smooth Muscle Proliferation ◽  
Hmb 45

Pulmonary lymphangiomyomatosis (PLAM) is a rare disease occurring exclusively in women of reproductive age. It involves the lungs, lymph nodes and lymphatic ducts. In the lungs, it is characterized by the proliferation of smooth muscle cells around lymphatics in the bronchovascular bundles, lobular septa and pleura The nature of smooth muscle proliferation in PLAM is still unclear. Recently, reactivity of the smooth muscle cells for HMB-45, a melanoma-related antigen has been reported by immunohistochemistry. The purpose of this study was the ultrastructural localization of HMB-45 immunoreactivity in these cells using gold-labeled antibodies.Lung tissue from three cases of PLAM, referred to our Institution for lung transplantation, was embedded in either Poly/Bed 812 post-fixed in 1% osmium tetroxide, or in LR White, without osmication. For the immunogold technique, thin sections were placed on Nickel grids and incubated with affinity purified, monoclonal anti-melanoma antibody HMB-45 (1:1) (Enzo Diag. Co) overnight at 4°C. After extensive washing with PBS, grids were treated with Goat-anti-mouse-IgG-Gold (5nm) (1:10) (Amersham Life Sci) for 1 hour, at room temperature.

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