scholarly journals The 110-kD protein-calmodulin complex of the intestinal microvillus (brush border myosin I) is a mechanoenzyme.

1989 ◽  
Vol 108 (6) ◽  
pp. 2395-2400 ◽  
Author(s):  
M S Mooseker ◽  
T R Coleman
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Average Rate ◽  
Skeletal Muscle Myosin ◽  
Intestinal Brush Border ◽  
Myosin I ◽  
Actin Cables ◽  
Muscle Myosin

The 110-kD protein-calmodulin complex (110K-CM) of the intestinal brush border serves to laterally tether microvillar actin filaments to the plasma membrane. Results from several laboratories have demonstrated that this complex shares many enzymatic and structural properties with myosin. The mechanochemical potential of purified avian 110K-CM was assessed using the Nitella bead motility assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature (Lond.). 303:31-35). Under low Ca2+ conditions, 110K-CM-coated beads bound to actin cables, but no movement was observed. Using EGTA/calcium buffers (approximately 5-10 microM free Ca2+) movement of 110K-CM-coated beads along actin cables (average rate of approximately 8 nm/s) was observed. The movement was in the same direction as that for beads coated with skeletal muscle myosin. The motile preparations of 110K-CM were shown to be free of detectable contamination by conventional brush border myosin. Based on these and other observations demonstrating the myosin-like properties of 110K-CM, we propose that this complex be named "brush border myosin I."

scholarly journals ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

1984 ◽  
Vol 99 (5) ◽  
pp. 1867-1871 ◽  
Author(s):  
M P Sheetz ◽  
R Chasan ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Quantitative Assay ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
In Vitro Characterization ◽  
Actin Cables ◽  
Muscle Myosin

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.

Brush border myosin-I microinjected into cultured cells is targeted to actin-containing surface structures

1994 ◽  
Vol 107 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
M. Footer ◽  
A. Bretscher
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Surface Structures ◽  
Specific Expression ◽  
Cultured Fibroblasts ◽  
Myosin I ◽  
Section Electron

The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When introduced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the consequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characterization of intestinal microvillar membrane disks: detergent-resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin).

1989 ◽  
Vol 109 (3) ◽  
pp. 1153-1161 ◽  
Author(s):  
M S Mooseker ◽  
K A Conzelman ◽  
T R Coleman ◽  
J E Heuser ◽  
M P Sheetz
Keyword(s):  
Brush Border ◽  
Membrane Fraction ◽  
Average Rate ◽  
Isolation And Characterization ◽  
Myosin I ◽  
Latex Beads ◽  
Flat Disk ◽  
Nonionic Detergent ◽  
Actin Cables

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.

scholarly journals Brush Border Myosin–I Structure and ADP-dependent Conformational Changes Revealed by Cryoelectron Microscopy and Image Analysis

1997 ◽  
Vol 139 (3) ◽  
pp. 683-693 ◽  
Author(s):  
James D. Jontes ◽  
Ronald A. Milligan
Keyword(s):  
Image Analysis ◽  
Conformational Changes ◽  
Light Chain ◽  
Brush Border ◽  
Actin Filaments ◽  
Catalytic Domain ◽  
Binding Domain ◽  
Cryoelectron Microscopy ◽  
Skeletal Muscle Myosin ◽  
Myosin I

Brush border myosin–I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751–753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain–binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The ∼31° swing corresponds to ∼63 Å at the end of the rigid light chain–binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.

scholarly journals Force-Velocity Relation of Sliding of Skeletal Muscle Myosin, Arranged on a Paramyosin Filament, on Actin Cables.

1998 ◽  
Vol 48 (2) ◽  
pp. 115-121 ◽  
Author(s):  
Tsukasa TAMEYASU ◽  
Tsuyoshi AKIMOTO ◽  
Yasuhisa HIROHATA ◽  
Ibuki SHIRAKAWA ◽  
Naoto YAMAMOTO ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Skeletal Muscle Myosin ◽  
Velocity Relation ◽  
Force Velocity ◽  
Actin Cables ◽  
Muscle Myosin

scholarly journals Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase.

1985 ◽  
Vol 260 (20) ◽  
pp. 11275-11285 ◽  
Author(s):  
A M Edelman ◽  
K Takio ◽  
D K Blumenthal ◽  
R S Hansen ◽  
K A Walsh ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Skeletal Muscle Myosin ◽  
Calmodulin Binding ◽  
Catalytic Domains ◽  
Muscle Myosin
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