Mapping of the microvillar 110K-calmodulin complex (brush border myosin I). Identification of fragments containing the catalytic and F-actin-binding sites and demonstration of a calcium ion-dependent conformational change

Biochemistry ◽  
1990 ◽  
Vol 29 (50) ◽  
pp. 11089-11094 ◽  
Author(s):  
Lynne M. Coluccio ◽  
Anthony Bretscher
Keyword(s):  
Conformational Change ◽  
Brush Border ◽  
Binding Sites ◽  
Calcium Ion ◽  
Actin Binding ◽  
Myosin I

Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains

1995 ◽  
Vol 108 (12) ◽  
pp. 3775-3786 ◽  
Author(s):  
C. Ruppert ◽  
J. Godel ◽  
R.T. Muller ◽  
R. Kroschewski ◽  
J. Reinhard ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Brush Border ◽  
Subcellular Fractionation ◽  
Actin Binding ◽  
Resting Cells ◽  
Medium Density ◽  
Subcellular Targeting ◽  
Myosin I ◽  
Punctate Pattern

Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin 1. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In subcellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density. The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are sufficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper subcellular localization.

scholarly journals Identification, characterization and cloning of myr 1, a mammalian myosin-I.

1993 ◽  
Vol 120 (6) ◽  
pp. 1393-1403 ◽  
Author(s):  
C Ruppert ◽  
R Kroschewski ◽  
M Bähler
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Brush Border ◽  
Binding Sites ◽  
Neuronal Development ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Tail Domain ◽  
Myosin I ◽  
Splice Forms

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.

scholarly journals Tropomyosin distinguishes between the two actin-binding sites of villin and affects actin-binding properties of other brush border proteins.

1987 ◽  
Vol 104 (1) ◽  
pp. 29-40 ◽  
Author(s):  
D R Burgess ◽  
K O Broschat ◽  
J M Hayden
Keyword(s):  
Epithelial Cell ◽  
Protein Interactions ◽  
Brush Border ◽  
Binding Sites ◽  
Intestinal Epithelial Cell ◽  
Actin Binding ◽  
Intestinal Epithelial ◽  
Binding Properties ◽  
Order Of Magnitude ◽  
Protein Components

The intestinal epithelial cell brush border exhibits distinct localizations of the actin-binding protein components of its cytoskeleton. The protein interactions that dictate this subcellular organization are as yet unknown. We report here that tropomyosin, which is found in the rootlet but not in the microvillus core, can bind to and saturate the actin of isolated cores, and can cause the dissociation of up to 30% of the villin and fimbrin from the cores but does not affect actin binding by 110-kD calmodulin. Low speed sedimentation assays and ultrastructural analysis show that the tropomyosin-containing cores remain bundled, and that 110-kD calmodulin remains attached to the core filaments. The effects of tropomyosin on the binding and bundling activities of villin were subsequently determined by sedimentation assays. Villin binds to F-actin with an apparent Ka of 7 X 10(5) M-1 at approximate physiological ionic strength, which is an order of magnitude lower than that of intestinal epithelial cell tropomyosin. Binding of villin to F-actin presaturated with tropomyosin is inhibited relative to that to pure F-actin, although full saturation can be obtained by increasing the villin concentration. Villin also inhibits the binding of tropomyosin to F-actin, although not to the same extent. However, tropomyosin strongly inhibits bundling of F-actin by villin, and bundling is not recovered even at a saturating villin concentration. Since villin has two actin-binding sites, both of which are required for bundling, the fact that tropomyosin inhibits bundling of F-actin under conditions where actin is fully saturated with villin strongly suggests that tropomyosin's and one of villin's F-actin-binding sites overlap. These results indicate that villin and tropomyosin could compete for actin filaments in the intestinal epithelial cell, and that tropomyosin may play a major role in the regulation of microfilament structure in these and other cells.

Multiple unconventional myosin domains of the intestinal brush border cytoskeleton

1994 ◽  
Vol 107 (12) ◽  
pp. 3535-3543 ◽  
Author(s):  
M.B. Heintzelman ◽  
T. Hasson ◽  
M.S. Mooseker
Keyword(s):  
Brush Border ◽  
Myosin Ii ◽  
Actin Binding ◽  
Myosin Vi ◽  
Myosin V ◽  
Terminal Web ◽  
Intestinal Brush Border ◽  
Myosin I ◽  
Biochemical Fractionation

Representatives of class V and class VI unconventional myosins are identified as components of the intestinal brush border cytoskeleton. With brush border myosin-I and myosin-II, this brings to four the number of myosin classes associated with this one subcellular domain and represents the first characterization of four classes of myosins expressed in a single metazoan cell type. The distribution and cytoskeletal association of each myosin is distinct as assessed by both biochemical fractionation and immunofluorescence localization. Myosin-VI exists in both the microvillus and terminal web although the terminal web is the predominant site of concentration. Myosin-V is present in the terminal web and, most notably, at the distal ends of the microvilli, thus becoming the first actin-binding protein to be localized to this domain as assessed by both immunohistochemical and biochemical methods. In the undifferentiated enterocytes of the intestinal crypts, myosin-VI is expressed but not yet localized to the brush border, in contrast to myosin-V, which does demonstrate an apical distribution in these cells. An assessment of myosin abundance indicates that while myosin-II is the most abundant in the cell and in the brush border, brush border myosin-I is only slightly less abundant in contrast to myosins-V and -VI, both of which are two orders of magnitude less abundant than the others. Extraction studies indicate that of these four myosins, myosin-V is the most tightly associated with the brush border membrane, as detergent, in addition to ATP, is required for efficient solubilization.

scholarly journals Localization and specificity of the phospholipid and actin binding sites on the tail of Acanthamoeba myosin IC

1992 ◽  
Vol 117 (6) ◽  
pp. 1241-1249 ◽  
Author(s):  
SK Doberstein ◽  
TD Pollard
Keyword(s):  
Binding Sites ◽  
Binding Assay ◽  
Lipid Binding ◽  
Actin Binding ◽  
Acidic Phospholipid ◽  
Myosin I ◽  
Head Groups ◽  
Acidic Phospholipids ◽  
Beta Galactosidase ◽  
Actin Binding Site

We used bacterially expressed beta-galactosidase fusion proteins to localize the phospholipid binding domain of Acanthamoeba myosin IC to the region between amino acids 701 and 888 in the NH2-terminal half of the tail. Using a novel immobilized ligand lipid binding assay, we determined that myosin I can bind to several different acidic phospholipids, and that binding requires a minimum of 5 mol% acidic phospholipid in a neutral lipid background. The presence of di- and triglycerides and sterols in the lipid bilayer do not contribute to the affinity of myosin I for membranes. We confirm that the ATP-insensitive actin binding site is contained in the COOH-terminal 30 kD of the tail as previously shown for Acanthamoeba myosin IA. We conclude that the association of the myosin IC tail with acidic phospholipid head groups supplies much of the energy for binding myosin I to biological membranes, but probably not specificity for targeting myosin I isoforms to different cellular locations.

scholarly journals Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

1994 ◽  
Vol 126 (2) ◽  
pp. 433-443 ◽  
Author(s):  
A McGough ◽  
M Way ◽  
D DeRosier
Keyword(s):  
Image Analysis ◽  
Smooth Muscle ◽  
Binding Sites ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Outer Face ◽  
Actin Binding Domain

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

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