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

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.

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Three-dimensional Structure of Acanthamoeba castellanii Myosin-IB (MIB) Determined by Cryoelectron Microscopy of Decorated Actin Filaments

The Journal of Cell Biology ◽

10.1083/jcb.141.1.155 ◽

1998 ◽

Vol 141 (1) ◽

pp. 155-162 ◽

Cited By ~ 21

Author(s):

James D. Jontes ◽

E. Michael Ostap ◽

Thomas D. Pollard ◽

Ronald A. Milligan

Keyword(s):

Actin Filaments ◽

Catalytic Domain ◽

Three Dimensional ◽

Acanthamoeba Castellanii ◽

Dimensional Structure ◽

Cryoelectron Microscopy ◽

Tail Domain ◽

Three Dimensional Structure ◽

Myosin I ◽

Carboxy Terminal

The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a “classical” myosin-I, Acanthamoeba myosin-IB (MIB), at ∼18 Å resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, ∼10°, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.

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

The Journal of Cell Biology ◽

10.1083/jcb.108.6.2395 ◽

1989 ◽

Vol 108 (6) ◽

pp. 2395-2400 ◽

Cited By ~ 63

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."

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Conformational changes due to calcium-induced calmodulin dissociation in brush border MyosinI-decorated F-actin revealed by cryoelectron microscopy and image analysis

Journal of Molecular Biology ◽

10.1006/jmbi.1997.1058 ◽

1997 ◽

Vol 269 (4) ◽

pp. 548-557 ◽

Cited By ~ 14

Author(s):

M Whittaker ◽

R.A Milligan

Keyword(s):

Image Analysis ◽

Conformational Changes ◽

Brush Border ◽

Cryoelectron Microscopy

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

Journal of Cell Science ◽

10.1242/jcs.107.6.1623 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1623-1631 ◽

Cited By ~ 3

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)

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

The Journal of Cell Biology ◽

10.1083/jcb.120.6.1393 ◽

1993 ◽

Vol 120 (6) ◽

pp. 1393-1403 ◽

Cited By ~ 71

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.

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