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 Isolation and partial characterization of a 110-kD dimer actin-binding protein.

1986 ◽  
Vol 103 (2) ◽  
pp. 621-630 ◽  
Author(s):  
T Ueno ◽  
E D Korn
Keyword(s):  
Myosin Ii ◽  
Deae Cellulose ◽  
Acanthamoeba Castellanii ◽  
Actin Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Myosin I ◽  
Soluble Supernatant ◽  
Low Shear

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and 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.

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

2006 ◽  
Vol 342 (1) ◽  
pp. 236-244 ◽  
Author(s):  
Hang Thi Thu Nguyen ◽  
Adda Berkane Amine ◽  
Daniel Lafitte ◽  
Abdul A. Waheed ◽  
Cendrine Nicoletti ◽  
...  
Keyword(s):  
Lipid Rafts ◽  
Brush Border ◽  
Intestinal Brush Border

scholarly journals Plastin 1 Binds to Keratin and Is Required for Terminal Web Assembly in the Intestinal Epithelium

2009 ◽  
Vol 20 (10) ◽  
pp. 2549-2562 ◽  
Author(s):  
Eva-Maria S. Grimm-Günter ◽  
Céline Revenu ◽  
Sonia Ramos ◽  
Ilse Hurbain ◽  
Neil Smyth ◽  
...  
Keyword(s):  
Brush Border ◽  
Actin Filaments ◽  
Keratin 19 ◽  
Terminal Web ◽  
Intestinal Brush Border ◽  
Important Regulator ◽  
Increased Sensitivity ◽  
Filament Network ◽  
Deficient Mice

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.

scholarly journals Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains.

1988 ◽  
Vol 107 (5) ◽  
pp. 1749-1757 ◽  
Author(s):  
J M Carboni ◽  
K A Conzelman ◽  
R A Adams ◽  
D A Kaiser ◽  
T D Pollard ◽  
...  
Keyword(s):  
Binding Site ◽  
Brush Border ◽  
Myosin Head ◽  
Myosin Ii ◽  
Structural Similarity ◽  
Actin Binding ◽  
Atp Binding Site ◽  
Calmodulin Binding ◽  
Tryptic Fragment ◽  
Binding Domains

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110-kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.

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