Abstract
The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 mumol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 mumol P(i)/mg-min), and low with Mg++ (0.01 mumol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.
Highlights
The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments
Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KC1 is highest with EDTA (i /xmol Pi/ mg-min; 37~ intermediate with Ca ++ (0.4 /xmol Pi/mg-min), and low with Mg++ (0.01 /~mol Pi/mg-min)
On the basis of the polarity of actin filaments in the microvillus and the nature of microvillar contraction observed in vitro in isolated brush borders [22, 23], that microvillar movements were mediated by myosin interactions with the basal ends of microvillar actin filaments in the terminal web
Summary
The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. If the paradigm of skeletal muscle holds for the production of force and the resultant movements observed in nonmuscle cells, we would predict that myosin is positioned so that interaction with actin filaments would be efficient This is important because there is not enough myosin to bind to all the available actin in nonmuscle cells [27]. This system is the apical cortex of intestinal epithelial cells, the brush border [22,23,24, 33] It was predicted, on the basis of the polarity of actin filaments in the microvillus and the nature of microvillar contraction observed in vitro in isolated brush borders [22, 23], that microvillar movements were mediated by myosin interactions with the basal ends of microvillar actin filaments in the terminal web. The results presented here demonstrate that two aspects of this prediction are correct: (a) the brush border contains myosin; and (b) this myosin is located exclusively in the terminal web
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