Terminal webs prepared from mouse intestinal epithelial cells were examined by the quick-freeze, deep-etch, and rotary-replication method. The microvilli of these cells contain actin filaments that extend into the terminal web in compact bundles. Within the terminal web these bundles remain compact; few filaments are separated from the bundles and fewer still bend towards the lateral margins of the cell. Decoration with subfragment 1 ($1) of myosin confirmed that relatively few actin filaments travel horizontally in the web. Instead, between actin bundles there are complicated networks of fine fibrils. Here we present two lines of evidence which suggest that myosin is one of the major cross-linkers in the terminal web. First, when brush borders are exposed to 1 mM ATP in 0.3 M KCI, they lose their normal ability to bind antimyosin antibodies as judged by immunofluorescence, and they lose the thin fibrils normally found in deep-etch replicas. Correspondingly, myosin is released into the supernatant as judged by SDS gel electrophoresis. Second, electron microscope immunocytochemistry with antimyosin antibodies followed by ferritin-conjugated second antibodies leads to ferritin deposition mainly on the fibrils at the basal part of rootlets. Deep-etching also reveals that the actin filament bundles are connected to intermediate filaments by another population of crosslinkers that are not extracted by ATP in 0.3 M KCI. From these results we conclude that myosin in the intestinal cell may not only be involved in a short range sliding-filament type of motility, but may also play a purely structural role as a long range cross-linker between microvillar rootlets.The brush border of the intestinal epithelial cell has been a favorite subject for study of the organization and biochemistry of actin fdaments and their related proteins in nonmuscle cells. This is in part due to the ease with which this portion of the cell can be isolated, in part due to the quantities which can be made, and in part due to the fact that microvilli occur on the surface of nearly all nonmuscle cells of the body, and thus, by studying the brush border, one can investigate a very basic cellular differentiation. Furthermore, there have been two in vivo studies (39, 40), albeit poorly substantiated, and two in vitro studies (27,37) that together indicate that microviUi on intestinal cells may move or wave about. Also it is now clear that within the brush border are all the components necessary for movement, including actin, myosin, tropomyosin, a-actinin, calmodulin, a myosin light chain kinase, 1 and a calcium-regulated actin cross-linking protein, villin (3-5, 8, 11, 16, 20, 29). KeUer, T. C. S., III, C. L. Howe, and M. S. Mooseker. 1981. J. Cell Biol. 91(2, Pt. 2):305a (Abstr.). Nevertheless, it is still not clear if in fact microvilli move in vivo and if they do, what type of motion they undergo. Likewise, it is still unclear exactly how the in vitro movements described in previous reports (27, 37) are actually generated. Several models ...

Actin was isolated from erythrocyte ghosts. It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM). When erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized. Spectrin has a larger molecular weight than muscle myosin. When salt is added to the EDTA extract, a branching filamentous polymer is formed. However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted. Thus, spectrin seems to inhibit the polymerization of actin. If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments. The addition of HMM or trypsin to erythrocyte ghosts results in filament formation in situ. These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polymerization of one or both proteins to occur. Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane. This network presumably functions in restricting the lateral movement of membrane-penetrating particles.The molecular biology of membranes is an area of extremely active research at the present time, as can be attested to by the large number of reviews and books published on this subject over the past few years (see Singer, 1974, for references). The erythrocyte membrane has been a favorite source of experimental material because (a) it is readily available, (b) it is relatively homogeneous; and (c) it can be prepared without contamination of other membrane fragments or cytoplasm. The literature concerning the proteins in this membrane has been extensively reviewed; no less than 10 reviews have appeared in the last two years (see Steck, 1974).About 20-25% of the total protein in erythrocyte membranes is made up of a pair of proteins called spectrin (bands 1 and 2 on SDS polyacrylamide gels) by Marchesi and Steers (1968). These proteins have molecular weights of 250,000 and 220,000 (Fairbanks et al.,

When Pisaster, Asterias, or Thyone sperm are treated with the ionophore A23187 or X537A, an acrosomal reaction similar but not identical to a normal acrosomal reaction is induced in all the sperm. Based upon the response of the sperm, the acrosomal reaction consists of a series of temporally related steps. These include the fusion of the acrosomal vacuole with the cell surface, the polymerization of the actin, the alignment of the actin filaments, an increase in volume, an increase in the limiting membrane, and changes in the shape of the nucleus. In this report, we have concentrated on the first two steps in this sequence. Although fusion of the acrosomal vacuole with the cell surface requires Ca ++, we found that the polymerization of actin instead appears to be dependent upon an increase in intracellular pH. This conclusion was reached by applying to sperm A23187, X537A, or nigericin, ionophores which all carry H § at high affinity, yet vary in their affinity for other cations. When sperm are suspended in isotonic NaCI, isotonic KCI, calcium-free seawater, or seawater, all at pH 8.0, and the ionophore is added, the actin polymerizes explosively and an efflux of H § from the cell occurs. However, if the pH of the external medium is maintained at 6.5, the presumed intracellular pH, no effect is observed. And, finally, if egg jelly is added to sperm (the natural stimulus for the acrosomal reaction) at pH 8.0, H § is also released. On the basis of these observations and those presented in earlier papers in this series, we conclude that a rise in intracellular pH induces the actin to disassociate from its binding proteins. Now it can polymerize. KEY WORDS actin polymerization acrosomal reaction H + Ca +* 9 intraceUular pH 9 sperm exocytosis In most nonmuscle ceils, in contrast to skeletal muscle, organized arrays of actin filaments are transitory, appearing at the appropriate place and time only to disappear at a later developmental stage. Frequently, these stages are separated by only a minute or two as, for example, during cytokinesis in animal cells (see reference 25). Thus, the cell must be capable of controlling the rapid assembly and disassembly of actin. It must $36J. CELL BIOLOGY 9 The Rockefeller University Press 9

In Limulus sperm an actin filament bundle 55 pm in length extends from the acrosomal vacuole membrane through a canal in the nucleus and then coils in a regular fashion around the base of the nucleus. The bundle expands systematically from 15 filaments near the acrosomal vacuole to 85 filaments at the basal end . Thin sections of sperm fixed during stages in spermatic! maturation reveal that the filament bundle begins to assemble on dense material attached to the acrosomal vacuole membrane . In micrographs of these early stages in maturation, short bundles are seen extending posteriorly from the dense material . The significance is that these short, developing bundles have about 85 filaments, suggesting that the 85-filament end of the bundle is assembled first. By using filament bundles isolated and incubated in vitro with G actin from muscle, we can determine the end "preferred" for addition of actin monomers during polymerization . The end that would be associated with the acrosomal vacuole membrane, a membrane destined to be continuous with the plasma membrane, is preferred about 10 times over the other, thicker end. Decoration of the newly polymerized portions of the filament bundle with subfragment 1 of myosin reveals that the arrowheads point away from the acrosomal vacuole membrane, as is true of other actin filament bundles attached to membranes. From these observations we conclude that the bundle is nucleated from the dense material associated with the acrosomal vacuole and that monomers are added to the membrane-associated end. As monomers are added at the dense material, the thick, first-made end of the filament bundle is pushed down through the nucleus where, upon reaching the base of the nucleus, it coils up . Tapering is brought about by the capping of the peripheral filaments in the bundle .A common feature of actin filaments in nonmuscle cells is their association with membranes, in general the plasma membrane . This association is essential for such basic phenomena as cytokinesis, filopodial and microvillar movements, elongation of cell processes (e .g ., the acrosomal process), and retraction of cell extensions such as occurs during clotting . To understand how the motility is generated and controlled we need to determine the polarity of the actin filaments, because this defines their possible direction of movement . We also want to know how the cell establishes this polarity with respect to the plasma membrane and how the filaments elongate . Are they, for example, nucleated from some membrane-associated protein, or are the filaments assembled from a cytoplasmic organelle and secondarily connected to a site on the membrane? Do these recently nucleated actin filaments, once connected to the membrane, elongate by the addition of monomers to the mem-THE JOURNAL OF CELL BIOLOGY " VOLUME 90 AUGUST 1981 485-494 © The Rockefeller University Press -0021-9525/81/08/0485/15 $1 .00 brane-associated end of the filament, or do they elongate by the addition of monomers to the cytoplasmic end of the...