Reassociation of microvillar core proteins: making a microvillar core in vitro

Intestinal epithelia have a brush border membrane of numerous microvilli each comprised of a cross-linked core bundle of 15-20 actin filaments attached to the surrounding membrane by lateral cross-bridges; the cross-bridges are tilted with respect to the core bundle. Isolated microvillar cores contain actin (42 kD) and three other major proteins: fimbrin (68 kD), villin (95 kD), and the 110K-calmodulin complex. The addition of ATP to detergent-treated isolated microvillar cores has previously been shown to result in loss of the lateral cross-bridges and a corresponding decrease in the amount of the 110-kD polypeptide and calmodulin associated with the core bundle. This provided the first evidence to suggest that these lateral cross-bridges to the membrane are comprised at least in part by a 110-kD polypeptide complexed with calmodulin. We now demonstrate that purified 110K-calmodulin complex can be readded to ATP-treated, stripped microvillar cores. The resulting bundles display the same helical and periodic arrangement of lateral bridges as is found in vivo. In reconstitution experiments, actin filaments incubated in EGTA with purified fimbrin and villin form smooth-sided bundles containing an apparently random number of filaments. Upon addition of 110K-calmodulin complex, the bundles, as viewed by electron microscopy of negatively stained images, display along their entire length helically arranged projections with the same 33-nm repeat of the lateral cross-bridges found on microvilli in vivo; these bridges likewise tilt relative to the bundle. Thus, reconstitution of actin filaments with fimbrin, villin, and the 110K-calmodulin complex results in structures remarkably similar to native microvillar cores. These data provide direct proof that the 110K-calmodulin is the cross-bridge protein and indicate that actin filaments bundled by fimbrin and villin are of uniform polarity and lie in register. The arrangement of the cross-bridge arms on the bundle is determined by the structure of the core filaments as fixed by fimbrin and villin; a contribution from the membrane is not required.     

Rings of intermediate (100 A) filament bundles in the perinuclear region of vascular endothelial cells. Their mobilization by colcemid and mitosis

Vascular endothelial cells cultured from guinea pig aorta or portal vein contain naturally occurring bundles of 100 A (diameter) filaments that completely encircle the nucleus. These rings are phase lucent and birefringent when examined with the light microscope. Perinuclear bundles of 100 A filaments were also seen in endothelial cells in vivo, indicating that they are a normal cytoplasmic component. These filaments did not decorate with S-1, and were not disrupted by glyceination. With these cells, experiments were designed to answer the following questions: (a) does Colcemid have an effect on these naturally occuring bundles? And (b) do these filaments remain during cell division? Endothelial cells grown in the presence of Colcemid were followed over 24 h. The perinuclear ring coiled into a juxtanuclear cap that consisted of disorganized arrays of 100 A filaments. This "coiling" effect was not blocked by cycloheximide, an inhibitor of protein synthesis. In another experiment, dividing cells were examined. During division the bundle of filaments is passively pulled in half into the daughter cells. These bundles did not disappear during the mitosis when mitotic spindle microtubules assemble. These studies suggest that Colcemid may exert a direct effect on 100 A filaments, independent of microtubules. Since these filaments do not disappear during mitosis, it is possible that in these cells the 100 A filaments and tubulin do not share a common pool of precursor proteins.     

The Z lattice in canine cardiac muscle

Filtered images of mammalian cardiac Z bands were reconstructed from optical diffraction patterns from electron micrographs. Reconstructed images from longitudinal sections show connecting filaments at each 38-nm axial repeat in an array consistent with cross-sectional data. Some reconstructed images from cross sections indicate two distinctly different optical diffraction patterns, one for each of two lattice forms (basket weave and small square). Other images are more complex and exhibit composite diffraction patterns. Thus, the two lattice forms co-exist, interconvert, or represent two different aspects of the same details within the lattice. Two three-dimensional models of the Z lattice are presented. Both include the following features: a double array of axial filaments spaced at 24 nm, successive layers of tetragonally arrayed connecting filaments, projected fourfold symmetry in cross section, and layers of connecting filaments spaced at intervals of 38 nm along the myofibril axis. Projected views of the models are compared to electron micrographs and optically reconstructed images of the Z lattice in successively thicker cross sections. The entire Z band is rarely a uniform lattice regardless of plane of section or section thickness. Optical reconstructions strongly suggest two types of variation in the lattice substructure: (a) in the arrangement of connecting filaments, and (b) in the arrangement of units added side-to-side to make larger myofilament bundles and/or end-to-end to make wider Z bands. We conclude that the regular arrangement of axial and connecting filaments generates a dynamic Z lattice.     

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges connecting spermatogenic cells in the black snail, Semisulcospira libertina (Mollusca, Mesogastropoda)

Striated microfilament bundles attaching to the plasma membrane of cytoplasmic bridges between spermatogenic cells are described in the black snail, Semisulcospira libertina. The bundles were occasionally observed in bridges connecting spermatogonia, spermatocytes and typical spermatids. Relations between bundles and centrioles could not be detected. The bundle had electron dense cross bands with a periodicity of approximately 200 nm, and attached to the membrane with almost right angle at the cross linker level. Phalloidin cytochemistry revealed that the bundle contained F-actin. In a case, a bundle connected two cytoplasmic bridges.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Coalignment of microtubules, cytokeratin intermediate filaments, and collagen fibrils in a collagen-secreting cell system

The distribution of microtubules and intermediate filaments in the collagen-secreting scleroblasts of the goldfish scale was investigated by immunofluorescence and electron microscopy. Many of the microtubules and cytokeratin type intermediate filaments formed bundles that were aligned with the underlying, parallel collagen fibrils. The intermediate filament bundles were evenly spaced and located adjacent to the basal plasma membrane. The microtubules, on the other hand, were located further away from the membrane, although many were found very close to the intermediate filament bundles. No detectable change was observed in scleroblast microtubules when cells on scales were treated with colchicine or cooled (greater than or equal to 0 degrees C) for up to 1 h. Cells had to be cooled overnight before the microtubules were affected. The final number and length of the microtubules in the cell depended only on the final steady-state temperature and not the temperature history of the scale cell, and steady state was reached more slowly at colder temperatures. The microtubules but not the intermediate filaments rapidly (within 5 min) and reversibly depolymerized when cells were chilled to -2 approximately -4 degrees C. When chilled cells were warmed, the microtubules polymerized back, within 15 min at room temperature, to the same pattern of parallel coalignment with the underlying collagen. They appeared to repolymerize via two different pathways: (1) a radial growth outwards from the microtubule organizing center followed by a progressive realignment with the underlying collagen and (2) a gradual and simultaneous polymerization along cold-stable, antitubulin staining fibers. These fibers were also aligned with the collagen fibrils and may be related to the aligned intermediate filaments.     

Organization of neuronal microtubules in the nematode Caenorhabditis elegans

We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

Endothelial cell connecting filaments anchor endothelial cells to the subjacent elastic lamina in the developing aortic intima of the mouse

The ultrastructural association of endothelial cells with the subjacent elastic lamina was investigated in the developing mouse aorta by electron microscopy. In the 5-day postnatal aorta, extensive filament bundles extend along the subendothelial matrix connecting the endothelial cells to the underlying elastic lamina. The connecting filaments form lateral associations with the abluminal surface of the endothelial cells in regions of membrane occupied by membrane-associated dense plaques. On the intracellular face of each plaque, the termini of stress fibers penetrate and anchor to the cell membrane in alignment with the extracellular connecting filaments. Both the stress fibers and the connecting filaments are oriented parallel to the longitudinal axis of the vessel. High magnification electron micrographs of individual endothelial cell connecting filaments reveal features similar to those of elastin-associated microfibrils. Each connecting filament consists of a 9–10 nm linear core with an electron-lucent center and peripheral spike-like projections. From the filaments, small thread-like extensions span laterally, linking the filaments into a loose bundle and anchoring them to the endothelial cell membrane and the surface of the elastic lamina. The filaments also appear heavily coated with electron-dense material; often with some degree of periodicity along the filament length. During development, the number of endothelial cell connecting filaments decreases as the elastic lamina expands and the subendothelial matrix is reduced. In the aortic intima of mature mice, the elastic lamina is closely apposed to the abluminal surface of the endothelial cell and no connecting filaments are seen. These observations suggest that endothelial cell connecting filaments are developmental features of the aortic intima which, together with the intracellular stress fibers, aid to maintain the structural integrity of the endothelial cell layer during development by providing the cells with protection from intraluminal shear forces.     

Development of a differentiated microtubule structure: formation of the chicken erythrocyte marginal band in vivo

The microtubules of mature nucleated erythrocytes are organized into a marginal band that is confined to a single plane at the periphery and that contains essentially the same number of microtubule profiles in each individual cell. Developing erythrocytes can be isolated in homogeneous and synchronously developing populations from chicken embryos. For these reasons, these cells offer a particularly accessible system for study of the pathway leading to a specific microtubule structure in a normal, terminally differentiated animal cell. Along this developmental course, striking changes occur in the properties of the microtubules. Between the postmitotic cell and the formation of the band, a novel arrangement is found: bundles of laterally associated microtubules in each cell, coursing through the cytoplasm but not confined to the periphery. The microtubule organizing centers evident at early stages disappear by the time the band forms. The microtubules in early cells are readily depolymerized by drugs, but that drug sensitivity is lost in the mature cells. The microtubule arrangement of mature cells is faithfully recapitulated after reversible depolymerization, while that of the immature cells is not. Finally, as the band forms, the microtubules and microfilaments increasingly become coaligned. In sum, the microtubules of immature cells have many properties in common with those of cultured cells, but during maturation those properties change. The results suggest that lateral interactions become increasingly important in stabilizing and organizing the microtubules. The properties of marginal band microtubules, and comparable properties of axonal microtubules, may reflect differences between the requirements for cytoskeletal structures of cycling cells and terminally differentiated cells.     

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.     

Helical arrangement of filaments in microvillar actin bundles

Actin filament arrays in in vivo microvillar bundles of rat intestinal enterocyte were re-evaluated using electron tomography (ET). Conventional electron microscope observation of semi-thin cross sections (300nm thick) of high-pressure freeze fixed and resin embedded brush border has shown a whirling pattern in the center of the microvilli instead of hexagonally arranged dots, which strongly suggests that the bundle consists of a non-parallel array of filaments. A depth compensation method for the ET was developed to estimate the actual structure of the actin bundle. Specimen shrinkage by beam irradiation during image acquisition was estimated to be 63%, and we restored the original thickness in the reconstruction. The depth compensated tomogram displayed the individual actin filaments within the bundles and it indicated that the actin filaments do not lie exactly parallel to each other: instead, they are twisted in a clockwise coil with a pitch of ～120°/μm. Furthermore, the lattice of actin filaments was occasionally re-arranged within the bundle. As the microvillar bundle mechanically interacts with the membrane and is thought to be compressed by the membrane's faint tensile force, we removed the shrouding membrane using detergents to eliminate the mechanical interaction. The bared bundles no longer showed the whirling pattern, suggesting that the bundle had released its coiled property. These findings indicate that the bundle has not rigid but elastic properties and a dynamic transformation in its structure caused by a change in the mechanical interaction between the membrane and the bundle.     

Mechanics of Individual Keratin Bundles in Living Cells

Along with microtubules and microfilaments, intermediate filaments are a major component of the eukaryotic cytoskeleton and play a key role in cell mechanics. In cells, keratin intermediate filaments form networks of bundles that are sparser in structure and have lower connectivity than, for example, actin networks. Because of this, bending and buckling play an important role in these networks. Buckling events, which occur due to compressive intracellular forces and cross-talk between the keratin network and other cytoskeletal components, are measured here in situ. By applying a mechanical model for the bundled filaments, we can access the mechanical properties of both the keratin bundles themselves and the surrounding cytosol. Bundling is characterized by a coupling parameter that describes the strength of the linkage between the individual filaments within a bundle. Our findings suggest that coupling between the filaments is mostly complete, although it becomes weaker for thicker bundles, with some relative movement allowed.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

Microtubule disassembly enhances reversible cytochalasin-dependent disruption of actin bundles in characean internodes

Summary Parallel bundles of actin filaments at the cortex-endoplasm interface provide tracks for myosin-generated cytoplasmic streaming in characean internodes. These bundles resist disassembly or structural modification when exposed to 10 μM cytochalasin D (CD) even though this concentration of CD rapidly (within minutes) but reversibly arrests streaming. Unexpectedly, we discovered that prolonged treatment with lower concentrations of CD could partially disassemble the subcortical actin bundles. Actin bundles became discontinuous following one- to several-day treatment with concentrations (6 μM) that reduced but did not arrest streaming, and the residual fragments mostly remained parallel to the chloroplast files. When microtubules were concurrently disassembled with tubulin-specific drugs, however, low CD concentrations (2.5–3 μM) completely arrested bulk streaming, disrupted the largely 2-dimensional actin bundle array and caused the formation of a coarse, thick-meshed actin network that extended from the cortex to the endoplasm. Despite such massive reconstruction, drug removal enabled cells to recover continuous parallel bundles and streaming. Recovery was possible if both or just one of the drugs were removed. In recovered cells, the streaming pattern frequently redeveloped in new directions that did not follow the chloroplast files, and later, chloroplast files readjusted to the new polarity established by the actin bundles. This first report on the complete and reversible disassembly of characean actin bundles provides new insights into the mechanism of actin bundle assembly and organization and supports the idea of indirect interactions between actin filaments and microtubules.     

Mechanics and dynamics of actin-driven thin membrane protrusions

Motile cells explore their surrounding milieu by extending thin dynamic protrusions, or filopodia. The growth of filopodia is driven by actin filament bundles that polymerize underneath the cell membrane. We compute the mechanical and dynamical features of the protrusion growth process by explicitly incorporating the flexible plasma membrane. We find that a critical number of filaments are needed to generate net filopodial growth. Without external influences, the filopodium can extend indefinitely up to the buckling length of the F-actin bundle. Dynamical calculations show that the protrusion speed is enhanced by the thermal fluctuations of the membrane; a filament bundle encased in a flexible membrane grows much faster. The protrusion speed depends directly on the number and spatial arrangement of the filaments in the bundle and whether the filaments are tethered to the membrane. Filopodia also attract each other through distortions of the membrane. Spatially close filopodia will merge to form a larger one. Force-velocity relationships mimicking micromanipulation experiments testing our predictions are computed.     

Filament arrangements in negatively stained cultured cells: the organization of actin

Treatment of spread, cultured cells with Triton X-100 followed by negative staining reveals the organization of the unextracted intracellular filamentous elements: actin, microtubules and the 100 angstrom filaments. The present report describes the organization of the actin-like filaments in human skin fibroblasts and mouse 3 T 3 cells. As shown in earlier studies, the cytoplasmic stress fibres were seen to be composed of bundles of colinear actin-like filaments. In addition to these large stress fibres much smaller bundles of thin filaments as well as randomly oriented thin filaments were also observed. A thick bundle of thin filaments, 0.2 microm to 0.5 microm in diameter, was found to delimit the concave cell edges most prominent in well-spread stationary cells. The leading edge and ruffled border of human skin fibroblasts appeared as a broad web, of meshwork of diagonally oriented thin filaments interconnecting radiating, linear bundles of thin filaments about 0.1 microm in diameter. These bundles corresponding to the microspikes described earlier ranged from about 1.5 microm in length and were separated by 1 microm to 3 microm laterally. The leading edge of 3 T 3 cells showed a similar organization but with fewer radiating thin filament bundles. Both the filaments in the bundles and in the meshwork formed arrowhead complexes with smooth muscle myosin subfragment - 1 which were unipolar and directed towards the main body of the cell. The findings are discussed in relation to the mechanisms of non-muscle cell motility.     

Control of shape and pattern during the assembly of a large microtubule bundle

Microtubules are packed and linked together in a well defined hexagonal arrangement in the cytopharyngeal microtubule bundles of the ciliate Nassula. Early stages in the morphogenesis of these bundles have been examined. Elements which nucleate assembly of bundle microtubules are apparently closely associated before tubule assembly commences. These nucleating elements seem to be bound together in highly ordered arrays to form microtubule-nucleating-templetes. Each array of elements is attached to the proximal end of a basal body and appears to establish the pattern of tubule packing and cross-sectional shape of a tubule bundle. A self-assembly procedure which accounts for the anisometric growth and shaping of a template and its microtubule bundle is proposed.     

The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin

The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles large enough to be visualized by light microscopy. The size of these bundles depends on the size of the containing vessel. The actin filaments in these bundles are tightly packed in some areas while in others they are more disperse. The bundles form a continuous reticulum that fills the container, since the filaments from a particular bundle may interdigitate with filaments from other bundles at points where they intersect. The same phenomena are seen when rabbit muscle aldolase rather than alpha-actinin is used as the crosslinker. We propose that actophorin promotes bundling by shortening the actin filaments enough to allow them to rotate into positions favorable for lateral interactions with each other via alpha-actinin. The network of bundles is more rigid and less thixotropic than the corresponding network of single actin filaments linked by alpha-actinin. One explanation may be that alpha-actinin (or aldolase) normally in rapid equilibria with actin filaments may become trapped between the filaments increasing the effective concentration of the crosslinker.