Structural basis of cytoplasmic streaming in Characean internodal cells. A hydrodynamic analysis

Summary Hydrodynamic equations were derived which relate the velocity profile of endoplasmic streaming with the motive force generated by active sliding of endoplasmic organelles in Characean internodal cells, under two implicit assumptions that (1) the sliding velocity of putative organelles is comparable to the streaming velocity of endoplasm, and (2) subcortical endoplasm is far less viscous than bulk endoplasm.The equations were extended so as to calculate the velocity profile in flattened or perfused internodal cells. Calculated profiles were basically consistent with reported patterns of streaming under these conditions.Utilizing published data, we deduce some hydrodynamic parameters of streaming, and predict the dimensions of putative organelles expected to drive entire cytoplasm. A revision for published values of the motive force of streaming is proposed.Hydrodynamic analyses made earlier on the spherical organelles are repeated. The results show that the organelles may generate streaming, depending on the configurationin vivo of fine filaments protruding from the body of the organelles.     

Studies on the growth and development of protoplasts of the moss,Physcomitrella patens,and its control by light

Protoplasts prepared from protonemal cultures of the moss Physcomitrella patens begin to regenerate a new cell wall within 1 h of removal from cellulase. The wall is seen as a gradually thickening mat of fibres when examined by scanning electron microscopy. Development of filaments from protoplasts takes place in the majority of cases only after one or more cell divisions have occurred. The direction of emergence of filaments is random in uniform light, but strongly negatively phototropic in bright unidirectional horizotal light. Filament growth is also strongly negatively phototropic. The influence of unidirectional light can be destroyed by incubating protoplasts in the presence of colchicine. Filaments growing in unidirectional light have cytoplasmic microtubules running along their long axes and in close association with large organelles. These results are discussed in terms of the potential for this system for the study of polarity in plants.     

Bacterial cell shape regulation: testing of additional predictions unique to the two-competing-sites model for peptidoglycan assembly and isolation of conditional rod-shaped mutants from some wild-type cocci.

The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsiella pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42 degrees C were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.     

Microfilament Orientation Constrains Vesicle Flow and Spatial Distribution in Growing Pollen Tubes

The dynamics of cellular organelles reveals important information about their functioning. The spatio-temporal movement patterns of vesicles in growing pollen tubes are controlled by the actin cytoskeleton. Vesicle flow is crucial for morphogenesis in these cells as it ensures targeted delivery of cell wall polysaccharides. Remarkably, the target region does not contain much filamentous actin. We model the vesicular trafficking in this area using as boundary conditions the expanding cell wall and the actin array forming the apical actin fringe. The shape of the fringe was obtained by imposing a steady state and constant polymerization rate of the actin filaments. Letting vesicle flux into and out of the apical region be determined by the orientation of the actin microfilaments and by exocytosis was sufficient to generate a flux that corresponds in magnitude and orientation to that observed experimentally. This model explains how the cytoplasmic streaming pattern in the apical region of the pollen tube can be generated without the presence of actin microfilaments.     

Morphological Aspects of Ciliary Motility

In Elliptio complanatus lateral cilia, two distinct patterns of filament termination can be discerned. In one case, all nine filaments are present and all are single; in the second, at least one filament is missing but doublets are still present. These probably represent different configurations within one cilium in different stroke positions; to get from one to the other, some peripheral filaments must move with respect to others. The data are consistent with the hypothesis that the filaments themselves do not change length, but rather slide past one another to accommodate increasing curvature. The bent regions of the cilium are in the form of circular arcs. In a few cases, apparent displacement of filaments at the tip (Δl) can be shown to be accounted for if we assume that all differences are generated within these arcs. The displacement per degree of bend is 35 A. Regions of bent arc are initially confined to the base of the cilium but move up the shaft as straight regions appear below them. From the relationship between arc length and radius of curvature, a shaft length that is the unit that initially bends and slides may be defined. Quantal displacements of the length of one 14S dynein may perhaps occur at sites between filaments at opposite sides of such a unit as sliding occurs.     

Distinctive features of cilia in metazoans and their significance for systematics.

A comparative study of epidermal cilia in the Turbellaria and Nemertea has revealed features in these organelles that are specific to certain taxonomic groups. Turbellarians of the order Acoela, in particular, have a characteristic pattern of axonemal filament termination in the distal tips of their cilia and a characteristic ciliary rootlet system that is not seen in other turbellarian orders nor in other metazoans. Each epidermal cilium in acoels has a typical 9 + 2 axonemal pattern through the main part of its length, but near its distal tip there is an abrupt shelf-life narrowing at which filaments 4-7 terminate; filaments 1, 2, 8 and 9 continue into the thinner distal-most part of the shaft along with singlet microtubules from the axonemal center. The rootlet system in acoel cilia involves an interconnecting pattern with lateral connectives. The unique structure of these cilia has systematic and phylogenetic significance for the Acoela, and it is argued that ultrastructural characters in general, including characters of organelles, can be validly applied to the phylogeny and systematics of the Metazoa.     

Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.     

The relationship of echinate inclusions and coated vesicles on wound healing inNitella flexilis (Characeae)

Summary Wound healing in internodal cells of the freshwater algaNitella flexilis (Characeae) was studied in the light and electron microscope. Immediately after punctation of the cell wall a wound plug is formed which stops outflow of cytoplasm. The plug consists of echinate inclusions which are normally located in the central vacuole. A wound wall consisting of pectin and cellulose microfibrils is formed beneath the plug within one to several hours. During that time the wound shows intensive fluorescence when treated with chlorotetracycline indicating transmembrane Ca²⁺ fluxes. Numerous coated pits and vesicles are found at the plasmalemma. The glycosomes undergo pronounced structural changes. Neither plug nor wound wall formation depend on actin filaments or microtubules as shown by inhibitor experiments with cytochalasin and amiprophos-methyl. The function of the coated vesicles and their interrelationship with other cell organelles is discussed.     

Lateral hydrodynamic effects of rotating filaments

On the basis of the filament rotation model that was elaborated for interpretations in cell motility, the lateral hydrodynamic effects of rotating filaments have been investigated by large-scale model experiments. Helices were rotated by small electric motors in a medium of high viscosity (honey or polyethyleneglycol). The observed effects, hitherto not investigated in detail by hydrodynamics, show some features that were attributed to the indefinable formative power or vital force of the past. The main effects generated by the rotating filaments are (1) flows and flow patterns with impact zones where flows collide, (2) regions of excessive pressure and negative pressure (corner effect) along a wall, (3) grooves and smoothly shaped ridges on a free fluid surface, and (4) rolling motions of freely hanging filaments. All effects and flow patterns depend on the appropriate distribution of rotating and counterrotating filaments. Each change of the rotational direction means a dramatic alteration. The application of the observed effects explains largely the function of the microtubule/microfilament hoops or helices during the cytokinesis of a plant cell. Interpretations or simulations are described for events as the formation of secondary wall thickenings, the orientation of their microfibrils, the motion of the preprophase band microtubules, the formation of the phragmosome, the migration, stationary position and shape of the preprophase nucleus, the girdle-, septum- and H-piece formation of cell walls in algae and some events of morphogenesis inMicrasterias. Further interpretations are related to the lateral flows and to invaginations of free cell membranes, to lateral filament motions, to the right-left problem, to the selfintertwining of filaments, to the rotation of a cell body by its flagellum, to the repulsion of chromatids during meiosis and to the tetragonal and hexagonal arrangement of filaments.Dedicated to Prof. DrLothar Geitler on the occasion of the 90th anniversary of his birthday.     

Structural aspects of bulge formation during root hair initiation

Using light and electron microscopy, the early stages of root hair initiation were investigated under control conditions and in a situation where F-actin polymerization was effectively inhibited by latrunculin B. Trichoblasts in their early stage of bulge formation possessed large vacuole traversed by cytoplasmic strands and enclosed within a narrow peripheral layer of cytoplasm. The nucleus was settled at the inner periclinal cell wall, typically opposite the site of bulge formation. Within the bulging area, dense cytoplasm and numerous ER elements, and other organelles were accumulated, together with pleiomorphic membrane-bound structures, the identity and nature of which will require further studies. These unusual structures, which were associated with the outer cell wall, contained material similar to that of the cell wall. Similar cell wall-like bodies were observed also in the cytoplasm and sometimes within vacuoles. The possible role of these novel organelles of plant cells in cell wall thinning/degradation or in the turgor pressure maintenance are discussed. Latrunculin B treatment allowed bulge formation but prevented the switch from the slow and diffuse expansion of bulge into the rapid tip-growth characteristic of the emerging root hair. Moreover, the cytoplasm of the bulging domain became extensively vacuolated and lacked abundant ER elements and other organelles including the membrane-bound structures. These results indicate important roles of F-actin in the switch from diffuse to highly polarized tip growth.     

Disturbance of endomembrane trafficking by brefeldin A and calyculin A reorganizes the actin cytoskeleton of <Emphasis Type="Italic">Lilium longiflorum</Emphasis> pollen tubes

We investigated the effect of brefeldin A on membrane trafficking and the actin cytoskeleton of pollen tubes of Lilium longiflorum with fluorescent dyes, inhibitor experiments, and confocal laser scanning microscopy. The formation of a subapical brefeldin A-induced membrane aggregation (BIA) was associated with the formation of an actin basket from which filaments extended towards the tip. The orientation of these actin filaments correlated with the trajectories of membrane material stained by FM dyes, suggesting that the BIA-associated actin filaments are used as tracks for retrograde transport. Analysis of time series indicated that these tracks (actin filaments) were either stationary or glided along the plasma membrane towards the BIA together with the attached membranes or organelles. Disturbance of the actin cytoskeleton by cytochalasin D or latrunculin B caused immediate arrest of membrane trafficking, dissipation of the BIA and the BIA-associated actin basket, and reorganization into randomly oriented actin rods. Our observations suggest that brefeldin A causes ectopic activation of actin-nucleating proteins at the BIA, resulting in retrograde movement of membranes not only along but also together with actin filaments. We show further that subapical membrane aggregations and actin baskets supporting retrograde membrane flow can also be induced by calyculin A, indicating that dephosphorylation by type 2 protein phosphatases is required for proper formation of membrane coats and polar membrane trafficking.     

Immunofluorescence evidence for cytoskeletal rearrangement accompanying pigment redistribution in goldfish xanthophores

Immunofluorescence and phase-contrast microscopic studies of goldfish xanthophores with aggregated or dispersed pigment show two unusual features. First, immunofluorescence studies with anti-actin show punctate structures instead of filaments. These punctate structures are unique for the xanthophores and are absent from both goldfish dermal non-pigment cells and a dedifferentiated cell line (GEM-81) derived from a goldfish xanthophore tumor. Comparison of immunofluorescence and phase-contrast microscopic images with electron microscopic images of thin sections and of Triton-insoluble cytoskeletons show that these punctate structures represent pterinosomes with radiating F-actin. The high local concentration of actin around the pterinosomes results in strong localized fluorescence such that, when the images have proper brightness for these structures, individual actin filaments elsewhere in the cell are too weak in their fluorescence to be visible in the micrographs. Second, whereas immunofluorescence images with anti-tubulin show typical patterns in xanthophores with either aggregated or dispersed pigment, namely, filaments radiating out from the microtubule organizing center, immunofluorescence images with anti-actin or with anti-intermediate filament proteins show different patterns in xanthophores with aggregated versus dispersed pigment. In cells with dispersed pigment, the punctate structures seen with anti-actin are relatively evenly distributed in the cytoplasm, and intermediate filaments appear usually as a dense perinuclear band and long filaments elsewhere in the cytoplasm. In cells with aggregated pigment, both intermediate filaments and pterinosomes with associated actin are largely excluded from the space occupied by the pigment aggregate, and the band of intermediate filaments surrounds not only the nucleus but also the pigment aggregate. The patterns of distribution of the different cytoskeleton components, together with previous results from this laboratory, indicate that formation of the pigment aggregate depends at least in part on the interaction between pigment organelles and microtubules. The possibility that intermediate filaments may play a role in the formation/stabilization of the pigment aggregate is discussed.     

Single microtubules from squid axoplasm support bidirectional movement of organelles

Single filaments, dissociated from the extruded axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, transport organelles bidirectionally. Organelles moving in the same or opposite directions along the same filament can pass each other without colliding, indicating that each transport filament has several tracks for organelle movement. In order to characterize transport filaments, organelle movements were first examined by video microscopy, and then the same filaments were examined by electron microscopy after rapid-freezing, freeze-drying, and rotary-shadowing. Transport filaments that supported bidirectional movement of organelles are 22 nm to 27 nm in diameter and have a substructure indicative of a single microtubule. Immunofluorescence showed that virtually all transport filaments contain tubulin. These results show that single microtubules can serve as a substratum for organelle movement, and suggest that an interaction between organelles and microtubules is the basis of fast axonal transport.     

Immunolocalization of actin in root statocytes of Lens culinaris L

Lentil root statocytes show a strict structural polarity of their organelles with respect to the g vector. These cells are involved in the perception of gravity and are responsible for the orientation of the root. Actin filaments take part in the positioning of their organelles and could also be involved in the transduction of the gravitropic signal. A pre-embedding immunogold silver technique was carried out with a monoclonal antibody in order to study the distribution of actin cytoskeleton in the statocytes at the electron microscopic level. Some areas were never labelled (cell wall, vacuole, nucleoplasm, mitochondria, starch grains of the amyloplasts) or very slightly labelled (stroma of the amyloplasts). The labelling was scattered in the cytoplasm always close to, or on the nuclear and amyloplast envelopes and the tonoplast. Associations of 2 to 6 dots in file were observed, but these short files were not oriented in one preferential direction. They corresponded to a maximum distance of 0.9 micron. This work demonstrated that each statocyte organelle was enmeshed in an actin web of short filaments arranged in different ways. The images obtained by rhodaminephalloidin staining were in accordance with those of immunogold labelling. The diffuse fluorescence of the cytoplasm could be explained by the fact that the meshes of the web should be narrow. The vicinity of actin and of the amyloplasts envelope could account for the movement of these organelles that was observed in spatial microgravity.     

Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body

We have investigated the relationship between lamellipodium protrusion and forward translocation of the cell body in the rapidly moving keratocyte. It is first shown that the trailing, ellipsoidal cell body rotates during translocation. This was indicated by the rotation of the nucleus and the movement of cytoplasmic organelles, as well as of exogenously added beads used as markers. Activated or Con A-coated fluorescent beads that were overrun by cells were commonly endocytosed and rotated with the internal organelles. Alternatively, beads applied to the rear of the cell body via a micropipette adhered to the dorsal cell surface and also moved forward, indicating that both exterior and underlying cortical elements participated in rotation. Manipulation of keratocytes with microneedles demonstrated that pushing or restraining the cell body in the direction of locomotion, and squeezing it against the substrate, which temporarily increased the intracellular pressure, did not effect the rate of lamellipodium protrusion. Rotation and translocation of the cell body continued momentarily after arrest of lamellipodium protrusion by cytochalasin B, indicating that these processes were not directly dependent on actin polymerization. The cell body was commonly flanked by phase-dense "axles," extending from the cell body into the lamellipodium. Phalloidin staining showed these to be comprised of actin bundles that splayed forward into the flanks of the lamellipodium. Disruption of the bundles on one side of the nucleus by traumatic microinjection resulted in rapid retraction of the cell body in the opposite direction, indicating that the cell body was under lateral contractile stress. Myosin II, which colocalizes with the actin bundles, presumably provides the basis of tension generation across and traction of the cell body. We propose that the basis of coupling between lamellipodium protrusion and translocation of the cell body is a flow of actin filaments from the front, where they are nucleated and engage in protrusion, to the rear, where they collaborate with myosin in contraction. Myosin-dependent force is presumably transmitted from the ends of the cell body into the flanks of the lamellipodium via the actin bundles. This force induces the spindle-shaped cell body to roll between the axles that are created continuously from filaments supplied by the advancing lamellipodium.     

Mechanotransduction in root gravity sensing cells

The analysis of the dose-response curve of the gravitropic reaction of lentil seedling roots has shown that these organs are more sensitive when they have been grown in microgravity than when they have been grown on a 1 g centrifuge in space before gravistimulation. This difference of gravisensitivity is not due to the volume or the density of starch grains of statoliths, which are about the same in both conditions (1 g or microgravity). However, the distribution of statoliths within the statocyte may be responsible for this differential sensitivity, since the dispersion of these organelles is greater in microgravity than in 1 g. When lentil roots grown in microgravity or in 1 g are stimulated at 0.93 g for 22 min, the amyloplasts sediment following two different trajectories. They move from the proximal half of the statocytes toward the lower longitudinal wall in the microgravity grown sample and from the distal half toward the longitudinal wall in the 1 g grown sample. At the end of the stimulation, they reach a similar position within the statocytes. If the roots of both samples are left in microgravity for 3 h, the amyloplasts move toward the cell centre in a direction that makes an average angle of 40 degrees with respect to the lower longitudinal wall. The actin filaments, which are responsible for this movement, may have an overall orientation of 40 degrees with respect to this wall. Thus, when roots grown in microgravity are stimulated on the minicentrifuge the amyloplasts slide on the actin filaments, whereas they move perpendicular to them in 1 g grown roots. Our results suggest that greater sensitivity of seedling roots grown in microgravity should be due to greater dispersion of statoliths, to better contacts between statoliths and the actin network and to greater number of activated mechanoreceptors. One can hypothesize that stretch activated ion channels (SACs) located in the plasma membrane are responsible for the transduction of gravistimulus. These SACs may be connected together by elements of the cytoskeleton lining the plasma membrane and to the actin filaments. They could be stimulated by the action of statoliths on the actin network and/or on these elements of the cytoskeleton which link the mechanoreceptors (SACs).     

Movement of organelles along filaments dissociated from the axoplasm of the squid giant axon

Cytoplasmic filaments, separated from the axoplasm of the squid giant axon and visualized by video-enhanced differential interference contrast microscopy, support the directed movement of organelles in the presence of ATP. All organelles, regardless of size, move continuously along isolated transport filaments at 2.2 +/- 0.2 micron/sec. In the intact axoplasm, however, movements of the larger organelles are slow and saltatory. These movements may reflect a resistance to movement imposed by the intact axoplasm. The uniform rate of all organelles along isolated transport filaments suggests that a single type of molecular motor powers fast axonal transport. Organelles can attach to and move along more than one filament at a time, suggesting that organelles have multiple binding sites for this motor.     

Short-range and long-range context effects on coliphage T4 endonuclease II-dependent restriction.

Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency. The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage. The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently. This, along with the effects of substitutions at one or two of the less-conserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence. The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced. This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region. Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent. Models and possible biological implications of these findings are discussed.     

Fine structure of the rhombencephalic tela of the bullfrog,Rana catesbeiana

Summary The posterior rhombencephalic tela choroidea of the bullfrog was examined by electron microscopy. This membrane, the pia-ependymal roof of the caudal hindbrain, contains a large central region characterized by cuboidal ependymal cells which surround sizable microscopic apertures — the interependymal pores.Ultrastructurally ependymal cells of this area are characterized by infrequent apical microvilli and cilia. They contain irregularly shaped nuclei and few cytoplasmic organelles that are largely apical in position. The most striking feature is an abundance of cytoplasmic filaments forming an extensive cytoskeleton. Laterally these cells are joined by numerous elaborate desmosomes. The majority of the ependymal cells have a basal lamina consisting of single, double, or triple laminae lying parallel to the basal plasma membrane.Several unusual specializations are seen at the margins of the interependymal pores. The ependymal cells have lateral cytoplasmic processes that form the actual border of each pore. These processes originate from the apical surface of the cell and partially enclose an elaborate network of basal lamina associated with the interependymal pores.These findings demonstrate microscopic apertures in the roof of the fourth ventricle in the bullfrog that are associated with an unusual form of supportive ependyma.     

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.