Actin versus tubulin configuration in arbuscule-containing cells from mycorrhizal tobacco roots

The involvement of the cytoskeleton in symbiotic interactions such as arbuscular mycorrhizas has received little attention. In this paper, we examine the organization of actin in tobacco mycorrhizal roots and compare actin and tubulin patterns within arbuscule-containing cells.
Our results show drastic reorganization of microfilaments and microtubules upon fungal infection and how those new cytoskeletal patterns relate to the host cytoplasm rearrangement and the intracellular fungal structures. Whereas in uninfected cells a network of cortical and perinuclear actin filaments was observed, in infected cells actin filaments closely follow the fungal branches and envelop the whole arbuscule in a dense coating network. Microtubules are less closely connected with the fungus surface. They run across the whole arbuscule mass, linking branches to each other and to the host cell cortex and nucleus.
These major differences between the two cytoskeletal components are used to advance some suggestions concerning their contribution to structural functions in the plant–fungus interactions during the mycorrhizal symbiosis.     

Model for the alignment of actin filaments in endothelial cells subjected to fluid shear stress

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article. we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.     

Cytoskeletal arrangement and its intercellular connec-tion in wheat young leaf cells

By using the techniques of partial digestion of cell wall and selective extraction,we examined the cytoskeleton of wheat yong leaf cells under scanning electron microscope(SEM).A 3-dimensional cytoskeletal system,showing some new features,was observed.The cortical network located beneath the cross wall was an anastomosing organization.The association of nucleus with the cell wall by some skeletal filaments was also found.It is notice able that there were cytoskeletal filaments,which passed through cell wall and connected together with cytoskeletal arrays of adjacent cells,Thus,it is possible that an integral skeletal network existed within the yong leaf tissue of wheat.     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Scanning Electron Microscopic Observations of Cytoskeletal Arrangement in Wheat Young Leaf Cells and Its Intercellular Connection

By using the techniques of partial digestidon of cell wall and selective extraction authors examined the cytoskeleton in wheat (Triticum aestivum L.) young leaf cells under scanning electron microscope. A three-dimensional cytoskeletal system showing some new features was observed. The cortical network locating beneath the cross wall is an anastomosing organization. Association between nucleus and cell wall by omc skeletal filaments was observed. It is noticeable that there are cytoskeletal filaments, which pass through cell wall, combine adjacent cells cytoskeletal arrays together. Thus it is po sible that an integral skeletal network exist within wheat young leaf tissue.     

Neurofilament protein synthesis and phosphorylation

Neurofilament proteins, a major intermediate filament component of the neuronal cytoskeleton, are organized as 10 nm thick filaments in axons and dendrites. They are large, abundantly phosphorylated proteins with numerous phosphate acceptor sites, up to 100 in some cases, organized as numerous repeat motifs. Together with other cytoskeletal components such as microtubules, MAPs, actin and plectin-like linking molecules, they make up a dynamic lattice that sustains neuronal function from neuronal “birthday” to apoptotic cell death. The activity of the neuronal cytoskeleton is regulated by phosphorylation, dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Factors regulating multisite phosphorylation of NFs are topographically localized, with maximum phosphorylation of NF proteins consigned to axons. Phosphorylation defines the nature of NF interactions with one another and with other cytoskeletal components such as microtubules, MAPs and actin. To understand how these functional interactions are regulated by phosphorylation we attempt to identify the relevant kinases and phosphatases, their specific targets and the factors modulating their activity. As an initial working model we propose that NF phosphorylation is regulated topographically in neurons by compartment-specific macromolecular complexes of substrates, kinases and phosphatases. This implies that axonal complexes differ structurally and functionally from those in cell bodies and dendrites. Such protein assemblies, by virtue of conformational changes within proteins, facilitate ordered, sequential multisite phosphorylations that modulate dynamic cytoskeletal interactions.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Cytoskeleton and plant salt stress tolerance

The plant cytoskeleton is a highly dynamic component of plant cells and mainly based on microtubules (MTs) and actin filaments (AFs). The important functions of dynamic cytoskeletal networks have been indicated for almost every intracellular activity, from cell division to cell movement, cell morphogenesis and cell signal transduction. Recent studies have also indicated a close relationship between the plant cytoskeleton and plant salt stress tolerance. Salt stress is a significant factor that adversely affects crop productivity and quality of agricultural fields worldwide. The complicated regulatory mechanisms of plant salt tolerance have been the subject of intense research for decades. It is well accepted that cellular changes are very important in plant responses to salt stress. Because the organization and dynamics of cytoskeleton may play an important role in enhancing plant tolerance through various cell activities, study on salt stress-induced cytoskeletal network has been a vital topic in the subject of plant salt stress tolerance mechanisms. In this article, we introduce our recent work and review some current information on the dynamic changes and functions of cytoskeletal organization in response to salt stress. The accumulated data point to the existence of highly dynamic cytoskeletal arrays and the activation of complex cytoskeletal regulatory networks in response to salt stresses. The important role played by cytoskeleton in mediating the plant cell''s response to salt stresses is particularly emphasized.Key words: cytoskeleton, microtubules (MTs), microfilaments (MFs), salt stress, response mechanisms, plant tolerance     

Cytoskeletal mechanisms of neuronal morphogenesis

The cytoskeleton is the major intracellular structure that determines the morphology of a neuron. Thus, mechanisms that ensure a precisely regulated assembly of cytoskeletal elements in time and space have an important role in the development from a morphologically simple neuronal precursor cell to a complex polarized neuron that can establish contacts to several hundreds of other cells. Here, cytoskeletal mechanisms that underlie the formation of neurites, directed elongation and stabilization of neuronal processes are summarized. It has become evident that different cytoskeletal elements are highly crosslinked with each other by several classes of specific linker proteins. Of these, microtubule-associated proteins (MAPs) appear to have an important role in connecting the microtubule skeleton to other cytoskeletal filaments and plasma membrane components during neuronal morphogenesis. Future experiments will have to elucidate the function and the regulation of the neuronal cytoskeleton in an authentic nervous system environment during development. Recent approaches are discussed at the end of this article.     

Subpopulations of tau interact with microtubules and actin filaments in various cell types

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-2, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP-2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.     

Modeled microgravity causes changes in the cytoskeleton and focal adhesions, and decreases in migration in malignant human MCF-7 cells

Because cells are sensitive to mechanical forces, microgravity might act on stress-dependent cell changes. Regulation of focal adhesions (FAs) and cytoskeletal activity plays a role in cell maintenance, cell movement, and migration. Human MCF-7 cells were exposed to modeled microgravity (MMG) to test the hypothesis that migration responsiveness to microgravity is associated with cytoskeleton and FA anomalies. MMG acts on MCF-7 cells by disorganizing cytoskeleton filaments (microfilaments and microtubules). Microfilaments in MMG did not display their typical radial array. Likewise, microtubules were disrupted in MCF-7 cells within 4 h of initiation of MMG and were partly reestablished by 48 h. FAs generated in microgravity were less mature than those established in controls, shown by reduced FAs number and clustering. In parallel, MMG decreased kinases activity (such as FAK, PYK2, and ILK) of FAs in MCF-7 cells. The expression of both integrinβ1 and integrinβ4 were downregulated by MMG. We conclude that cytoskeletal alterations and FAs changes in MMG are concomitant with cell invasion and migration retardation. We suggest that reduced migration response in MCF-7 cells following MMG is linked to changes of cytoskeleton and FAs.     

Cutting edge: integration of human T lymphocyte cytoskeleton by the cytolinker plectin.

Chemokine-induced polarization of lymphocytes involves the rapid collapse of vimentin intermediate filaments (IFs) into an aggregate within the uropod. Little is known about the interactions of lymphocyte vimentin with other cytoskeletal elements. We demonstrate that human peripheral blood T lymphocytes express plectin, an IF-binding, cytoskeletal cross-linking protein. Plectin associates with a complex of structural proteins including vimentin, actin, fodrin, moesin, and lamin B in resting peripheral blood T lymphocytes. During chemokine-induced polarization, plectin redistributes to the uropod associated with vimentin and fodrin; their spatial distribution indicates that this vimentin-plectin-fodrin complex provides a continuous linkage from the nucleus (lamin B) to the cortical cytoskeleton. Overexpression of the plectin IF-binding domain in the T cell line Jurkat induces the perinuclear aggregation of vimentin IFs. Plectin is therefore likely to serve as an important organizer of the lymphocyte cytoskeleton and may regulate changes of lymphocyte cytoarchitecture during polarization and extravasation.     

The cytoskeletal framework and poliovirus metabolism

The cytoskeletal framework prepared by detergent lysis of suspension-grown HeLa cells is compared to the structure obtained from poliovirus-infected cells. This framework, which retains major features of cell morphology and carries the cellular polyribosomes as well as the major structural filaments, is profoundly reorganized following virus infection. This reorganization underlies, at least in part, the morphological changes termed the “cytopathic effect.” These cytoskeletal changes appear related to the involvement of the framework with viral-specific metabolism.Extensive cytoskeleton alterations occur even when guanidine inhibits viral replication, and thus result from small amounts of early viral products. The normally spheroidal nucleus deforms, allowing a modified region of the cytoplasm to occupy a central position in the cell, and many membrane-enclosed vesicles peculiar to the infected cell are elaborated here. The skeleton preparation reveals that this region contains intermediate filaments arranged in a pattern unique to infected cells. Further changes occur when viral replication is permitted. The central region filaments become coated with darkly staining material which may be viral RNA. Numerous small particles appear on the filaments which resemble partially assembled virions. Mature virions, however, have no affinity for the cytoskeleton and appear to be free in the cytoplasm.Host cell messenger RNA, normally attached to the skeletal framework, is released in infected cells and is replaced by the viral-specific polyribosomes. The trabecular network which carries polyribosomes appears to be rearranged; the viral polyribosomes are located principally at the cell periphery and are excluded from the central region. The viral replication complex with its double-stranded RNA is also attached to the skeletal framework and may comprise the dark staining material coating the filaments of the central cell region.     

Cytoskeletal structure of myoblasts with the mitochondrial DNA 3243A-->G mutation and of osteosarcoma cells with respiratory chain deficiency

The cytoskeleton, mainly composed of actin filaments, microtubules, and intermediate filaments, is involved in cell proliferation, the maintenance of cell shape, and the formation of cellular junctions. The organization of the intermediate filaments is regulated by phosphorylation and dephosphorylation. We examined cell population growth, apoptotic cell death, and the morphology of cytoskeletal components in myoblast cultures derived from patients with the 3243A-->G mutation in mitochondrial DNA (mtDNA) and from control subjects by means of assays detecting cellular nucleic acids, histone-associated DNA fragments and by immunolabeling of cytoskeletal components. Population growth was slower in the 3243A-->G myoblast cultures, with no difference in the amount of apoptotic cell death. The organization of vimentin filaments in myoblasts with 3243A-->G was disturbed by randomization of filament direction and length, whereas no disturbances were observed in the other cytoskeletal proteins. Vimentin filaments formed large bundles surrounding the nucleus in mtDNA-less (rho(0)) osteosarcoma cells and in osteosarcoma cells after incubation with sodium azide and nocodazole. We conclude that defects in oxidative phosphorylation lead to selective disruption of the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases.     

Structural interaction of cytoskeletal components

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament “branches” are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the “microtrabecular network” (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).     

The cytoskeleton and epidermal morphogenesis in C. elegans

During Caenorhabditis elegans development, the process of epidermal elongation converts the bean-shaped embryo into the long thin shape of the larval worm. Epidermal elongation results from changes in the shape of epidermal cells, which in turn result from changes in the epidermal cytoskeleton, the extracellular matrix, and in cell-matrix adhesion junctions. Here, we review the roles of cytoskeletal filament systems in epidermal cell shape change during elongation. Genetic and cell biological analyses have established that all three major cytoskeletal filament systems (actin microfilaments, microtubules, and intermediate filaments (IFs)) play distinct and essential roles in epidermal cell shape change. Recent work has also highlighted the importance of communication between these systems for their integrated function in epidermal elongation. Epidermal cells undergo reciprocal interactions with underlying muscle cells, which regulate the position and function of IF-containing cell-matrix adhesion structures within the epidermis. Elongation thus exemplifies the reciprocal tissue interactions of organogenesis.     

Actin and Microtubules in Cell Motility: Which One is in Control?

The cytoskeleton is composed of three distinct elements: actin microfilaments, microtubules and intermediate filaments. The actin cytoskeleton is thought to provide protrusive and contractile forces, and microtubules to form a polarized network allowing organelle and protein movement throughout the cell. Intermediate filaments are generally considered the most rigid component, responsible for the maintenance of the overall cell shape. Cytoskeletal elements must be coordinately regulated for the cell to fulfill complex cellular functions, as diverse as cell migration, cell adhesion and cell division. Coordination between cytoskeletal elements is achieved by signaling pathways, involving common regulators such as the Rho guanosine-5'-triphosphatases (GTPases). Furthermore, evidence is now accumulating that cytoskeletal elements participate in regulating each other. As a consequence, although their functions seem well defined, they are in fact overlapping, with actin playing a role in membrane trafficking and microtubules being involved in the control of protrusive and contractile forces. This cytoskeletal crosstalk is both direct and mediated by signaling molecules. Cell motility is a well-studied example where the interplay between actin and microtubules appears bidirectional. This leads us to wonder which, if any, cytoskeletal element leads the way.     

Modulation of milk protein synthesis through alteration of the cytoskeleton in mouse mammary epithelial cells cultured on a reconstituted basement membrane

Recent studies indicate that the cytoskeleton may be involved in modulating tissue-specific gene expression in mammalian cells. We have studied the role of the cytoskeleton in regulating milk protein synthesis and secretion by primary mouse mammary epithelial cells cultured on a reconstituted basement membrane that promotes differentiation. After 8 days in culture, cells were treated with cytochalasin D (CD) (0.5-1 micrograms/ml) to alter actin filaments or acrylamide (Ac) (5 mM) to alter intermediate filaments (cytokeratins). CD inhibited synthesis of most proteins in a concentration-dependent manner, with beta-casein being the first affected. In contrast, Ac increased protein synthesis and secretion by 17-31% after a 12 hr treatment. Polyacrylamide gel electrophoresis of total secreted proteins indicates that synthetic rates of most proteins were increased equally by Ac treatment. This increase is apparently controlled at the level of translation, because control and Ac-treated cells contained the same amount of poly-A+ RNA, and neither CD nor Ac altered mRNA levels for beta-casein. There was also no indication that either CD or Ac can induce the expression of milk proteins in quiescent cells cultured on a plastic substratum. In conjunction with the biochemical studies, changes in cytoskeletal morphology caused by the drug treatments were analyzed by immunofluorescence microscopy. As has been observed in other cell types, low concentrations of CD caused cells to round up by disrupting actin filaments. Ac treatment slightly decreased the intensity of actin staining, but no changes in microfilament organization were observed. Ac-treated cells showed slight disorganization of the cytokeratin filaments, with some peripheral interfibrillar bundling, but the cytokeratin network did not collapse and no retraction of cell extensions or breakdown of cell-cell contacts was observed. These results confirm previous reports that the actin cytoskeleton may play a role in regulating tissue-specific protein synthesis. How Ac stimulates protein synthesis is unknown, but it is unlikely that this effect is directly mediated through intermediate filaments.     

Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments

The progressive cytoskeletal alterations of frog virus 3-infected baby hamster kidney (BHK) and fathead minnow (FHM) cells were studied by immunofluorescence and electron microscopy. The virus assembly sites, which contain viral genomes and viral proteins, were detected in the cytoplasm at 4 h (FHM) or 6 h (BHK) and mature virions appeared 2 h later. When infected cells were treated with Triton X-100, the assembly sites were found in association with the cytoskeleton. In infected cells, the number of microtubules progressively decreased but a few microtubules traversing in the vicinity of the assembly sites remained intact. Early in infection, the intermediate filaments retracted from the cell periphery, delimited the forming assembly sites, and remained there throughout infection. We suggest that intermediate filaments are involved in the formation of assembly sites. In addition, the filaments either by themselves or in conjunction with microtubules may anchor the assembly sites near the nucleus. The microfilament bundles (stress fibers) disappeared with the formation of assembly sites, and late in infection many projections containing microfilaments and virus particles appeared at the cell surface. The observation suggests a role for microfilaments in virus release. Taken together, these results provide the first example of a virus-infected cell in which all three cytoskeletal filaments show profound organizational changes and suggest an active participation of the host cytoskeleton in viral functions.     

Differential effects of calcium deprivation on the cytoskeleton of non-tumorigenic and tumorigenic rat liver cells in culture

Normal rat liver T51B epithelial cells and Morris no. 7795 hepatoma cells growing exponentially were exposed for 24 h to standard medium containing low (0.02 mM) calcium, a concentration which drastically reduces the proliferation of normal but not tumour cells. Cell surface morphology was examined by scanning electron microscopy (SEM); and the distribution and organization of microtubules, cytokeratin and vimentin filaments, and microfilaments were analysed by indirect immunofluorescence microscopy using specific antibodies. Calcium deprivation caused the loss of intercellular cohesion in both cell types and the appearance of some microvilli and blebs, particularly on tumour cells. However, marked differential (normal vs tumour cells) effects on the organizational integrity of the cytoskeleton fibrillar network were observed. Extracellular calcium deprivation led to a particular rearrangement of microtubules, and a perinuclear accumulation of cytokeratin and vimentin filaments in normal, but not in tumour cells. A massive concentration of actin-containing microfilaments was observed in the cell periphery and blebs of hepatoma cells. In the light of the possible involvement of calcium in controlling cytoskeleton assembly, the differing cytoskeletal changes of the two cell types may be linked to their diffferent proliferative capabilities in low-calcium medium.