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).     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.     

Structural interactions of actin filaments and endoplasmic reticulum in honeybee photoreceptor cells

Summary Fluorescent phallotoxins and heavy meromyosin were used to reveal the organization of the actin cytoskeleton in honeybee photoreceptor cells, and the relationship of actin filaments to the submicrovillar, palisade-like cisternae of the endoplasmic reticulum (ER). Bundles of unipolar actin filaments (pointed end towards the cell center) protrude from the microvillar bases and extend through cytoplasmic bridges that traverse the submicrovillar ER. Within the cytoplasmic bridges, the filaments are regularly spaced and tightly apposed to the ER membrane. In addition, actin filaments are deployed close to the microvillar bases to form a loose web. Actin filaments are scarce in cell areas remote from the rhabdom; these areas contain microtubule-associated ER domains. The results suggest that the actin system of the submicrovillar cytoplasm shapes the submicrovillar ER cisternae, and that the distinct ER domains interact with different cytoskeletal elements.     

Immunoelectron microscopic studies of intermediate filaments in cultured cells

Indirect immunoferritin labelling has been used to show the localization of the 57 000 D cytoskeletal protein vimentin in intermediate filaments. Labelling could be shown using either detergent-extracted cytoskeletons prepared from unfixed cultured cells or cells subjected to light fixation and rendered permeable to antibodies by treatment with saponin. Immunoferritin and immunoperoxidase labelling methods were combined with stereo electron microscopy to describe the three-dimensional organization of the intermediate filament system in cultured cells of fibroblastic morphology. The filaments form a complex three-dimensional network throughout the cytoplasm and are frequently found to be associated with microfilament bundles near the upper and lower plasma membranes. The filaments also surround the nucleus and may therefore play a role in anchoring this organelle in the cytoplasm.     

Cytoskeletal elements in migrating and tentacle-integrated nematocytes of a marine hydrozoan

Summary— Actively migrating nematocytes of the marine polyp Stauridiosarsia producta are converted into completely immotile cells as soon as they become integrated in the ectodermal tissue of the tentacles. Immunocytochemical and electron microscopical methods revealed that cytoskeletal elements composed of actin, tubulin, centrin and a still unidentified protein are interwoven within the complete cell. While the organization of the sensory pole of the nematocyte, containing the cnidocil complex, the pseudovillar system and the distal half of a microtubular basket surrounding the nematocyst, is not affected by the transition from a motile to an immotile cell, the cytoskeletal elements in the basal portion of the cell are re-organized. Thus, the basolateral cytoplasm of migrating cells contains less organized microtubular arrays and bundles of about 10 nm-thick filaments. In the tentacle-integrated state, the 10-nm filaments are concentrated within a stalk-like foot which is stabilized by some rigid microtubular arrays derived from the microtubular basket. By elongation of the microtubular basket towards the cellular basis, the nematocyst becomes indirectly anchored at the mesoglea. As indicated by pharmacological treatments, the stiffness of the stalk depends on its microtubular content only.     

Ultrastructure and function of nurse cells in phthirapterans. Possible function of ramified nurse cell nuclei in the cytoplasm transfer

The structure of nurse cells as well as the distribution of cytoskeletal elements (actin filaments, microtubules) in three representatives of phthirapterans: the pig louse, Haematopinus suis (Anoplura) and bird lice, Eomenacanthus stramineus, Columbicola columbae (Mallophaga) were investigated. All three species have polytrophic-meroistic ovaries which means that each oocyte remains connected with a group of nurse cells via specialized cytoplasmic canals-intercellular bridges (ring canals). Throughout vitellogenesis, various macromolecules as well as organelles (mitochondria, endoplasmic reticulum vesicles, ribosomes) are transferred from the nurse cells to the oocyte. During this flow, the nurse cell nuclei do not enter the oocyte and are retained in the cell centers. In holometabolous insects (e.g. Drosophila, hymenopterans), the central position of nurse cell nuclei is maintained by cytoskeletal elements (actin filaments or microtubules). In the investigated species, the nurse cells are equipped with large, highly extended (irregularly lobed) nuclei. The inner nuclear membrane is lined with a relatively thick layer of nuclear lamina. Ultrastructural analysis and staining with rhodamine-labeled phalloidin revealed that the nurse cell cytoskeleton is poorly developed and represented only by: (1) single microtubules in the perinuclear cytoplasm; and (2) the F-actin layer in the cortical cytoplasm. In the light of this, we postulate that in phthirapterans the position of nurse cell nuclei during the cytoplasm transfer is maintained not by the cytoskeletal elements, but by a largely extended shape of the nuclei (i.e. their elongated extensions).     

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.     

Regulated Expression of Human Filaggrin in Keratinocytes Results in Cytoskeletal Disruption, Loss of Cell–Cell Adhesion, and Cell Cycle Arrest

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.     

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.     

Statistics of active transport in Xenopus melanophores cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.     

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland : Expression of cytokeratins and desmosomal plaque proteins in unusual arrays

Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed.     

The cortical cytoskeletal network and cell-wall dynamics in the unicellular charophycean green alga Penium margaritaceum

Background and Aims

Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division.

Methods

Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy.

Key Results

The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division.

Conclusions

The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.     

Breaking the WAVE complex: the point of Arabidopsis trichomes

Actin filaments comprise an essential cytoskeletal array that organizes the cytoplasm during growth and cell division. In growing cells, actin filaments carry out many functions. Actin filaments position the endomembrane system and act as a substrate on which organelle motility occurs. Other actin-filament arrays appear to be more dynamic and to reorganize in response to growth signals and external cues. The diverse cellular functions of the actin cytoskeleton are mediated by actin-binding proteins that nucleate, destabilize, and bundle actin filaments. The distorted trichome morphology mutants provide a simple genetic system in which to study mechanisms of actin-dependent morphogenesis. Recent results from several groups indicate that 'distorted group' genes encode subunits of the actin-related protein (Arp)2/3 and WAVE complexes, and function in a cell morphogenesis pathway.     

The cytoskeletal and contractile apparatus of smooth muscle: contraction bands and segmentation of the contractile elements

Confocal laser scanning microscopy of isolated and antibody-labeled avian gizzard smooth muscle cells has revealed the global organization of the contractile and cytoskeletal elements. The cytoskeleton, marked by antibodies to desmin and filamin is composed of a mainly longitudinal, meandering and branched system of fibrils that contrasts with the plait-like, interdigitating arrangement of linear fibrils of the contractile apparatus, labeled with antibodies to myosin and tropomyosin. Although desmin and filamin were colocalized in the body of the cell, filamin antibodies labeled additionally the vinculin- containing surface plaques. In confocal optical sections the contractile fibrils showed a continuous label for myosin for at least 5 microns along their length: there was no obvious or regular interruption of label as might be expected for registered myosin filaments. The cytoplasmic dense bodies, labeled with antibodies to alpha-actinin exhibited a regular, diagonal arrangement in both extended cells and in cells shortened in solution to one-fifth of their extended length: after the same shortening, the fibrils of the cytoskeleton that showed colocalization with the dense bodies in extended cells became crumpled and disordered. It is concluded that the dense bodies serve as coupling elements between the cytoskeletal and contractile systems. After extraction with Triton X-100, isolated cells bound so firmly to a glass substrate that they were unable to shorten as a whole when exposed to exogenous Mg ATP. Instead, they contracted internally, producing integral of 10 regularly spaced contraction nodes along their length. On the basis of differences of actin distribution two types of nodes could be distinguished: actin-positive nodes, in which actin straddled the node, and actin-negative nodes, characterized by an actin-free center flanked by actin fringes of 4.5 microns minimum length on either side. Myosin was concentrated in the center of the node in both cases. The differences in node morphology could be correlated with different degrees of coupling of the contractile with the cytoskeletal elements, effected by a preparation-dependent variability of proteolysis of the cells. The nodes were shown to be closely related to the supercontracted cell fragments shown in the accompanying paper (Small et al., 1990) and furnished further evidence for long actin filaments in smooth muscle. Further, the segmentation of the contractile elements pointed to a hierarchial organization of the myofilaments governed by as yet undetected elements.     

Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro

Fibroblastic cells of human origin have a limited lifespan in culture. One of the senescence-associated phenotypic changes is an increase in the abundance of cytoplasmic filaments. Human skin fibroblasts (strain 0011), derived from an 8-week-old male fetus, were passaged according to a predetermined schedule and examined at successive population doubling levels. In young rapidly growing cultures, fluorescence microscopy with NBD-Phallacidin shows a normal organization of the actin-containing fibers, microtubules and intermediate filaments, as has been described previously. At stages close to the end of the in vitro lifespan of the cell strain, large flat fibroblasts are the predominant cell type in culture. These large senescent fibroblasts contain numerous prominent actin fibers traversing the entire long axis of the cytoplasm. The fibers are often located adjacent to each other and appear to form a sheet on the ventral side of the cytoplasm. Staining of senescent cells with anti-tubulin antibody reveals an increase in the abundance of microtubules per cell and the distribution pattern is altered through the increase in the number of organization centers. Intermediate filaments are also more abundant and display tightly packed fibrillar sheets or bundles. Electron microscopic studies have confirmed the increased organization of microfilaments into bundles in senescent cells. These results suggest that during in vitro senescence, the increase in cell size is correlated with increased organization of the cytoskeleton. The presence of a rigid cytoskeletal structure may contribute in part to the inability of the senescent cell to replicate.     

Three-dimensional study of epithelioid cells by a quick-freezing and deep-etching method in muramyl dipeptide-induced granulomas

The three-dimensional ultrastructure of epithelioid cells was studied by the quick-freezing and deepetching (QF-DE), as well as the freeze-substitution (QF-FS) methods. The granulomas were induced in rats by injecting muramyl dipeptide (MDP) into the hind footpads. At 3 weeks after the injection, the footpads were perfused with a fixative, excised, and quickly frozen to prepare the replica membranes. Some unfixed footpads were also quickly frozen and freeze-substituted. Dense networks of intermediate filaments, connected with the nuclei, mitochondria and other vesicular cell organelles, were observed throughout the cytoplasm of epithelioid cells by the QF-DE method. A few actin filaments were located in filopodia and just beneath the cell membranes. Interdigitation of the cell membranes between adjacent cells was clearly demonstrated by the QF-FS method and clathrin-coated pits were identified at the base of interdigitating filopodia. In addition, the exact moment of fusion between endosomes and lysosomes was ascertained by the same method. These results suggest that the cytoskeletal organization of epithelioid cells resembles that of epithelial cells rather than actively motile macrophages.     

Entamoeba histolytica: fibrilar aggregates in dividing trophozoites

Entamoeba histolytica trophozoite cytokinesis is dependent upon cytoskeletal elements such as filamentous actin and myosin. Here we present confocal and transmission electron microscopy studies of this process. A sequence in the formation of the contractile ring was shown with rhodamine-phalloidine staining. Ultrastructural analysis revealed the presence of fibrilar aggregates in the cytoplasm of dividing trophozoites. Among them two filaments of different diameter were identified. These aggregates presented repeating assemblies of thin and thick filaments that in cross section revealed a muscle-like appearance. Our results suggest that these aggregates constitute the contractile ring responsible for the separation of daughter cells.     

Controlling cytoskeleton structure by phosphoinositide-protein interactions: phosphoinositide binding protein domains and effects of lipid packing

Cell movement and resistance to mechanical forces are largely governed by the cytoskeleton, a three-dimensional network of protein filaments that form viscoelastic networks within the cytoplasm. The cytoskeleton underlying the plasma membrane of most cells is rich in actin filaments whose assembly and disassembly are regulated by actin binding proteins that are stimulated or inhibited by signals received and transmitted at the membrane/cytoplasm interface. Inositol phospholipids, or phosphoinositides, are potent regulators of many actin binding proteins, and changes in the phosphorylation of specific phosphoinositide species or in their spatial localization are associated with cytoskeletal remodeling in vitro. This review will focus on recent studies directed at defining the structural features of phosphoinositide binding sites in actin binding proteins and on the influence of the physical state of phosphoinositides on their ability to interact with their target proteins.