In vitro formation of filaments from calf brain microtubule protein.

Electron micrographs of filaments formed by purified calf brain tubulin show structures with a uniform cross-section diameter of 29 ± 3 nm, similar to that of native microtubules. Exposure to low temperature led to the disappearance of the filaments.     

10 nm filaments in normal and transformed cells.

An antibody was raised against an electrophoretically homogeneous protein from cultured fibroblasts and shown to be directed against 10 nm filaments. The antiserum did not stain microtubules or actin microfilaments. The distribution of 10 nm filaments in normal cells was studied during growth, spreading, locomotion, mitosis, and after treatment with colchicine and cytochalasin B. The 58,000 dalton subunit protein is apparently all polymerized in the filaments which are insoluble in nonionic detergent. The distribution of 10 nm filaments is altered by colchicine treatments which disrupt microtubules. The organization of 10 nm filaments is altered in transformed cells.     

The cytoskeleton of cryofixed Purkinje cells of the chicken cerebellum

Summary Cerebella of 3- to 6-week-old chickens were cryofixed in a nitrogen-cooled propane jet, deep-etched and rotary-shadowed. The use of a brief perfusion of 0.32 M sucrose improved the quality of the cryofixation and allowed the study of the deeper layers of the cerebellar cortex. It is reported that the cytoskeleton of the Purkinje cells (PC) shows distinct domains and composition of filamentous structures in the different regions of the cell cytoplasm, such as the perikaryon, the cytoplasm of dendrites and the axoplasm. The perikaryon is occupied by a meshwork of fine filaments, 4–7 nm in diameter, that extends from the nuclear outer membrane to the cell membrane. In this zone the cell organelles (e.g., endoplasmic reticulum, mitochondria) adopt a circular arrangement around the nucleus. All structures are anchored by microfilaments to the cytoplasmic network. The dendrites show a dense cytoplasmic network including bundles of microtubules, neurofilaments and microfilaments. Numerous aggregated globular components are attached to this cytoskeleton. The cytoskeleton of the dendritic spines shows axially oriented 10-nm bundles of filaments, which are interconnected and anchored also to the cell membrane and the components of the agranular endoplasmic reticulum by cross-linkers. As described in peripheral nerves, the axoplasm of axons in the central nervous system exhibits predominantly neurofilaments and microtubules aligned along the axis of the neuntes in a three-dimensional arrangement and interconnected by cross-linker filaments and filamentous structures.     

Organization of filaments underneath the plasma membrane of developing chicken skeletal muscle cells in vitro revealed by the freeze-dry and rotary replica method

Summary Cytoskeletal organization and its association with plasma membranes in embryonic chick skeletal muscle cells in vitro was studied by the freeze-drying and rotary-shadowing method of physically ruptured cells. The cytoskeletal filaments underlying the plasma membranes were sparse in myogenic cells at the stage when cells exhibited great lipid fluidity in plasma membranes (fusion competent mononucleated myoblasts and recently fused young myotubes). Myotubes at more advanced stages of development possessed a highly interconnected dense filamentous network just underneath the cell membrane. This subsarcolemmal network was composed predominantly of 8–10 nm filaments; they were identified as actin filaments because of their decoration with myosin subfragment-1. Fine fibrils having a diameter of 3–5 nm were found on the protoplasmic surface of the plasmalemma at both the early and advanced stages of development. They were associated with the subsarcolemmal cytoskeletal filaments. Short 2–5 nm cross-linking filaments were occasionally seen between filaments in the subsarcolemmal network. We conclude that, although the subsarcolemmal cytoskeletal network contains many actin filaments, this domain appears to play some role in preserving the cell shape in the form of the membrane skeleton rather than membrane mobility.     

Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves

Changes in the population of microtubules and filaments within the cytoplasm of maturing axons and astrocytes have been studied during the early postnatal development of rat optic nerves. At birth, all of the axons are unmyelinated; most have a diameter of 0.2–0.3 µ and contain 4–10 microtubules. Neurofilaments do not occur with any frequency until about 5 days postnatal when they appear as individual groups, each containing 4–12. Subsequently, the neurofilaments of each group disperse so that they become more evenly distributed in mature axons. Developing astrocytes show similar but rather more dramatic changes. Most astrocytic processes contain only microtubules at birth, but during maturation filaments begin to appear in increasing numbers while microtubules become less common. This process continues until, in the mature fibrous astrocytes, filaments pack the cytoplasm and microtubules are rare. These observations suggest that the filaments within axons and astrocytes may be formed by the breakdown of microtubules.     

Ultrastructure of the flagellar apparatus of Oxyrrhis marina

Summary— Oxyrrhis marina, like all dinoflagellates, possesses one transverse and one longitudinal flagellum, which show structural differences. The transverse flagellum contains a small fibre, 20 nm in diameter, associated with doublet no.7, whereas the longitudinal flagellum is substantially by a large (200–300 nm) hollows structure closely resembling the paraflagellar rod described by several authors in kinetoplastidae and in euglenoids. This structure is made up of a hemicylindrical network of filaments which are often linked on one side to the outer doublet no. 4, and on the other side to a dense plate. Another thinner filamentous network closes this hemicyclinder. In cross-section, the wall of this structure is made up of 8 filaments 2–4 nm in diameter that show a thicker periodic structure. In longitudinal section the same filaments appear arranged in periodic rhombus meshes or a helicoidal pattern, depending on the orientation of the section relative to the axoneme.     

MICROFILAMENTS AND CELL LOCOMOTION

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro. Such cells are found to contain two types of microfilament systems: First, a sheath of 50–70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes. Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell. The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network. Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited. Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis. Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity. The microfilament network is, therefore, indispensable for locomotion.     

Disruption of cytoplasmic microtubules by ultraviolet radiation.

Ultraviolet (UV) irradiation of cultured human skin fibroblasts causes the disassembly of their microtubules. Using indirect immunofluorescence microscopy, we have now investigated whether damage to the microtubule precursor pool may contribute to the disruption of microtubules. Exposure to polychromatic UV radiation inhibits the reassembly of microtubules during cellular recovery from cold treatment. In addition, the ability of taxol to promote microtubule polymerization and bundling is inhibited in UV-irradiated cells. However, UV irradiation of taxol-pretreated cells or in situ detergent-extracted microtubules fails to disrupt the microtubule network. These data suggest that damage to dimeric tubulin, or another soluble factor(s) required for polymerization, contributes to the disassembly of microtubules in UV-irradiated human skin fibroblasts.     

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.     

Fluid shear stress induction of COX-2 protein and prostaglandin release in cultured MC3T3-E1 osteoblasts does not require intact microfilaments or microtubules.

Cultured osteoblasts express three major types of cytoskeleton: actin microfilaments, microtubules, and intermediate filaments. The cytoskeletal network is thought to play an important role in the transmission and conversion of a mechanical stimulus into a biochemical response. To examine a role for the three different cytoskeletal networks in fluid shear stress-induced signaling in osteoblasts, we individually disrupted actin microfilaments, micro-tubules, and intermediate filaments in MC3T3-E1 osteoblasts with multiple pharmacological agents. We subjected these cells to 90 min of laminar fluid shear stress (10 dyn/cm(2)) and compared the PGE(2) and PGI(2) release and induction of cyclooxygenase-2 protein to control cells with intact cytoskeletons. Disruption of actin microfilaments, microtubules, or intermediate filaments in MC3T3-E1 cells did not prevent a significant fluid shear stress-induced release of PGE(2) or PGI(2). Furthermore, disruption of actin microfilaments or microtubules did not prevent a significant fluid shear stress-induced increase in cyclooxygenase-2 protein levels. Disruption of intermediate filaments with acrylamide did prevent the fluid shear stress-induced increase in cyclooxygenase-2 but also prevented a PGE(2)-induced increase in cyclooxygenase-2. Thus none of the three major cytoskeletal networks are required for fluid shear stress-induced prostaglandin release. Furthermore, although neither actin microfilaments nor microtubules are required for fluid shear stress-induced increase in cyclooxygenase-2 levels, the role of intermediate filaments in regulation of cyclooxygenase-2 expression is less clear.     

The tektin family of microtubule-stabilizing proteins

Tektins are insoluble alpha-helical proteins essential for the construction of cilia and flagella and are found throughout the eukaryotes apart from higher plants. Being almost universal but still fairly free to mutate, their coding sequences have proved useful for estimating the evolutionary relationships between closely related species. Their protein molecular structure, typically consisting of four coiled-coil rod segments connected by linkers, resembles that of intermediate filament (IF) proteins and lamins. Tektins assemble into continuous rods 2 nm in diameter that are probably equivalent to subfilaments of the 10 nm diameter IFs. Tektin and IF rod sequences both have a repeating pattern of charged amino acids superimposed on the seven-amino-acid hydrophobic pattern of coiled-coil proteins. The length of the repeat segment matches that of tubulin subunits, suggesting that tektins and tubulins may have coevolved, and that lamins and IFs may have emerged later as modified forms of tektin. Unlike IFs, tektin sequences include one copy of a conserved peptide of nine amino acids that may bind tubulin. The 2 nm filaments associate closely with tubulin in doublet and triplet microtubules of axonemes and centrioles, respectively, and help to stabilize these structures. Their supply restricts the assembled lengths of cilia and flagella. In doublet microtubules, the 2 nm filaments may also help to organize the longitudinal spacing of accessory structures, such as groups of inner dynein arms and radial spokes.     

THE EFFECTS OF HIGH HYDROSTATIC PRESSURE ON THE MICROTUBULES OF TETRAHYMENA PYRIFORMIS

Exposure of Tetrahymena pyriformis to 7,500 or 10,000 psi of hydrostatic pressure for 2, 5, or 10 min intervals results in a change in cell shape and ciliary activity. Shape changes occur concurrently with a degradation of longitudinal microtubules in a posterior to anterior direction. High pressure also causes a disruption of ciliary activity. Fine structural analysis reveals a breakdown (presumably microtubule depolymerization) of the central ciliary microtubules. The depolymerization begins at the junction of the central ciliary microtubules with the axosome and progresses distally along the ciliary shaft for a distance of about 0.5 µ.     

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties

Myosin Va (myoVa) motors transport membrane-bound cargo through three-dimensional, intracellular actin filament networks. We developed a coarse-grained, in silico model to predict how actin filament density (3-800 filaments) within a randomly oriented actin network affects fluid-like liposome (350 nm vs. 1750 nm) transport by myoVa motors. Five thousand simulated liposomes transported within each network adopted one of three states: transport, tug-of-war, or diffusion. Diffusion due to liposome detachment from actin rarely occurred given at least 10 motors on the liposome surface. However, with increased actin density, liposomes transitioned from primarily directed transport on single actin filaments to an apparent random walk, resulting from a mixture of transport and tug-of-wars as the probability of encountering additional actin filaments increased. This phase transition arises from a percolation phase transition at a critical number of accessible actin filaments, N_c. N_c is a geometric property of the actin network that depends only on the position and polarity of the actin filaments, transport distance, and the liposome diameter, as evidenced by a fivefold increase in liposome diameter resulting in a fivefold decrease in N_c. Thus in cells, actin network density and cargo size may be regulated to match cargo delivery to the cell’s physiological demands.     

In vivo identification of Tetrahymena actin probed by DMSO induction of nuclear bundles

We report the first successful identification of actin, an ubiquitous contractile protein, in Tetrahymena pyriformis (strain W). We employed dimethyl sulfoxide (DMSO) as a probe to induce the formation of actin bundles in the cell nucleus [1, 2] through disruption of cytoplasmic microfilament organization [3, 4]. The cells were incubated for 30 min at 22 °C in the inorganic medium of Prescott & James [5] containing 10% DMSO, and observed under a transmission electron microscope (TEM). Microfilarment bundles were formed in interphase macronuclei, and these microfilaments, approx. 6 nm in diameter, could be decorated by rabbit skeletal muscle heavy meromyosin (HMM) in the glycerinated model. In many cases, the bundles formed closely parallel to natively existing bundles of microtubules. Interestingly, these microtubules had prominent striation with 15–16 nm periodicity. SDS-polyacrylamide gel electrophoresis was designed to show the low actin content of Tetrahymena cells in comparison with that of Dictyostelium. Actin was suggested to comprise less than 1.7% of the total protein in Tetrahymena, whereas as much as 6% was actin in Dictyostelium cells. In assessing the physiological significance of the bundle formation, we further performed HMM and myosin subfragment-1 (S1)-binding studies to clarify the organization process and the polarity of the DMSO-induced nuclear actin filaments by using the tannic acid staining technique [6]. Randomly oriented short filaments appeared in the nucleus treated with 10% DMSO for 10 min. These filaments became elongated and associated with each other to form loose bundles in the following 10 min. With 30-min treatment, the filaments were organized and large bundles with single axes developed. With these well-developed bundles, the Student's t-test was performed on 172 pairs of neighboring filaments and the probability (p) of the deviation from random polarity was 0.08, suggesting that the filaments were organized in an anti-parallel manner. The results show that the DMSO induction of nuclear actin is a powerful tool to demonstrate the existence of cellular actin in vivo and to study the mechanism of microfilament organization in relation to cell physiological activities.     

Microtubules with different diameter, protofilament number and protofilament spacing in Ornithogalum umbellatum ovary epidermis cells

Microtubules present in the epidermis of Ornithogalum umbellatum ovary in the area of lipotubuloids (i.e. aggregates of lipid bodies surrounded by microtubules) are 25-51 nm in diameter. They consist mainly of 10 and 11, sometimes 9 and 12 protofilaments. An average diameter of microtubule consisting of 9 subunits is about 32 nm, of 10-35 nm, of 11-38 nm and of 12-43 nm, however, individual microtubules in each category significantly vary in size. These differences result from varying distance between protofilaments in microtubule walls and diameters of protofilaments: in thin microtubules they are densely packed and smaller while in thicker ones they are loosely arranged and bigger. A hypothesis has been put forward that changes in microtubule diameter depend on structural changes associated with their functional status and are executed by modifications of protofilament arrangement density and their diameters in microtubule wall. The above hypothesis seems to be in agreement with the opinion formed on the basis of in vitro image of microtubules, that lateral contact between tubulin subunits in neighboring protofilaments indicates some flexibility and changeability during microtubule function.     

Cortical microtubules and cortical microfilaments in the green alga,Micrasterias pinnatifida

Summary Cortical microtubules and cortical microfilaments were visualized in cells ofMicrasterias pinnatifida treated by freeze-substitution, and the pattern of their distribution was reconstructed from serial sections. Most cortical microtubules accompanied the long microfilaments that ran parallel to the microtubules. Cortical microfilaments not accompanied by the microtubules were also found. They were short and slightly curved. Both types of cortical microfilament were not grouped into bundles, and were 6–7 nm in diameter, a value that corresponds to the diameter of filaments of F-actin.     

Coupled oscillations of a protein microtubule immersed in cytoplasm: an orthotropic elastic shell modeling

Revealing vibration characteristics of sub-cellular structural components such as membranes and microtubules has a principal role in obtaining a deeper understanding of their biological functions. Nevertheless, limitations and challenges in biological experiments at this scale necessitates the use of mathematical and computational models as an alternative solution. As one of the three major cytoskeletal filaments, microtubules are highly anisotropic structures built from tubulin heterodimers. They are hollow cylindrical shells with a ∼ 25 nm outer diameter and are tens of microns long. In this study, a mechanical model including the effects of the viscous cytosol and surrounding filaments is developed for predicting the coupled oscillations of a single microtubule immersed in cytoplasm. The first-order shear deformation shell theory for orthotropic materials is used to model the microtubule, whereas the motion of the cytosol is analyzed by considering the Stokes flow. The viscous cytosol and the microtubule are coupled through the continuity condition across the microtubule–cytosol interface. The stress and velocity fields in the cytosol induced by vibrating microtubule are analytically determined. Finally, the influences of the dynamic viscosity of the cytosol, filament network elasticity, microtubule shear modulus, and circumferential wave-number on longitudinal, radial, and torsional modes of microtubule vibration are elucidated.     

The structural organization of the pathogenic protozoan Tritrichomonas foetus as seen in replicas of quick frozen,freeze-fractured and deep etched cells

Summary— The quick-freezing and freeze-etching technique was used to analyse the cytoskeleton of Tritrichomonas foetus, a pathogenic protozoan of the urogenital tract of cattle. The cytoplasm presented a network of filamentous structures interacting with each other, with the surface of the hydrogenosomes and the nuclear membrane. Two nm wide filamentous structures were found in the luminal space of the Golgi complex, connecting the two faces of each cisterna. The microtubules of the pelta-axostyle system were connected by bridges 30–40 nm long and 10 nm wide, regularly spaced with an interval of 25 nm. The costa is a structure formed by a complex array of filaments and globous structures. It seems to be connected to the recurrent flagellum through a complex network formed by 15 and 10 nm wide filaments which emerge from the peripheral region of the costa and penetrate into the surface projections of the protozoan body to which the recurrent flagellum is attached. Other filaments were seen connecting the surface of these projections with the surface of the flagellum.     

Lateral Motion and Bending of Microtubules Studied with a New Single-Filament Tracking Routine in Living Cells

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells.     

Effects of various treatments on microtubules and axial units of lung-fluke spermatozoa

Summary Sperm of the frog lung-fluke, Haematoloechus medioplexus, were treated in various ways and their microtubules and axial units were subsequently studied in sectioned and negatively-stained material. Microtubules and axial units were generally unaffected by exposure to colchicine, cold, and KCl, although with KCl certain lateral projections from doublet tubule A appeared more prominent in negatively-stained preparations. Both mercaptoethanol and urea have a dissociative effect on doublet tubules and microtubules, with doublet tubules being the more sensitive. Pepsin-HCl initially digests the dense region associated with the A tubule of a doublet pair and the core of the axial unit. Microtubules and B tubules of doublet units are later digested; in microtubules, there appears to be a proteinaceous material in the lucent central region which is digested before disappearance of the wall of the microtubule. Further evidence is presented indicating that the characteristically helical wall of the microtubules is made up of spherical subunits about 50 Å in diameter, with about 8 subunits in one turn of the helix. Under certain conditions, the helical structure may be altered to form a wall comprised of longitudinal filaments. It is emphasized that not all microtubules are structurally and chemically equivalent, and it follows that all microtubules do not share a common function.This research was supported by U.S. Public Health Service Grant AI-06448 and an institutional grant from the American Cancer Society.