Instability of pleomorphic tubulin paracrystals artificially induced by Vinca alkaloids in tissue-cultured cells

The processes of tubulin paracrystal induction in Chinese hamster ovary cells treated with a Vinca alkaloid, ie, vinblastine or vincristine, and treated simultaneously with one of the Vinca alkaloids and colcemid or colchicine were followed by four different microscopic techniques, in particular by tubulin-immunofluorescence. Vinca alkaloid alone, in lower concentrations, induced basically tactoid or needle-shaped (N-shaped) paracrystals. However, the formation of crystalloid was greatly enhanced by increasing the concentration of Vinca alkaloid. Square or barrel-shaped (S-shaped) and hexagonal paracrystals were also commonly induced by simultaneous treatment with a Vinca alkaloid and colcemid or colchicine. Large rectangular paracrystals often displayed fibrillar or lamellar fine structures which ran perpendicular to the long axis but tended to cleave into fragments by spontaneous splitting. Electron micrographs revealed the fine structure of crystalloids to be aggregates of numerous filaments. The growth of paracrystals, particularly N-shaped crystals, was markedly inhibited when cells were exposed to drug(s) at a low temperature (4 degrees C). We confirmed that both N- and S-shaped paracrystals dissociated rapidly after the culture medium was replaced with fresh, drug-free medium. Glutaraldehyde-fixed paracrystals treated with RNase solution were stained with acridine orange, showing a weak orange color. Possible factors involved in the assembly and disassembly of tubulin paracrystals are discussed.     

Interaction of tropomyosin with troponin components.

1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1 M KC1 at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules. 2. The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgC1(2) solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-I at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0. 3. The TN-C component does not precipitate tropomyosin in 0.1 M KC1. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgC12.     

Isoforms of tau protein from mammalian brain and avian erythrocytes: Structure, self-assembly, and elasticity

Previous studies on tau protein showed that the protein forms paracrystals which are unusually elastic. The paracrystals were obtained from a mixture of isoforms prepared from brain tissue, and the protein was in a mixed state of phosphorylation. Subsequently we showed that the structure and elasticity was related to the state of phosphorylation. However, this left open the possibility that the isotype composition played a role as well. We have now addressed this question by separating the individual isoforms and analyzing their structure. The paracrystals from all isoforms are similar to one another and to those of the native mixture; the same holds for the elasticity. Thus the tendency to self-associate, the apparent structure, and the elasticity are determined by those regions of tau which all isoforms have in common. In addition we compare tau paracrystals from three different sources. Apart from the porcine brain tau described earlier we have prepared paracrystals from bovine brain tau because its sequence is now known (Himmler et al., 1989). The structure and elasticity is indistinguishable from porcine tau. Second, we have prepared tau from avian erythrocytes where it is found in the membrane-associated marginal band microtubules ( Murphy and Wallis, 1985). Its isoform composition differs from mammalian brain tau, but again the structural properties are similar. A notable difference is that the shift in electrophoretic mobility induced by phosphorylation with CaM kinase, typical of all brain tau isotypes, is not found in the marginal band tau. Tau shows a strong tendency of longitudinal self-association which is apparent not only in the crystallization buffer but also in standard microtubule reassembly buffer. This leads to rod-like tau oligomers, fibers, and three-dimensional networks. This property, coupled with tau's elasticity, suggests a role in the organization of the cytoplasm beyond the stabilization of microtubules.     

Polymorphism of actin paracrystals induced by polylysine

We describe a method for the induction of different polymorphic forms of actin filament paracrystals. This polymorphism is probably based on differences in the stagger and/or polarity of adjacent filaments in single-layered paracrystals and by superposition of different layers in multilayered paracrystals. The helical parameters defining the filament geometry are indistinguishable for the different polymorphic forms observed and for the four different actins used. Analysis of these paracrystals, some of which are ordered to better than 2.5 nm, should provide a reference structure suitable for alignment and orientation within the actin filament of high resolution models of the actin monomer obtained from crystal data.     

Light meromyosin paracrystal formation

Studies of paracrystal formation by column purified light meromyosin (LMM) prepared in a variety of ways led to the following conclusions: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.     

Ultrastructure of paracrystals of a lipoprotein from the outer membrane of Escherichia coli.

The highly purified lipoprotein of the outer membrane of Escherichia coli forms paracrystals. The ultrastructures of these paracrystals were examined by electron microscopy. The needle-shaped paracrystals show several different band patterns, depending on conditions of paracrystallization. Models are presented to explain possible arrangements of the lipoprotein molecules within the paracrystals.     

Paracrystal Formation in Cell Cultures Infected with Adenovirus Type 2

Large rod-shaped structures corresponding to paracrystals were seen in the nucleus, cytoplasm, or both of adenovirus type 2 (Ad2)-infected cells by immunofluorescence staining with antibody prepared against purified Ad2. In exception to this, Ad2-induced crystals did not stain with either hexon or fiber antibody. The crystalline structures were first observed in Ad2-infected Vero cells at 28 hr with a maximum number at 70 hr postinoculation. The kinetics of paracrystalline formation closely paralleled the experimental synthesis of infectious progeny virus. Acridine-orange staining revealed the lack of nucleic acids associated with the crystal. Also, the paracrystals stained intensely with phenanthrenequinone, suggesting that they are composed of basic proteins. Interferon induced by Newcastle disease virus from African green monkey kidney cell cultures was used to pretreat Vero cells prior to Ad2 infection. This resulted in inhibiting the formation of viral-induced paracrystals in 97% of the cells and reduced virus yields by 95%. The African green monkey kidney cell culture interferon did not reduce Ad2 yields in HeLa cell cultures or display any virus inhibitory activity in rabbit kidney cell cultures. Staining procedures, fluorescent-antibody tests with whole virus, hexon or fiber antibody, and interferon studies suggested that the paracrystals were viral-directed and composed of basic proteins (possibly core proteins).     

Effect of H-protein on the formation of myosin filaments and light meromyosin paracrystals

H-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.     

Hybrid Reverse Monte Carlo and electron phase contrast image simulations of amorphous silicon with and without paracrystals

Atomic networks of as-implanted and relaxed amorphous silicon solids were simulated using a Hybrid Reverse Monte Carlo algorithm constrained by high-resolution electron diffraction data. No significant structural distinction was observed between the two forms of amorphous silicon. A nanometer-sized crystallite was inserted into the as-implanted structure, to model medium-range order due to paracrystals, and the atomic network was energetically relaxed whilst maintaining consistency with experiment. Experimental pair–pair correlations were then simulated using a stochastic generalised Debye sum of fourth order. The idealised pair–pair correlation calculations were not able to readily distinguish between models with and without paracrystals. On the other hand, wave mechanical simulations surprisingly showed that paracrystals could be experimentally imaged using phase contrast transmission electron microscopy and/or nanoscale electron diffraction on a contemporary aberration-corrected microscope.     

Vinblastine-induced paracrystals and unusually large microtubules (macrotubules) in rat renal cells

Summary Vinblastine sulfate was administered to adult rats by intravenous injections. Kidney cortex was fixed after 1, 2, or 5 hours of treatment and studied by routine transmission electron microscopy.In control animals, cells of distal convoluted tubules possessed numerous microtubules with an average diameter of 280 Å. In treated animals, the microtubules of these cells were reduced in number, and paracrystalline inclusions characteristic of vinblastine treatment were common. Macrotubules (570 Å average diameter) were also present and often were seen close to, or in apparent continuity with, paracrystals. Since the work of others indicates that vinblastine-induced paracrystals contain microtubular protein (tubulin), observation of continuities between paracrystals and macrotubules is interpreted as evidence that macrotubules are also composed of tubulin and that macrotubules may become incorporated into paracrystals.Unlike the ordinary microtubules of cells of the distal tubules, vinblastine-induced macrotubules exhibited cross-striations in longitudinal view and subunit structure in cross section.Macrotubules and paracrystals were also observed in cells of the proximal convoluted tubule, mesangium, glomerular endothelium, parietal epithelium of Bowman's capsule, and visceral epithelium of Bowman's capsule. Continuities between macrotubules and paracrystals, although relatively common in occurrence in distal tubule cells, were only rarely seen in the other kinds of cells examined. Acknowledgements. The authors gratefully acknowledge the technical help of Mrs. Dawn Bockus, Miss Judy Groombridge, Mrs. Jeri Hunter, Mrs. Jolan Pinter, Miss Franque Remington, Miss Mary Stewart, Miss Louise Young, Mr. Reginald Pickering, and Mr. W. J. Masten. This research was supported by N.I.H. grants AM 16 236, GM 00 100, and HE 03 174, by Institutional Cancer Grant IN-26L from the American Cancer Society, and by the Graduate School Research Fund of the University of Washington.     

Comparative electron microscopic study on projectin and titin binding to F-actin

Using the system of F-actin paracrystals, we have obtained electron microscopic evidence that projectin from synchronous flight muscles of Locusta migratoria binds to actin filaments in the same fashion as skeletal titin. Control actin paracrystals formed in the presence of Mg(2+) ions have great width and length and blunted ends. The addition of either projectin or titin results in disruption of compact ordered packing of F-actin in paracrystals and leads to the formation of loose filament bundles with smaller diameters and tapered ends. It is also accompanied with the appearance of individual actin filaments in considerable amounts. The effect becomes more pronounced with the increase in concentrations of added projectin or titin. Possible physiological implications of projectin-actin interactions are discussed.     

Effects of acidic pH on the formation of vinblastine-induced paracrystals in Chinese hamster ovary cells

Summary— The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5×10^?6 M and 10^?5 M). At higher concentrations of VLB (5×10^?5 M and 10^?4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH.     

Electron microscopic analysis of tropomyosin paracrystals.

Negatively stained images of divalent cation-induced tropomyosin paracrystals show polymorphic patterns which are almost bipolar. Although these bipolar forms are naturally due to antiparallel arrays of molecules, the precise molecular arrangements have not been clarified yet except in the case of one type of these polymorphic paracrystals by Stewart and McLachlan [(1976) J. Mol. Biol. 103, 251--269]. In the previous paper we showed that the lead-induced polar paracrystal is a parallel and in-register array of tropomyosin molecules. Moreover, we have made it possible to locate a given residue on the staining pattern. By overlapping two photographic transparencies of the polar paracrystal antiparallel, directly observed images of polymorphic bipolar paracrystals could be synthesized photographically with fidelity. The overlap length between N-terminals of antiparallel pairs of molecules could be easily determined without any assumptions. Next, we considered the stabilizing forces involved in the morphogenesis of such polymorphic paracrystals. The cation-bridged attractive forces already proposed by some groups were insufficient to account for the stability of some specific forms of tropomyosin paracrystals. From the primary amino acid sequence of tropomyosin, we calculated the changes of repulsive forces between the basic residues with changes of molecular overlap length between the N-terminals of antiparallel pairs. By setting the values of charge appropriately, we could account well for the stability of the polymorphic structures observed by electron microscopy.     

Helical disorder and the filament structure of F-actin are elucidated by the angle-layered aggregate

Angle-layered aggregates of F-actin are net-like structures induced by Mg2+ concentrations below that used to form paracrystals. These aggregates incorporate the angular disorder of subunits, which has been described elsewhere for isolated actin filaments. Because this disorder is incorporated into the aggregates in solution at the time they are formed, the possibility of negative stain preparation being responsible for the disorder is excluded. The simple two-layered geometry of the angle-layered aggregate provides information about the shape of the component actin filaments free from the superposition of large numbers of layers. A model for the filament shape, derived from single filaments and confirmed by the angle-layered aggregate, is different from those that have previously emerged from paracrystal studies. An understanding of the interfilament bond in both the angle-layered aggregate and the paracrystal allows one to reconcile these different models. We have found a bipolar bonding rule, with staggered crossover points in the angle-layered aggregate, which we suggest is also responsible for Mg2+ paracrystals. This bonding rule can explain the apparent alignment of crossover points in adjacent filaments in paracrystals as a consequence of the superposition of staggered filaments.     

Prismatic cristae and paracrystalline inclusions in mitochondria of myocardial cells of the oyster Crassostrea virginica Gmelin

Summary Several types of unusual mitochondrial configurations were found in myocardial cells of the oyster Crassostrea virginica Gmelin. These mitochondria include, in order of frequency, prismatic cristae, filamentous paracrystals in honeycomb and herringbone configurations, and paracrystals composed of rows of electron dense particles. The long, parallel, evenly spaced prismatic cristae are square or rhomboidal in cross section. In the space between the prismatic cristae are rodlike structures (4–6 nm in diameter) that are regularly spaced about 12nm apart and appear to pass between adjacent cristae. Filamentous paracrystals are observed in slender, elongated mitochondria. The filament spacing and form of these paracrystals suggest that they are composed of the intercristal rods. Alternatively, filamentous paracrystals might be tangential sections of prismatic cristae and intercristal rods. Particulate paracrystals which consist of dense lines or rows of particles are the least frequent type of unusual configuration. The particles are triangular, possibly pyramidal, in shape; their bases are 10–12 nm thick and repeat in rows every 17–18 nm. There is a close association between particulate paracrystals and prismatic cristae plus intercristal rods. Although similar mitochondrial configurations have been associated with disease or altered metabolism in a number of species, we have found no such association in the oyster as yet.Supported in part by the Mississippi-Alabama Sea Grant Consortium, through NOAA, Dept. of Commerce under grant no. NA 79AA-D-0049We wish to thank Ms. Barbara M. Hyde, Ms. Patricia A. Vermiere and Mr. Robert Allen for their technical assistance     

Vinblastine paracrystals from cultured cells are calcium-stable

Vinblastine has two distinct tubulin-related effects in 3T6 fibroblasts and J774.2 macrophages. It depolymerizes microtubules and it induces the formation of paracrystals in the cytoplasm. These paracrystals are retained in cytoskeletons prepared by Triton extraction and are stable to treatment with calcium. The direct addition of vinblastine to cytoskeletons does not alter the organization of microtubules. The two effects of vinblastine are concentration-dependent, as assayed by binding of [3H]taxol and tubulin immunofluorescence. At low concentrations, vinblastine depolymerizes cellular microtubules; at high concentrations the drug induces the formation of paracrystals.     

Comparative studies of light meromyosin paracrystals derived from red, white, and cardiac muscle myosins

Tryptic and chymotryptic light meromyosin paracrystals from red and cardiac muscles of rabbit show a negative and positive staining pattern with uranyl acetate and phosphotungstate that sharply differs from that of white muscle light meromyosin paracrystals. The main periodicity of about 430 A is the same regardless of the source of light meromyosin. The results are discussed in terms of the molecular structure and the functional properties of various myosins.     

Organization and expression of Drosophila tropomyosin genes

It has been shown (Jockusch &; Isenberg, 1981) that vinculin (130K protein) binds to actin and induces actin filaments to form bundles even at low ionic strength. Here, we present structural details on the vinculin molecule itself and on its interaction with actin. In negatively stained preparations, vinculin appeared as a globular protein with an average diameter of 85 Å. The ability of vinculin to form actin filament bundles was confirmed using shadowing techniques and gel analysis of sedimented material. Analysis of vinculin-induced paracrystals by optical diffraction and computer processing revealed their structural similarity to Mg-induced paracrystals. The lateral position of vinculin on surface-exposed actin filaments of such paracrystals was demonstrated directly in electron micrographs and indirectly by labelling vinculin with ferritin-coupled anti-vinculin F(ab′) fragments. Polymerization of actin in the presence of vinculin-coated polystyrene beads did not result in an “end-on” binding of filaments to the beads. Rather, actin bundles were laterally associated with the whole surface of the beads, from where they radiated in a star-like pattern. The growth of actin filaments onto myosin subfragment-I decorated, vinculin-incubated. fixed filament fragments was not inhibited, as was shown directly by electron microscopy and monitored viscometrically in a nucleation assay. These results suggest that in vivo at the site of an adhesion plaque vinculin may link actin filaments together into a suitable configuration to interact with the plasma membrane.     

The axial repeats in paracrystals of light meromyosin and its complex with C-protein

We examined the axial repeats in electron micrographs of three types of negatively stained paracrystals (two tactoid- and one sheet-like type) of rabbit light meromyosin (LMM) and its complex with C-protein characterized previously by similar axial period of about 43.0 nm. Assuming for the axial repeat in type II tactoids the value of 42.93 +/- 0.05 nm as it was determined by X-ray diffraction technique (Yagi and Offer 1981), we found average axial repeats in type I tactoid and in sheet-like paracrystal of 42.93 +/- 0.75 nm and 43.50 +/- 0.62 nm respectively. Analyzing the micrographs where the two types paracrystals are located side-by-side we determined rather accurately the average ratio of axial repeat in sheet-like paracrystal to that in type I tactoid (1.013 +/- 0.002). Taking 42.93 nm as the axial repeat in type I tactoid, the axial repeat in sheet-like paracrystal was found to be 43.50 +/- 0.08 nm. C-protein binds to LMM with the period of the underlying LMM paracrystals and independently of the value of their axial repeats. Two different axial repeats (42.9 nm and 43.5 nm) revealed for LMM paracrystals in this study precisely coincide with the average repeat periods of myosin crossbridges along the thick filaments found for different physiological states of skeletal muscles (Lednev and Kornev 1987). Molecular basis for the appearance of two structural states in LMM paracrystals and in the shafts of thick filaments are discussed.     

Arthrin: a new actin-like protein in insect flight muscle

There are one or more proteins of 50,000 to 60,000 Mr in the thin filaments of insect flight muscle. A protein of 55,000 Mr has been isolated from insect fibrillar flight muscle and called arthrin. Despite its higher molecular weight, arthrin is in many ways like actin. The amino acid composition of arthrin was similar to that of actin. There were similarities in the peptides produced by digesting the denatured proteins and mild digestion of polymerized proteins cleaved similar-sized fragments from arthrin and actin. Polymerized arthrin activated the Mg2+ ATPase of myosin to the same extent as actin and the ATPase was regulated by rabbit or Lethocerus troponin and tropomyosin. Arthrin did not itself act as troponin-T. Electron microscopy of negatively stained specimens showed that arthrin and actin filaments were similar in structure and that arthrin could be decorated by rabbit subfragment-1 to form normal-looking arrowheads. Arthrin formed paracrystals at an optimum concentration of MgCl2 (25 mM) that was somewhat lower than the optimum for actin paracrystals. Optical diffraction showed that the structure of the paracrystals was similar to those formed from actin. The mass of arthrin and actin filaments relative to phage fd was measured by scanning transmission electron microscopy; the relative mass of arthrin and actin was 1.33, in agreement with molecular weight estimations. Therefore arthrin has the properties of a heavy form of actin. The proportion of actin, arthrin and troponin-T in Lethocerus myofibrils was six moles of actin to one mole of arthrin and one mole of troponin-T. The function of arthrin is not known.