Bioinformatics Analysis of Actin Molecules: Why Quantity Does Not Translate Into Quality?

It is time to review all the available data and find the distinctive characteristics of actin that make it such an important cell molecule. The presented double-stranded organization of filamentous actin cannot explain the strong polymorphism of actin fibrils. In this work, we performed bioinformatics analysis of a set of 296 amino acid actin sequences from representatives of different classes of the Chordate type. Based on the results of the analysis, the degree of conservatism of the primary structure of this protein in representatives of the Chordate type was determined. In addition, 155 structures of rabbit actin obtained using X-ray diffraction analysis and electron microscopy have been analyzed over the past 30 years. From pairwise alignments and the calculation of root-mean-square deviations (RMSDs) for these structures, it follows that they are very similar to each other without correlation with the structure resolution and the reconstruction method: the RMSDs for 11,781 pairs did not exceed 3 Å. It turned out that in rabbit actin most of the charged amino acid residues are located inside the protein, which is not typical for the protein structure. We found that two of six exon regions correspond to structural subdomains. To test the double-stranded organization of the actin structure, it is necessary to use new approaches and new techniques, taking into account our new data obtained from the structural analysis of actin.

Amiodarone InhibitsTrypanosoma cruziInfection and Promotes Cardiac Cell Recovery with Gap Junction and Cytoskeleton ReassemblyIn Vitro

ABSTRACTWe present the results of the first detailed study of the antiproliferative and ultrastructural effects of amiodarone onTrypanosoma cruzi, the causative agent of Chagas' disease. Moreover, we report the effects of this compound on the recovery of F-actin fibrils, connexin43, and contractility inT. cruzi-infected cardiac myocytes. Amiodarone is the most prescribed class III antiarrhythmic agent and is frequently used for the symptomatic treatment of Chagas' disease patients with cardiac compromise. In addition, recent studies identified its antifungal and antiprotozoal activities, which take place through Ca2+homeostasis disruption and ergosterol biosynthesis blockade. We tested different concentrations of amiodarone (2.5 to 10 μM) on infected primary cultures of heart muscle cells and observed a dose- and time-dependent effect on growth of the clinically relevant intracellular amastigote form ofT. cruzi. Ultrastructural analyses revealed that amiodarone had a profound effect on intracellular amastigotes, including mitochondrial swelling and disorganization of reservosomes and the kinetoplast and a blockade of amastigote-trypomastigote differentiation. Amiodarone showed no toxic effects on host cells, which recovered their F-actin fibrillar organization, connexin43 distribution, and spontaneous contractility concomitant with the drug-induced eradication of the intracellular parasites. Amiodarone is, therefore, a promising compound for the development of new drugs againstT. cruzi.

Aldolase-localization in cultured cells: Cell-type and substrate-specific regulation of cytoskeletal associations

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20–2000 µM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20–60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin–aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.Key words: aldolase, cytoskeleton, electron microscopy, viscosimetry.

Restoration of movement and apical growth in the angiosperm pollen tube following cytochalasin-induced paralysis

Cytochalasin D (CD) at 5 pg ml- 1 arrested growth and vectorial movement in pollen tubes of Narcissus pseudonarcissus and Endymion nonscriptus and caused the mainly longitudinally oriented actin fibrils in the vegetative cells to coalesce and form massive, more randomly oriented, cables. As extension growth was arrested, the tubes formed apical bulbs and abnormal wall thickenings. During recovery from a 10 min treatment period in E. nonscriptus , an essentially normal fibril system was reconstituted by partial dissociation of the thick cables formed during the exposure to CD. As this progressed movement was restored in the vegetative cells. Some 80 % of the blocked tubes initiated new growing points, either by producing randomly oriented swellings in sites where the wall was thinner, or by erosion and penetration of thicker zones. Contrary to expectation, the sites of the prospective growing points were not indicated in advance by any special disposition of the actin cytoskeleton. With the transition to cylindrical growth in the secondary tubes the standard stratification of the tube wall reappeared, with outer pectocellulosic and inner callosic layers. Normal movement pathways were established concomitantly, together with the apical zonation of organelles and other cytoplasmic inclusions characteristic of the extending tube. CD-treatment brought about rapid contraction of the vegetative nuclei with the loss of the elastic extensions of the nuclear envelopes. The extended form was resumed as the actin cytoskeleton was restored during recovery, and vegetative nuclei and generative cells moved into the secondary tubes where they continued to track the apex as in the normal tube.

Spreading of trypsinized cells: cytoskeletal dynamics and energy requirements

The spreading of trypsinized XTH-2 cells (a line derived from Xenopus laevis tadpole heart endothelia) on glass was investigated. Three phases can be distinguished: (1) blebbing of rounded cells, first attachment to a solid substratum and formation of a broad smooth contact area; (2) organization of a peripheral zone of actin fibrils and reinforcement of the basal cytoplasm by a stress fibre-like pattern; (3) extension of lamellae. The first phase seems to be independent of a supply of metabolic energy, while the others clearly depend on it. This is concluded from the close relationship between cellular projection area and energization of mitochondria as revealed by (a) the fluorescence intensity of cells vitally stained with the mitochondria-specific fluorochrome DASPMI (2–4-(dimethylamino)-styryl-1-methylpyridinium-iodine); (b) the degree of spreading in the presence of inhibitors of respiration; (c) effective amelioration of spreading (phases (2) and (3] under conditions of high ATP content. In phase (2) the extension of the central part of the cells becomes stabilized, the cell body settles on the basal cytoplasmic layer and further expansion of the projection area is achieved by lamella formation (phase (3]; motile and stabile regions of the cells become separated. This sequence of events is interpreted as a self-organizing process based on the development of internal hydraulic pressure, actin polymerization and contraction of the newly developed actomyosin network. During trypsinization, depolymerization of actin does not occur but rather on addition of Ca2(+)-containing media. Cellular ATP content drops as well on trypsinization, as on addition of Ca2+. Manganese promotes spreading by decreasing F-actin disassembly and maintaining a high level of cytosolic ATP, most probably because it is not accepted by the calcium pumps. Regarding the association of glycolytic enzymes with F-actin and their influence on actin assembly, lactate dehydrogenase has been inhibited with oxamic acid. This treatment improves the correlation between F-actin content and the degree of spreading; however, the total amount of F-actin remains smaller and the cells spread more.

Myosin associated with the surfaces of organelles, vegetative nuclei and generative cells in angiosperm pollen grains and tubes

Myosin, detected by immunofluorescence using an antibody to bovine skeletal and smooth muscle myosin, has been localised on individual identifiable organelles from the grasses Alopecurus pratensis and Secale cereale, and on the surfaces of vegetative nuclei and generative cells from pollen and pollen tubes of Hyacinthus orientalis and Helleborus foetidus. Taken in conjunction with recent evidence showing that the growing pollen tube contains an actin cytoskeleton consisting of numerous mainly longitudinally oriented microfilament bundles, and that isolated pollen-tube organelles show ATP-dependent movement along the actin bundles of the giant cells of the characeous algae, this finding suggests that an actomyosin motility system is present in pollen tubes, and indicates that the movements of the different classes of inclusions are driven by interaction of the surface myosin with the actin fibrils at the zones of contact.

Evidence Against a Direct Role for Cortical Actin Arrays in Saltatory Organelle Motility in Hyphae of the Fungus Saprolegnia Ferax

Actin stained by rhodamine-labelled phalloidin in growing hyphae of the oomycete fungus Saprolegnia ferax is restricted to an approximately 0.25 μm deep layer of the cell periphery where it forms an apical cap of fine filaments and a subapical array of coarser longitudinal fibrils interspersed with plaques. The functions of this actin are unknown, but because actin-rich fibrils in other cells are involved in organelle motility I have sought evidence for a similar role in these hyphae. The most prominent motile structures are a population of spherical, predominantly sub-micrometre diameter, refractile vesicles of unknown function, which show typical saltatory movements along the hyphae. The motility of these vesicles is statistically identical in both the central cytoplasm, remote from the actin fibrils, and the peripheral cytoplasm adjacent to the fibrils. Treatment of hyphae with specific concentrations and durations of the detergents Tween 20 and Brij 58 causes extensive reorganization of the actin arrays with no effect on vesicle motility, whereas Triton X-100 severely inhibits motility with little detectable effect on the actin filaments. These observations show that normal vesicle motility does not depend on the proximity of a normal population of actin filaments and, therefore, suggest that the latter have some function other than a direct role in saltatory organelle motility.