cytochalasin-d and phallacidin

Although changes of cytoskeleton (CSK) stiffness and friction can be induced by diverse interventions, all mechanical changes reported to date can be scaled onto master relationships that appear to be universal. To assess the limits of the applicability of those master relationships, we focused in the present study on actin and used a panel of actin-manipulating drugs that is much wider than any used previously. We focused on the cultured rat airway smooth muscle (ASM) cell as a model system. Cells were treated with agents that directly modulate the polymerization (jasplakinolide, cytochalasin D, and latrunculin A), branching (genistein), and cross linking (phallacidin and phalloidin oleate) of the actin lattice. Contractile (serotonin, 5-HT) and relaxing (dibutyryl adenosine 3',5'-cyclic monophosphate, DBcAMP) agonists and a myosin inhibitor (ML-7) were also tested for comparison, because these agents may change the structure of actin indirectly. Using optical magnetic twisting cytometry, we measured elastic and frictional moduli before and after treatment with each agent. Stiffness increased with frequency as a weak power law, and changes of friction paralleled those of stiffness until they approached a Newtonian viscous limit. Despite large differences in the mechanism of action among the interventions, all data collapsed onto master curves that depended on a single parameter. In the context of soft glassy systems, that parameter would correspond to an effective temperature of the cytoskeletal matrix and reflect the effects of molecular crowding and associated molecular trapping. These master relationships demonstrate that when the mechanical properties of the cell change, they are constrained to do so along a special trajectory. Because mechanical characteristics of the cell shadow underlying molecular events, these results imply special constraints on the protein-protein interactions that dominate CSK mechanical properties.

Topics: Actins; Animals; Azepines; Biomechanical Phenomena; Bridged Bicyclo Compounds, Heterocyclic; Bucladesine; Cells, Cultured; Cross-Linking Reagents; Cytochalasin D; Genistein; Muscle Contraction; Myocytes, Smooth Muscle; Myosins; Naphthalenes; Peptides, Cyclic; Phalloidine; Rats; Rheology; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Thiazoles; Thiazolidines; Trachea

We found previously that in living cells of Oedogonium cardiacum and O. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (< 2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore of Oedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.

Topics: Actins; Boron Compounds; Cell Nucleus; Chlorophyta; Cytochalasin D; Dinitrobenzenes; Fluorescent Dyes; Herbicides; Kinetochores; Microscopy, Fluorescence; Microtubules; Nucleic Acid Synthesis Inhibitors; Peptides, Cyclic; Spindle Apparatus; Sulfanilamides; Time Factors

The effects of hypoxia and reoxygenation on the lateral mobility of membrane lipids were studied in primary cultures of rat proximal tubule epithelial cells at a subconfluent stage. The lipid lateral diffusion coefficient (DL) of the fluorescent probe 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazol)-aminocaproyl phosphatidylethanolamine was determined in membranes of attached cells by the technique of fluorescence redistribution after photobleaching, using confocal imaging to differentiate apical from basal surfaces. Fluidity was greater in basolateral membranes than in apical membranes [DL(apical) = 3.15 +/- 0.8 and DL (basolateral) = 6.56 +/- 1.2 x 10(-10) cm2/s]. In apical membranes, 60 min of hypoxia plus 30 min of reoxygenation increased DL to 10.04 +/- 2.1. When cells were pretreated with the antioxidants superoxide dismutase, catalase, or alpha-tocopherol, there was no clear-cut effect on the hypoxia-reoxygenation-induced increase in apical membrane DL, although t-butyl hydroperoxide treatment of cells did increase DL. Disruption of the cytoskeleton with cytochalasin D or exposure to Ca(2+)-free medium markedly increased DL. However, when cells were pretreated with phallacidin to stabilize cytoskeletal actin microfilaments, the hypoxia-reoxygenation-induced change in lipid lateral mobility was completely prevented. Thus the cytoskeleton is an important regulator of the translational motion of membrane phospholipids. Hypoxia-reoxygenation may have increased DL because of cytoskeletal dysfunction.

Topics: Animals; Antioxidants; Cell Hypoxia; Cytochalasin D; Cytoskeleton; Diffusion; Fluorescent Dyes; Kidney Tubules, Proximal; Lipid Metabolism; Oxygen; Peptides, Cyclic; Phosphatidylethanolamines

Filaments about 6-7 nm in diameter were seen associated with germ cell intercellular bridges in detergent-permeabilized cells treated with tannic acid. Approximately 40-50 filaments were present subjacent to the bridge density. Filaments encircled the bridge channel in a manner similar to contractile ring actin filaments of dividing cells. NBD-phallacidin and myosin S-1 subfragments were employed to demonstrate that the filaments observed at intercellular bridges are actin. Intratesticular injection of a single dose of cytochalasin D, a specific inhibitor of actin filaments, caused certain intercellular bridges of spermatids to open within 3 hr after injection, leading to the production of symplasts. During bridge opening, remnants of bridge densities were gradually incorporated into the lateral aspect of the plasma membrane of the symplast. Thus actin, present in bridge structures, appeared to participate in maintaining certain intercellular bridges. A model of intercellular bridge structure is presented.

Topics: Actins; Amanitins; Animals; Cytochalasin D; Cytochalasins; Epithelium; Intercellular Junctions; Male; Microscopy, Electron; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Models, Biological; Peptides, Cyclic; Rats; Rats, Inbred Strains; Sciuridae; Spermatids; Spermatocytes; Testis