cytochalasin-d and 4-4-difluoro-4-bora-3a-4a-diaza-s-indacene

There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE) membrane system. However, this interaction has yet to be characterised in living interphase cells.. Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY) we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies).. We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

Topics: Actins; Animals; Binding Sites; Biopolymers; Boron Compounds; Bridged Bicyclo Compounds, Heterocyclic; Circular Dichroism; Cytochalasin D; Depsipeptides; Fluorescent Dyes; HeLa Cells; Humans; Liver; Nuclear Envelope; Rabbits; Rats; Thiazoles; Thiazolidines

We tested the hypothesis that bacterial lipopolysaccharide (LPS) must be internalized to facilitate endotoxin-dependent signal activation in cardiac myocytes. Fluorescently labeled LPS was used to treat primary cardiomyocyte cultures, perfused heart preparations, and the RAW264.7 macrophage cell line. Using confocal microscopy and spectrofluorometry, we found that LPS was rapidly internalized in cardiomyocyte cultures and Langendorff-perfused hearts. Although LPS uptake was also observed in macrophages, only a fraction of these cells were found to internalize endotoxin to the extent seen in cardiomyocytes. Colocalization experiments with organelle or structure-specific fluorophores showed that LPS was concentrated in the Golgi apparatus, lysosomes, and sarcomeres. Similar intracellular localization was demonstrated in cardiomyocytes by transmission electron microscopy using gold-labeled LPS. The internalization of LPS was dependent on endosomal trafficking, because an inhibitor of microfilament reorganization prevented uptake in both cardiomyocytes and whole hearts. Inhibition of endocytosis specifically restricted early activation of extracellular signal-regulated kinase proteins and nuclear factor-kappaB as well as later tumor necrosis factor-alpha production and inducible nitric oxide synthase expression. In conclusion, we have demonstrated that bacterial endotoxin is internalized and transported to specific intracellular sites in heart cells and that these events are obligatory for activation of LPS-dependent signal transduction.

Topics: Animals; Biological Transport; Boron Compounds; Cell Line; Cytochalasin D; Endocytosis; Fluorescent Dyes; Golgi Apparatus; Lipopolysaccharides; Lysosomes; Microscopy, Confocal; Microscopy, Electron; Mitogen-Activated Protein Kinases; Myocardium; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phosphorylation; Rats; Rats, Wistar; Signal Transduction; Tumor Necrosis Factor-alpha

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