Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. We hypothesized that the integration of environmental factors might induce specific cytoskeletal architecture patterns, characterized by quantitative image analysis. Human breast cancer cells MCF-7, flown in space in a photon capsule, were fixed after 1.5, 22, and 48 h in orbit. Cells subjected to weightlessness were compared with 1g in-flight and ground controls. Postflight, fluorescent labelings were performed to visualize cell proliferation (Ki-67), signal transduction (phosphotyrosine), three cytoskeleton components (microtubules, microfilaments, and intermediate filaments), and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network, and chromatin structure. In weightlessness, phosphotyrosine signal transduction was lower, more cells were cycling, and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade. Microtubules were altered in many cells. The perinuclear cytokeratin network was more loosely 'woven', and chromatin structure was modified. The prolongaion of mitosis can be explained by an alteration of microtubule self-organization in weightlessness, involving reaction-diffusion processes. The loosening of the perinuclear cytokeratin network and modification of chromatin distribution are in agreement with basic predictions of cellular tensegrity.

Abstract. Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extraceUular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for/3-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [~25I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au~sm) and the receptor (labeled with LHR38-Ausm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Aus~ on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors. C ELL surface receptors for various ligands differ in their localization and their mechanisms and pathways of internalization. These receptors may be: (a) either specifically included in the coated pits (i.e., LDL-receptors [3,4], transferrin receptors [18][19][20]); (b) expressed only outside the coated pits on the membrane (i.e., B-adrenergic receptors) (37); or (c) randomly exposed on the cell surface, coated pits included (i.e., EGF-receptors) (11,12). Under the effect of the ligand (or sometimes constitutively) (16, 46) the receptor is internalized and may follow one of the four major endocytic pathways which have been described: (a) the ligand-receptor complex dissociates at the endosomal level; the receptor is recycled to the surface whereas the ligand is degraded in lysosomes (i.e., LDL receptor) (7); (b) the ligand-receptor complex is recycled to the cell surface, dissociates and the receptor is reused (i.e., transferrin receptor) (20); (c) the ligand-receptor complex is delivered by transcytosis to the opposite front of polarized cells where the ligand is released intact and the receptor partlally degraded (i.e., IgA receptor) (31); and (d) both partners are transported to lysosomes and degraded (i.e., EGFreceptor) (12).The hCG/LH receptor is involved in the regulation of steroidogenes...

Interphasic nuclear organization has a key function in genome biology. We demonstrate that p21 WAF-1 , by inf luencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a major role as a nuclear organizer. Transfection of U937 tumor cells with p21 WAF-1 resulted in expression of the HUMSIAH (human seven in absentia homologue), Rb, and Rbr-2 genes and strong suppression of the malignant phenotype. p21 WAF-1 drastically modified the compartmentalization of the nuclear genome. DNase I genome exposure and f luorescence in situ hybridization show, respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide potentially significant perspectives for the understanding of tumor suppression.

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