The relationship between cell shape and function has long been of interest. However, although the behaviour of the cytoskeleton during the cell cycle has been studied extensively variations in the shape and three-dimensional substructure of the nucleus are less well documented. The spatial distribution of chromatin has previously been studied by a mathematical analysis of the optical densities of stained nuclei, allowing an indirect derivation of the three-dimensional distribution of chromatin. More direct information on chromatin organization can be obtained from electron-microscopic serial sections, although this is very laborious. Using an iterative deconvolution algorithm, Agard and Sedat achieved a degree of optical sectioning in conventional fluorescence microscopy and reconstructed the three-dimensional arrangement of polytene chromosomes. We report here on the three-dimensional structure of cultured mammalian cells as visualized by confocal scanning laser microscopy (CSLM). The exceptionally short depth of field of this imaging technique provides direct optical sectioning which, together with its higher resolution, makes CSLM extremely useful for studying the three-dimensional morphology of biological structures.

Abstract. The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membraneassociated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The colocalization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular-located filaments.Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X-100 insoluble cytoskeleton.PIDERMAL growth factor (EGF) 1 is one of the most intensively studied polypeptide growth factors, and detailed knowledge has been obtained of the various effects of EGF in its target cells as well as of the molecular characteristics of EGF and EGF receptor (for reviews see references 7, 10, 27). Among the effects of EGF in its target cells are morphological changes, such as rounding up of cells (9) and induction of membrane ruffling and extension of filopodia (8). Furthermore, it has been shown that EGF causes alterations in the distribution of actin and tt-actinin (26). Since morphology and dynamics of the cell are largely maintained by an integrated action of cytoskeletal systems (2, 12), these observations suggest that EGF causes changes in the organization of the cytoskeleton via a direct or indirect coupling. Interestingly, an association of the EGF receptor kinase with the Triton X-100-insoluble cytoskeleton of A431 cells has been described recently (17). Furthermore, an enhanced phosphorylation of high molecular weight cytoskeletal proteins was determined as a consequence of EGFinduced kinase activity (17). In addition to the A431 cells, binding of EGF to purified cytoskeleton of the pheochromocytoma cell line PC12 has also been demonstrated (29).1. Abbreviation used in this paper: EGF, epidermal growth factor. So far, no morphological evidence at the ultrastructural level has been presented for an interaction of EGF receptors with the cytoskele...

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Acid phosphatase is present in two layers of the cell envelope of Saccharomyces cerevisiae. These are separated by another layer, which is free of acid phosphatase. We have evidence that the cell wall is built up in two stages, which are independent. In the first stage, the cell wall is built up during the formation of the bud. Glucanase vesicles are involved in this process. In the second stage, a thick layer is deposited at the inside against the new cell wall. This results in the thick, rigid wall of the mature yeast cell. This latter layer is probably assembled on the outer surface of the plasmalemma.