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Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

To gain insights into the possible guidance mechanisms used by Dictyostelium cells as they undergo morphogenesis, we have used time-lapse computational optical-sectioning microscopy to visualize and quantify the three-dimensional (3D) trajectories of both normal (Ax2) and myosin-II-null cells. To accomplish this, we typically collected 30-60 time-lapse 3D images every 2-3 min at the earliest multicellular stage, the mound. These time-lapse data were used to generate 3D movies of morphogenesis and to construct 3D trajectories for individual cells. In contrast to previous 2D time-lapse cinematography studies which revealed predominantly spiral trajectories of Ax2 cells in the mound, we have found a complex assortment of motile behaviors: some cells jiggled in place; others appeared to follow either linear or spiral trajectories; some cells reversed their directions; and others apparently converted from one motile behavior to another. These results suggest that a number of different, potentially competing cell-guidance mechanisms are operative in the mound. To assess one molecular mechanism underlying this assortment of motile behaviors, we have examined cell locomotion in a mutant, namely, in myosin-II-null cells which never develop beyond the mound. Previous studies had shown that these cells can crawl, albeit somewhat slowly, on a 2D substrate. We also found, at the earliest stages of myosin-II-null mound formation, some directed cell locomotion. But later, as the mound condensed into a tightly packed cell conglomerate, extended cell trajectories disappeared, and instead virtually all of the cells jiggled in place. Thus, our results suggest that myosin-II is absolutely essential for normal 3D ameboid locomotion.

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