Brian S. Spooner

An analysis of salivary gland morphogenesis: Role of cytoplasmic microfilaments and microtubules

The roles of microfilaments and microtubules as causative organelles in the cell shape changes required for in vitro morphogenesis of embryonic mouse salivary epithelium have been explored by use of the drugs cytochalasin B and colchicine. Cytochalasin inhibits morphogenesis, causes flattening of the epithelium and loss of the clefts that were present at the time of drug application. These effects correlate with a specific disruption of cytoplasmic microfilaments when viewed with the electron microscope. Removal of cytochalasin results in reappearance of ordered microfilaments and resumption of morphogenesis. Colchicine disrupts microtubules and halts morphogenesis, but does not cause flattening of the epithelium or loss of clefts. Treatment with cytochalasin followed by recovery in the presence of colchicine demonstrates that recovery clefts can form in the absence of microtubules. It is proposed that normal salivary gland morphogenesis includes microfilament participation via contractile activity, in addition to mitosis and to extracellular stabilization processes.

Heavy meromyosin binding to microfilaments involved in cell and morphogenetic movements

Abstract Certain microfilaments found in migratory single cells and in morphogenetically-active epithelial cells are shown to bind heavy meromyosin prepared from skeletal muscle. This ‘actin-like’ feature of the filaments is compatible with the hypothesis that such filaments are components of contractile systems in these cells.

Effects of papaverine and calcium-free medium on salivary gland morphogenesis☆

Abstract Papaverine reversibly inhibits morphogenesis of cultured mouse salivary glands. The drug does not alter the morphology of epithelial cell microfilament systems or other organelles. Incubation in calcium-free medium also reversibly inhibits morphogenesis, though effects upon tissue integrity are possible.

Thorotrast uptake and transit in embryonic glia, heart fibroblasts and neurons in vitro.

Abstract Thorotrast (colloidal ThO 2 ) is incorporated into coated vesicles, various agranular vesicles and sacs, and a surface-associated system of membranous channels in times as short as 1 min by single cultured glial and heart cells. Thorotrast appears in ‘C’-shaped bodies and in small, dense bodies of the lysosomal series within ca. 25 min. With longer chase periods, thorotrast ‘clears’ from all cytoplasmic organelles except the lysosomal series. The technique of applying thorotrast and using varying chase periods fails to distinguish a class of membranous organelles, located close to the cell periphery, that might serve as a source of new cell surface during locomotory activity. Similarly, thorotrast (colloidal ThO 2 ) is incorporated into almost all classes of membrane-bounded organelles of growth cones and axons of single nerve cells in vitro in times as short as 1 min. This includes elements of the smooth endoplasmic reticulum. No thorotrast enters the lysosomal granules in this short time. During various chase periods, the tracer disappears from the initial sites of incorporation and accumulates in dense bodies of the lysosome series within growth cones and axons. ‘C’-shaped bodies may be an intermediate in that process. No unique sites of endocytotic activity or of a complete absence of endocytosis were observed that could be correlated with growth cone function and axonal elongation, though the presence of the tracer in agranular sacs of the smooth endoplasmic reticulum in growth cones could reflect hypothesized cycling of cell surface (Bray, 1973).

Membrane fusion in the growth cone-microspike region of embryonic nerve cells undergoing axon elongation in cell culture

Abstract Fine structural analysis of embryonic nerve cells undergoing axon elongation in vitro has revealed structural evidence supportive of the time lapse cinematographic observation that extensive areas of active membrane fusion are present in the distal tip of the axon. Pre-fusion membrane alignment and post-fusion strings of vesicles characterize the putative fusions between microspikes, between microspikes and growth cones, and between growth cones and the distal axon. The restriction and possible significance of these apparent fusions are discussed.

Actin in embryonic organ epithelia

Abstract The presence of actin in morphogenetically active embryonic epithelia was assessed by SDS-polyacrylamide gel electrophoresis of pellets and low ionic strength extracts of acetone powders of isolated mouse embryo salivary and lung epithelia, and chick embryo epidermis. A polypeptide that co-electrophoresed with skeletal muscle actin was resolved in each of these systems. Approx. 80–95% of this protein was extracted from acetone powders by low ionic strength solutions, demonstrating solubility properties like those of muscle actin. Similar results were obtained with salivary, bronchial, and pancreatic mesenchyme. Cytoplasmic actin represented approx. 7 % of the protein in salivary epithelium and 13% of the protein in salivary mesenchyme. Electron microscopy demonstrated that incubation of glycerinated salivaries in low ionic strength solutions preferentially removed microfilaments from the epithelia, thus linking these heavy meromyosin-binding structures with the actin extracted from acetone powders and resolved on SDS-gels.