Secretory kinetics in the follicular cells of silkmoths during eggshell formation.

Abstract

ABSTRAGTProcedures for quantitative autoradiography were used for studying the process of secretion of eggshell (chorion) proteins in the follicular epithelium of silkmoths. The method was based on photometric measurements of the reflectance of vertically illuminated autoradiographic silver grains. Results were analyzed and plotted by computer. Secretory kinetics were also determined by analysis of labeled proteins in physically separated epithelium and chorion. Rapid accumulation of radioactivity into "clumps" visualized by light microscope autoradiography and evidence from preliminary electron microscope autoradiography indicate that, within 2 min from the time of synthesis, labeled chorion proteins move to Golgi regions scattered throughout the cytoplasm. The proteins begin to accumulate in the apical area 10-20 rain later and to be discharged from the cell. The time for half-secretion is 20-25 min, and discharge is essentially complete 30-50 min after labeling. At the developmental stages examined, the kinetics of secretion appear to be similar for all proteins. Within the chorion the proteins rapidly assume a characteristic distribution, which varies for different developmental stages. Two relatively slow steps have been identified in secretion, associated with residence in Golgi regions and in the cell apex, respectively. By contrast, translocation of proteins across the cell and deposition of discharged proteins in the chorion are rapid steps.KEY WORDS secretion 9 quantitative autoradiography intracellular transport kinetics 9 photometry 9Kinetic studies of the secretory pathway have been limited by the methods commonly used, subcellular fractionation and electron microscopic autoradiography. These methods are well suited for identifying the organelles involved but are somewhat cumbersome for detailed kinetic studies. In the present paper, we describe widely applicable methods for quantitative light microscopic autoradiography. These methods have expedited the quantitative analysis of large numbers of autoradiograms, making possible detailed studies of secretory kinetics in the follicular epithelium of silkmoths. Studies using separation of cells from extracellular secreted products, plus preliminary electron microscope autoradiography, corroborated and extended the results of light microscope autoradiography. The major steps involved in secretion were identified and the duration of each step was evaluated. The follicular epithelium is well suited for studying secretory kinetics. It forms a monolayer around each oocyte and during the terminal pe-J. CELL BIOLOGY 9 The Rockefeller University Press -0021-9525/78/0701-013151.00 131

Biochemical properties of the major proteins from Rhodnius prolixus eggshell

“…Ultrastructurally chorion consists of fibrous layers that run in parallel to the chorion surface (Smith et al, 1971;Kafatos et al, 1977;Mazur et al, 1982). At the end of morphogenesis the chorion structure is finalized by the formation of disulphide bonds (Blau and Kafatos, 1978).…”

“…This leads to the synthesis of proteins which will constitute the eggshell, the vitelline membrane (VM) and the chorionic layers of the egg as described for several insects (Kafatos et al, 1977;Margaritis et al, 1980;Orr-Weaver, 1991). Secretion of eggshell by follicle cells has already been studied in insects such as R. prolixus, Schistocerca gregaria, Drosophila melanogaster, Scaptomyza sp., Bombyx mori and Leptinotarsa decemlineata (Beament, 1946b;King, 1970;Blau and Kafatos, 1978;Kimber, 1980;Margaritis et al, 1980;Kambysellis, 1993;Leclerc and Regier, 1993;Regier et al, 1993;Pascucci et al, 1996;Papassideri et al, 2003). Insect eggshells are normally composed of three layers, the VM, endochorion and exochorion (passing from the oocyte outwards).…”

Isolation and characterization of human muscle cells.

“…Other major contractile proteins were identified on two-dimensional gels (13) by comigration of labeled proteins with human contractile proteins purified directly from adult human muscle (14). The distribution of acetylcholine receptors was assayed by autoradiographic analysis of the binding of '"I-labeled-a-bungarotoxin to intact myotubes in culture (15,16). Cells were labeled for 45 min in FM with '"I-labeled-a-bungarotoxin (specific activity, 95-140 Ci/mmol; New England Nuclear) at 50 nM, a concentration determined to be sufficient to saturate the receptors.…”

Hybridization-selected translation of Bombyx mori high-cysteine chorion proteins in Xenopus laevis oocytes.

“…Newly synthesized chorion proteins appear to have NH2-terminal signal peptides (11,29) that must be cleaved prior to their transport through the endoplasmic reticulum, Golgi apparatus, and secretory vesicles (35). Moreover, after removal of the signal peptide, certain chorion proteins of the wild silk moth Antheraea polyphemus are known to undergo charge modification by cyclization of an NH2-terminal glutamine (36).…”