Temporally regulated protein synthesis in cultured haemocytes of the Mediterranean fruit fly Ceratitis capitata during larval and prepupal development: Internalization of larval serum proteins into the haemocytes

Abstract

Detailed analysis of haemocyte proteins by one-dimensional polyacrylamide gel electrophoresis during the late larval stages and white pupae of the Mediterranean fruit fly Ceratitis capitata reveals more than 50 polypeptides. A numbering system is established based on molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Detailed examination leads to the conclusion that each protein resolved by our methods has its own characteristic kinetics of accumulation. The most abundant protein is unequivocally the 36-kDa band with an isoelectric point at 5.07, followed by the 47- and 54-kDa proteins. The 36-kDa band constitutes approximately 9% of total haemocyte protein. Comparison of the haemocyte protein pattern to that of cell-free haemolymph clearly shows that the protein bands at 81, 82, 85, and 87 kDa are common in both sources. We demonstrated by immunoblotting and pulse-labeled experiments followed by 2D SDS-PAGE that these protein bands which constitute the larval serum proteins were internalized into the haemocytes from the serum. By pulse labeling cells with [ 35S]methionine, separating polypeptides by one-dimensional PAGE electrophoresis, and comparing the rates of synthesis of over 50 individual polypeptides by fluorography, the following results were obtained: Three protein groups with distinct patterns of synthesis were noted: (1) proteins that are synthesized throughout the developmental period studied, (2) proteins whose synthesis begins at the wandering stage, and (3) proteins that are synthesized only during the feeding period. The 47-kDa protein shows the highest relative rate of synthesis, but since it is not the most accumulated band it must have a high turnover rate. Most of the labeled proteins were secreted into the incubation medium. The intracellular transit time was estimated to be about 11 min. Patterns of protein synthesis in haemocytes are regulated in a precise temporal sequence during the transformation of larvae to pupae. Their study yields a useful system for the analysis of molecular events in gene control.