A potential dual apical pathway in polarized regenerative cells of the midgut of Manduca sexta.

Two novel proteins with apparent molecular weight of 38 (Manduca sexta midgut MsM38) and 46kDa (MsM46) were isolated from midgut homogenates in wandering stage Manduca sexta larvae and both of them were found to be present exclusively in this tissue on Western blots. Immunocytochemical studies revealed that both proteins are expressed in the regenerative cells however, their distribution pattern is clearly different. MsM38 is localized in the cytoplasm of resting regenerative cells during the feeding period, and is accumulated in the calcospherits at the beginning of the wandering period. Along with the delamination of the larval epithelium, this protein is released apically from these vesicles. The antiserum labels an additional 76 kDa protein in the wandering larval midgut homogenates. The appearance of this 76 kDa protein coincides with the accumulation of the immunopositive material in the calcospherits. MsM46 is similarly distributed during the feeding period in the cytoplasm of regenerative cells. At the beginning of the wandering period it accumulates around the newly forming large apical vacuoles, that are released at the time of complete delamination of the larval epithelium. In parallel with this process MsM46, and another 40 kDa protein, that becomes labeled from this period on Western blots appeares on the apical microvillar projections. Thus both isolated proteins are directed apically from different compartments, that raises the possibility of a dual apical routing pathway in regenerative cells.

Localization of a cuticular protein during the postembryonal development of Manduca sexta.

The pattern of cuticle protein synthesis by the epidermis of insects changes during the last larval, pupal and adult development, leading to an alteration in cuticular stucture and feature. We have isolated a protein that had an apparent molecular mass of 33.1 kD from larval cuticle of Manduca sexta. Synthesis, transport and accumulation of MsCP33.1 were followed during metamorphosis by immunoblots and immunocytochemical methods using the antibody developed against this protein. Our data prove that the presence of MsCP33.1 in the larval cuticle is general while its appearance in the pupal or adult integument is restricted only in the cuticle of wings and apodemes. We established that the synthesis of 33.1 kD protein is negatively regulated by moulting hormone (20-hydroxyecdysone). Possible roles for this cuticular protein are discussed.

The immunoprotein scolexin and its synthesizing sites--the midgut epithelium and the epidermis.

Scolexin is one of the bacterial induced hemolymph proteins of tobacco homworm (Manduca sexta) larvae, that has hemocyte coagulation-provoking activity. The 72 kDa scolexin complex is composed of two 36 kDa subunits. To examine the protein secretory pathways in insect epithelia a polyclonal antibody was raised against the 36 kDa hemolymph protein. This MsH36 antibody recognised a 36 and a 72 kDa protein in tissue homogenates. On the basis of the characteristic labelling pattern observed on immunoblots and immunocytochemical sections we concluded that the 36 kDa protein in the hemolymph, in the midgut and in the epidermis was identical with the scolexin subunit. In present paper we report a labelling shift in the midgut epithelium between goblet and columnar cells that may be controlled by the hormonal system. A 72 kDa protein showed similar epitops and molecular weight to the scolexin complex and was detected in epidermis and in cuticle under both reducing and non-reducing conditions. Tissue localization of 36 kDa and 72 kDa MsH36 antibody labelling proteins indicated the possibility that the epidermal cells produce two kinds of scolexin-like proteins. The complex composed of 36 kDa subunits are transported basolaterally into the circulation and display hemocyte coagulation inducing activity while the 72 kDa form contains two subunits linked covalently secreted apically into the cuticle.

Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta).

Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fifth-stadium larvae was found exclusively in this tissue on Western blots. Presence of MsM41 protein was indicated in both anterior and posterior regions of the midgut during the whole fifth stadium. However, in the posterior region an additional 39-kDa protein was also detected during the feeding period of the last larval stage. Upon light-microscopic examination immunoreactivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during the feeding period was different in the anterior and posterior regions. In the anterior region immunoreactivity was localized only to the brush border membrane of columnar cells, while in the posterior region some cytoplasmic structures identified as large trans-Golgi vesicles, endoplasmic reticulum and small secretory vesicles were also labeled. Large, apical extrusions remained immunonegative. In vitro translation confirmed that our protein was expressed only in the posterior region of the midgut. The primary translation product was a 39-kDa protein. Putative post-translational modifications yielded the 41-kDa form, which was then secreted apically. Its presence in the region of the anterior part microvilli was probably due to the countercurrent flux of the ectoperitrophic fluid.

Insect cuticle, an in vivo model of protein trafficking.

In the course of this study more than 20 proteins have been isolated from the larval cuticle of Manduca sexta. Synthesis, secretion, transport and accumulation of four particular proteins, representative members of four characteristic groups, were followed during metamorphosis by immunoblot and immuncytochemical methods and are described in detail in this paper. We established that only some of the proteins of the soft cuticle of Lepidopteran larvae are synthesized in epidermal cells at the beginning of the larval stages and are digested during the moulting period (MsCP29). Other proteins (MsCP30/11) are secreted into the cuticle by the epidermal cells in different forms during various developmental stages. Some proteins are secreted apically during the feeding period, but before ecdysis they are then taken up by epidermal cells and transported in a basolateral direction back into the hemolymph and saved in an immunologically intact form by the fat body cells (MsCP12.3). Some cuticle proteins have a non-epidermal origin. They are transported from the hemolymph into the cuticle. Before and during ecdysis these molecules reappear in the hemolymph and are detectable again in the pupal cuticle (MsCP78). Our data prove that the cuticle is not a non-living part of the insect body: it is not only an inert, protective armor, but maintains a continuous and dynamic metabolic connection with the other organs of the organism.