Temporal and spatial distribution of V-ATPase and its mRNA in the midgut of moulting Manduca sexta.

Abstract

The spatial and temporal distribution of the plasma membrane V-ATPase and its encoding mRNA in the midgut of Manduca sexta were investigated during the moult from the fourth to the fifth larval instar. Digoxigenin-labelled RNA probes were used for in situ hybridization of V-ATPase mRNA of both peripheral and integrated subunits; monoclonal antibodies to subunits of the peripheral sector of the purified plasma membrane V-ATPase were used for immunocytochemistry. Extensive mRNA labelling was found in both mature columnar and goblet cells of intermoult and moulting larvae. Hybridization screening in several tissues suggested that only cells with increased V-ATPase biosynthesis were labelled by our hybridization method. Mature goblet cells contain a large amount of V-ATPase in the apical plasma membrane and were therefore expected to contain V-ATPase mRNA. The intense mRNA signal found in mature columnar cells was unexpected. However, after refining the techniques of tissue preparation, immunolabelling in apical blebs of columnar cells was demonstrated. Since this immunoreactivity did not appear to be membrane-associated, it suggested a cytosolic localization of peripheral V1 subunits. The mRNA encoding subunit A of the peripheral V1 sector was distributed unevenly in columnar cells with a strong apical preference, whereas the mRNA for the proteolipid of the integral V0 sector was evenly distributed in the cytosol. This spatial pattern reflected the distribution of free ribosomes and rough endoplasmic reticulum in the cell, supporting the view that V1 subunits are synthesized at free ribosomes, whereas the V0 subunits are synthesized at the rough endoplasmic reticulum. All undifferentiated cells exhibited intense mRNA signals for V-ATPase subunits of both holoenzyme sectors from the start of proliferation and thus precursors of columnar and goblet cells could not be distinguished.