Related Galpha subunits play opposing roles during Dictyostelium development.

Of the several known Dictyostelium G protein subunits, the Galpha4 and Galpha5 subunits are the most closely related pair based on phylogenetic analysis and expression patterns, but these subunits perform different roles during development. To investigate potential relationships between these subunits with respect to cell differentiation, chimeric organisms composed of strains lacking or overexpressing either subunit were created and examined for developmental morphogenesis and spore production. Chimeras of galpha4 null and galpha5 null strains or Galpha4 and Galpha5 overexpression strains displayed compensatory morphogenesis, implying that the subunits promote complementary developmental processes. However, chimeras composed of galpha4 null and Galpha5 overexpression strains or galpha5 null and Galpha4 overexpression strains displayed distorted tip morphogenesis, suggesting the strains of these chimeras share common developmental deficiencies. Cells lacking the Galpha5 subunit localized to the prespore region of chimeras similar to the pattern observed for cells overexpressing the Galpha4 subunit, and cells overexpressing the Galpha5 subunit displayed localization patterns similar to galpha4 null mutants. A strain overexpressing both subunits displayed a partial suppression of morphology, gene expression, and cell localization phenotypes associated with the overexpression of the individual Galpha subunit genes, suggesting that each Galpha subunits can inhibit signaling mediated by the other subunit. Overexpression of the Galpha5 subunit inhibited chemotaxis and cGMP accumulation in response to folic acid, indicating that the Galpha5 subunit can inhibit early steps in the Galpha4-mediated signal transduction pathway. The contrasting phenotypes of the Galpha mutants suggest the Galpha4 and Galpha5 subunits provide opposing functions in cell differentiation, localization, and chemotactic responses to folic acid.

Electron microscopic radioautographic detection of sites of protein synthesis and migration in liver.

The sites of synthesis of proteins and their subsequent migration in rat liver have been studied during a 75 min period after labeling of liver-slice proteins by exposure to leucine-H(3) for 2 min. Incorporation of the label into protein began after 1 min and was maximal by 4 min. Electron microscopic radioautography showed that synthesis of proteins in hepatocytes occurs mainly on ribosomes, particularly those in rough endoplasmic reticulum and, to some extent, in nuclei and mitochondria. Most of the newly formed proteins leave the endoplasmic reticulum in the course of 40 min, and concurrently labeled proteins appear in Golgi bodies, smooth membranes, microbodies, and lysosomes. A likely pathway for the secretion of some or all plasma proteins is from typical rough endoplasmic reticulum to a zone of reticulum which is partially coated with ribosomes, to the Golgi apparatus, and thence to the cell periphery. The formation of protein by reticuloendothelial cells was measured and found to be about 5% of the total protein formed by the liver.

An artefact in radioautography due to binding of free amino acids to tissues by fixatives.

The binding of labeled free amino acids to liver and to purified protein by commonly used fixatives was investigated. Glutaraldehyde caused 25% of free leucine to be bound to serum albumin in solution, whereas formaldehyde bound only 0.5%. Liver slices were incubated for 2 min in the presence of labeled leucine and of puromycin, which permits absorption of leucine into the cell but inhibits incorporation into protein. Both counting and radioautographic techniques showed that glutaraldehyde bound 30 times, and osmic acid six times, as much free amino acid as did formaldehyde. By comparing liver slices incubated with and without puromycin for 2 min, it was calculated that in radioautographs prepared after fixation with glutaraldehyde, osmic acid, or formaldehyde 63, 25, and 4% respectively of the grains were due to binding of free amino acid. Formaldehyde, freshly prepared from paraformaldehyde, gives good preservation and is the recommended fixative for radioautography. When levels of free substrate in a tissue are high at the time fixative is added, the amount of binding of free substrate induced by the fixative should be included as a control in radioautographic experiments.