Intracellular centrifugal separation of organelles in Phycomyces

Abstract

Live sporangiophores of Phycomyces blakesleeanus were centrifuged at 35,000 rpm. The cell contents sedimented into distinct layers, and each layer was studied with an electron microscope and with cytochemical methods. The following layers were found (their volumes and their densities are shown in Fig. 3): 1. polyphosphates; 2. polyphosphates and protein crystals; 3. glycogen; 4. yellow layer with ferritin; 5. ribosomes; 6. protein crystals; 7. mitochondria; 8. mitochondria and fibrils; 9. nuclei; 10. endoplasmic reticulum; 11. vesicles, membranes, and reticulum; 12. vacuole; 13. lipoproteins, membranes; 14. fat droplet. The densities of the various layers were determined by the injection of droplets of inert oils of known density into the sporangiosphores before centrifugation. Sedimented cell organelles could be isolated. Centrifuged nuclei of a lycopene-producing mutant were injected into the intact sporangiophore of an albino host where they induced color formation. The ensuing spores, when plated, gave a mixture of white and colored colonies. It was concluded that cell organelles, sedimented by centrifugation of living sporangiophores, remain alive and can be used for biochemical studies. Microspectrophotometric examination of the layers indicated the presence of cytochromes and flavines in the mitochondria and of cytochromes in the nuclei. No pigments corresponding to the action spectrum for the light growth response were found.