Very Cool Clathrin

Clathrin-coated vesicles are the shuttle containers within cells. The vesicles carry lipids and proteins between membrane-bound compartments. Clathrin forms a cage-like structure around the membrane-bound vesicle that is pinched off from the plasma membrane (in endocytosis) or a membranous component of the cytoplasm. Clathrin recruits cargo that is within a vesicle through intermediary proteins known as adaptors that help select membrane-anchored protein and form an interface between the clathrin cage and the membrane bilayer.

Phosphatidylinositol 3-kinase activity is required for early endosome fusion

The homotypic fusion between early endosomes from baby-hamster kidney cells is blocked by addition of the fungal metabolite wortmannin with an IC50 of approx. 15 nM. Over this concentration range, wortmannin has been regarded as a specific inhibitor of phosphatidylinositol (PI) 3-kinase. Further confirmation of the participation of a PI 3-kinase in the fusion reaction has been obtained by demonstrating a sensitivity to an additional, structurally unrelated, PI 3-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. Assays constructed such that only the membranous component has been incubated with wortmannin show in vitro fusion to be sensitive to treatment with the drug. Assays in which only the cytosolic component has been treated with wortmannin also showed inhibition of in vitro fusion, but to a lesser extent. PI 3-kinase action almost certainly involves direct regulation of membrane fusion, as no vesicular intermediate has been identified, despite previous extensive morphological examination of in vitro endosome fusions.

The cytoplasmic droplet of rat epididymal spermatozoa contains saccular elements with Golgi characteristics.

The cytoplasmic droplet of epididymal spermatozoa is a small localized outpouching of cytoplasm of the tail of unknown significance. EM revealed flattened saccular elements as the near exclusive membranous component of the droplet. Light and electron microscopic immunolabeling for Golgi/TGN markers showed these saccules to be reactive for antibodies to TGN38, protein affinity-purified alpha 2,6 sialyltransferase, and anti-human beta 1,4 galactosyltransferase. The saccules were isolated by subcellular fractionation and antibodies raised against this fraction immunolabeled the saccules of the droplet in situ as well as the Golgi region of somatic epithelial cells lining the epididymis. The isolated droplet fraction was enriched in galactosyltransferase and sialyltransferase activities, and endogenous glycosylation assays identified the modification of several endogenous glycopeptides. EM lectin staining in situ demonstrated galactose and N-acetyl galactosamine constituents in the saccules. Endocytic studies with cationic and anionic ferritin as well as HRP failed to identify the saccules as components of the endocytic apparatus. Epididymal spermatozoa were devoid of markers for the ER as well as the Golgi-associated coatamer protein beta-COP. It is therefore unlikely that the saccular elements of the droplet participate in vesicular protein transport. However, the identification of Golgi/TGN glycosylating activities in the saccules may be related to plasma membrane modifications which occur during epididymal sperm maturation.

The synthesis of messenger and ribosomal RNA in pea seedlings as detected by electrophoresis

This paper describes a method for the analysis of microsome fractions from pea seedling roots by electrophoresis on cellulose acetate membrane. The method separates the lipid membranous component, nucleoprotein and free RNA . The synthesis of RNA in the growing root has been studied by incubating the seedlings in radioactive phosphate, and analysing the isolated microsomal fractions by electrophoresis. A rapidly labelled nucleoprotein could be distinguished from the ribosomes, which remain unlabelled for nearly one hour. After longer incubation times, the ribosomes become labelled. Citrate buffer causes the dissociation of a trace of highly labelled RNA from the labelled ribosomes. It was found that the base composition of this RNA and of the RNA of the rapidly labelled nucleoprotein, differs from that of ribosomal or transfer RNA and is somewhat more like that of DNA .

The Fine Structure of Streptomyces coelicolor

Colonies and spore suspensions of Streptomyces coelicolor were fixed by the method of Kellenberger, Ryter, and Séchaud (1958) and embedded in methacrylate or araldite. Thin sections were cut with an A. F. Huxley microtome and examined in a Siemens' Elmiskop I. At all stages of development the hyphae of Streptomyces coelicolor have an extensive membranous component in the cytoplasm. The membranes are continuous with the plasma membrane and have a variety of configurations at different places in the hyphae. Tubular structures, vesicles, and parallel stacks of membranes are seen. In some areas concentric layers of membranes form whorled structures which are particularly frequent in the region of developing cross-walls and within maturing spores. In the spores membranous structures often lie embedded in the nuclear material. In disintegrating hyphae the intracytoplasmic membranes round off into small vesicles and remain when the rest of the cytoplasmic structure has gone. In the absence of typical mitochondria and other cytoplasmic membranous structures it is possible that the membranous component of the cytoplasm of Streptomyces coelicolor may perform the functions of the endoplasmic reticulum and/or the mitochondria of higher cells.