No abstract

We have used the pH-dependent fluorochrome fluorescein-dextran (FD) to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured macrophages and fibroblasts. FD was internalized by pinocytosis under conditions that allowed its selective localization in endosomes (1-to 5-min pulse) or in lysosomes (5-min pulse, 30-min chase). Fibroblasts were also exposed to FD at 20°C, at which temperature endosome-lysosome fusion is inhibited. Cells were homogenized and labeled organelles were separated by centrifugation in Percoll density gradients. The addition of ATP rapidly decreased the internal pH of both endosomes and lysosomes, as indicated by a decrease in fluorescence intensity. The pH gradient was dissipated by H+ ionophores and ammonium.chloride. Acidification was not affected by inhibitors of the mitochondrial F1,F0-ATPase or the Na+, K, K+-ATPase and did not require permeant anions, Na+, or KV. Of the inhibitors tested, only N-ethylmaleimide prevented the ATP-dependent acidification of both compartments. These findings provide direct support for the existence of an acidic prelysosomal compartment that may be acidified via the same type of H+ pump believed to operate in lysosomes and.secretory granules.Pinocytosis typically results in the accumulation and degradation of internalized macromolecules in secondary lysosomes (1). However, delivery to lysosomes requires the participation of at least two other classes of endocytic vacuoles. Pinocytic vesicles, often with clathrin-containing coats, form at the plasma membrane and constitute the primary endocytic compartment. These vesicles subsequently fuse with, or fuse together to form, a somewhat larger class of secondary endocytic vacuoles (0.2-1.0 ,um in diameter), referred to here as endosomes (1,2). Endosomes can be distinguished from lysosomes in several ways: (i) they have a relatively low buoyant density (3-8); (ii) they are labeled by pinocytic markers more rapidly than and prior to labeling of lysosomes (7,9); (iii) internalized macromolecules exhibit only a transient residence in endosomes, whereas lysosomes are typically the final destination (9); and (iv) endosomes are relatively devoid of acid hydrolases (3,(6)(7)(8).These differences notwithstanding, endosomes may be similar to secondary lysosomes in at least one significant characteristic, low internal pH. This has been suggested by recent studies in which receptor-bound ligands were coupled to fluorescein (whose fluorescence spectrum is a titratable function of pH) and found to be internalized into an acidic compartment prior to delivery to lysosomes (8,10). This was the case also with Semliki Forest virus (SFV), which requires a pH of <6 for fusion and penetrates into the cytosol from prelysosomal vacuoles (11). Here, we have used isolated endosomes labeled with the pH-sensitive fluorochrome fluorescein-dextran (FD) (12, 13) to study more directly the acidification of prelysosomal vacuoles. Two points are made: (i) both isolated endosomes and lysosomes c...

There is continued emphasis on increasing and improving genetics education for grades K-12, for medical professionals, and for the general public. Another critical audience is undergraduate students in introductory biology and genetics courses. To improve the learning of genetics, there is a need to first assess students' understanding of genetics concepts and their level of genetics literacy (i.e., genetics knowledge as it relates to, and affects, their lives). We have developed and evaluated a new instrument to assess the genetics literacy of undergraduate students taking introductory biology or genetics courses. The Genetics Literacy Assessment Instrument is a 31-item multiple-choice test that addresses 17 concepts identified as central to genetics literacy. The items were selected and modified on the basis of reviews by 25 genetics professionals and educators. The instrument underwent additional analysis in student focus groups and pilot testing. It has been evaluated using 400 students in eight introductory nonmajor biology and genetics courses. The content validity, discriminant validity, internal reliability, and stability of the instrument have been considered. This project directly enhances genetics education research by providing a valid and reliable instrument for assessing the genetics literacy of undergraduate students.

The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein.Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorpor.1te the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body. At least five proteins are essential for motor rotation (41, 47), but none of these have been found within the flagellar basal body (1,. 20), even though the basal body is considered a major part of the motor. Two of these proteins, MotA and MotB, are integral to the cell membrane (6, 37, 41). They are not necessary for assembly of the flagellum and do not copurify with it (20). They can be synthesized after flagellar assembly and used to activate the motor; paralyzed motA motB mutants acquire the ability to rotate their flagella after MotA and MotB synthesis under the direction of lambda-E. coli hybrid bacteriophage (41). Recently, it was shown (6) that, at least in the case of MotB, this acquisition of motility proceeds by quantum increases in flagellar rotation rate, presumably as a result of successive incorporation of subunits of MotB protein. The fact that motA and motB null mutants are nonetheless flagellated makes these genes different from all other flagellum-associated genes and will have * Corresponding author.

The organization of the endosome compartment in BHK-21 cells was studied by using horseradish peroxidase as a fluid-phase marker and Semliki Forest virus as an adsorptive marker. Stereo pairs of semithin sections (0.2-0.5 ,sm) and computer-aided reconstruction and tracing of serial thin sections (CARTOS) were used to obtain three-dimensional images of the labeled compartments. Two types of labeled organelles could be observed: (i) small vesicles and tubules (=z50 nm in diameter) and (i) large complex structures consisting of central vesicular elements (with diameters up to 0.5 ,um) and associated systems of radiating tubules. The large endosomes were located either in the peripheral cytoplasm or in the perinuclear region, and, importantly, they existed as independent organelles and not as an interconnected reticulum. Each endosomal vacuole had two to seven tubules oriented in random directions from the central vesicle. The tubules were 60-100 nm in diameter and up to 4 Im in length. Morphometric estimates indicated that 60-70% of the endosomal membrane was in the tubules, in contrast to 30-40% of the volume. No structural continuity between endosomes and Golgi cisternae was observed, although elements of the two systems were frequently found in close proximity.Endosomes constitute a ubiquitous prelysosomal compartment with critical functions in the endocytic pathway (1-3). They serve as intermediates in the transport of endocytosed ligands, fluid-phase components, and membrane components to lysosomes; they are involved in the dissociation of ligand-receptor complexes and the recycling ofreceptors and other plasma membrane components; and they constitute the major site for molecular sorting in the endocytic pathway (2-9). In some cell types, they also are thought to be involved in storage and processing of incoming ligands, regulation of receptor expression on the cell surface, and the maintenance of plasma membrane polarity (10,11). Endosomes are, in addition, the sites of entry for certain enveloped viruses and bacterial toxins (12-15). Many of these functions depend on the acidic endosomal pH generated, at least in part, by proton-translocating ATPases (16)(17)(18).Available data from thin-section and whole-mount preparations indicate that endosomes comprise an extensive system of vacuoles and membrane-bounded tubules (4-9, 19, 20). Here we have investigated the three-dimensional structure of endosomes and their overall organization in the cytoplasm by a combination of semithin-section electron microscopy and by computer-aided reconstruction of serial thin sections (CARTOS). MATERIALS AND METHODSCells, Labeling, and Electron Microscopy. Baby hamster kidney (BHK-21) cells were maintained in minimal essential medium, Glasgow variation (G-MEM), with 5% fetal calf serum and 10%6 tryptose phosphate broth as described (12). For electron microscopy, monolayers of cells were grown to confluence (>48 hr) on 35-mm plastic tissue culture dishes.Horseradish peroxidase (HRP; type II, Sigma) was used to label BHK-21...