Bulk Flow Revisited: Transport of a Soluble Protein in the Secretory Pathway

The C-terminal domain, Cp, of the Semliki Forest virus capsid protein, known for its rapid, efficient and chaperone-independent folding, was used to measure bulk fluid flow in the secretory pathway of Chinese hamster ovary cells. Being small, nonglycosylated, soluble and cytoplasmic in origin, Cp was not likely to interact with lectins, cargo receptors and retention factors. Using pulse-chase analysis, we observed that translocation into the endoplasmic reticulum resulted in rapid and efficient folding and transport of the newly synthesized Cp protein to the extracellular medium. The first Cp molecules were secreted 15 min after synthesis, which is the fastest transport of a protein so far recorded in mammalian cells. The rate constant of secretion was 1.2% per min, which amounts to an estimated bulk flow rate of about 155 coat protein II (COPII) vesicles per second. Transport was independent of expression level, and blocked by CI-976, brefeldin A and ATP depletion indicating that it depended on COPII vesicle formation, and followed the classical secretory pathway. In polarized Madin-Darby canine kidney cells, the secretion rate was similar but occurred mainly apically. The results demonstrated that fluid flow in the secretory pathway is fast, and can therefore play a significant role in the secretion of soluble secretory products.     

Intermediates in influenza induced membrane fusion.

Our results show that the mechanism by which influenza virus fuses with target membranes involves sequential complex changes in the hemagglutinin (HA, the viral fusion protein) and in the contact site between virus and target membrane. To render individual steps amenable to study, we worked at 0 degree C which decreased the rate of fusion and increased the efficiency. The mechanism of fusion at 0 degree C and 37 degrees C was similar. The process began with a conformational change in HA which exposed the fusion peptides but did not lead to dissociation of the tops of the ectodomain of the trimer. The change in the protein led to immediate hydrophobic attachment of the virus to the target liposomes. Attachment was followed by a lag period (4-8 min at 0 degree C, 0.6-2 s at 37 degrees C) during which rearrangements occurred in the site of membrane contact between the virus and liposome. After a further series of changes the final bilayer merger took place. This final fusion event was not pH dependent. At 0 degree C efficient fusion occurred without dissociation of the top domains of the HA trimer, suggesting that a transient conformation of HA is responsible for fusion at physiological temperatures. The observations lead to a revised model for HA mediated fusion.     

More than one glycan is needed for ER glucosidase II to allow entry of glycoproteins into the calnexin/calreticulin cycle

Nascent and newly synthesized glycoproteins enter the calnexin (Cnx)/calreticulin (Crt) cycle when two out of three glucoses in the core N-linked glycans have been trimmed sequentially by endoplasmic reticulum (ER) glucosidases I (GI) and II (GII). By analyzing arrested glycopeptides in microsomes, we found that GI removed the outermost glucose immediately after glycan addition. However, although GII associated with singly glycosylated nascent chains, trimming of the second glucose only occurred efficiently when a second glycan was present in the chain. Consistent with a requirement for multiple glycans to activate GII, pancreatic RNase in live cells needed more than one glycan to enter the Cnx/Crt cycle. Thus, whereas GI trimming occurs as an automatic extension of glycosylation, trimming by GII is a regulated process. By adjusting the number and location of glycans, glycoproteins can instruct the cell to engage them in an individually determined folding and quality control pathway.     

N-glycolyl GM1 ganglioside as a receptor for simian virus 40

Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.     

Pre-attachment Striga hermonthica resistance of New Rice for Africa (NERICA) cultivars based on low strigolactone production

Striga hermonthica (Striga) is an obligate hemiparasitic weed, causing severe yield losses in cereals, including rice, throughout sub-Saharan Africa. Striga germination depends on strigolactones (germination stimulants) exuded by the host roots. The interspecific New Rice for Africa (NERICA) cultivars offer a potentially interesting gene pool for a screen for low germination-inducing rice cultivars. Exudates were collected from all NERICA cultivars and their parents (Oryza sativa and Oryza glaberrima) for the analysis of strigolactones. In vitro and in situ Striga germination, attachment and emergence rates were recorded for each cultivar. NERICA 1 and CG14 produced significantly less strigolactones and showed less Striga infection than the other cultivars. NERICAs 7, 8, 11 and 14 produced the largest amounts of strigolactones and showed the most severe Striga infection. Across all the cultivars and parents, there was a positive relationship between the amount of strigolactones in the exudate and Striga germination, attachment and emergence rates. This study shows that there is genetic variation in Striga pre-attachment resistance in NERICA rice. Cultivars combining this pre-attachment resistance with post-attachment resistance (already identified) can provide a key component for durable integrated management of this noxious weed in cereal production systems in sub-Saharan Africa.     

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

Interactions of Semliki Forest virus spike glycoprotein rosettes and vesicles with cultured cells

Semliki Forest virus (SFV)-derived spike glycoprotein rosettes (soluble octameric complexes), virosomes (lipid vesicles with viral spike glycoproteins), and liposomes (protein-free lipid vesicles) have been used to investigate the interaction of subviral particles with BHK-21 cells. Cell surface binding, internalization, degradation, and low pH- dependent membrane fusion were quantitatively determined. Electron microscopy was used to visualize the interactions. Virosomes and rosettes, but not liposomes, bound to cells. Binding occurred preferentially to microvilli and was inhibited by added SFV; it increased with decreasing pH but was, in all cases, less efficient than intact virus. At 37 degrees C the cell surface-bound rosettes and virosomes were internalized via coated pits and coated vesicles. After a lag period of 45 min the protein components of the internalized ligands were degraded and appeared, as acid-soluble activity, in the medium. The uptake of rosettes and virosomes was found to be similar to the adsorptive endocytosis of SFV except that their average residence times on the cell surface were longer. The rosettes and the liposomes did not show low pH-induced membrane fusion activity. The virosomes, however, irrespective of the lipid compositions used, displayed hemolytic activity at mildly acidic pH and were able to fuse with the plasma membrane of cells with an efficiency of 0.25 that observed with intact viruses. Cell-cell fusion activity was not observed with any of the subviral components. The results indicated that subviral components possess some of the entry properties of the intact virus.     

Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus.

The matrix (M1) protein of influenza virus is a major structural component, involved in regulation of viral ribonucleoprotein transport into and out of the nucleus. Early in infection, M1 is distributed in the nucleus, whereas later, it is localized predominantly in the cytoplasm. Using immunofluorescence microscopy and the influenza virus mutant ts51, we found that at the nonpermissive temperature M1 was retained in the nucleus, even at late times after infection. In contrast, the viral nucleoprotein (NP), after a temporary retention in the nucleus, was distributed in the cytoplasm. Therefore, mutant M1 supported the release of the viral ribonucleoproteins from the nucleus, but not the formation of infectious virions. The point mutation in the ts51 M1 gene was predicted to encode an additional phosphorylation site. We observed a substantial increase in the incorporation of 32Pi into M1 at the nonpermissive temperature. The critical role of this phosphorylation site was demonstrated by using H89, a protein kinase inhibitor; it inhibited the expression of the mutant phenotype, as judged by M1 distribution in the cell. Immunofluorescence analysis of ts51-infected cells after treatment with H89 showed a wild-type phenotype. In summary, the data indicated that the ts51 M1 protein was hyperphosphorylated at the nonpermissive temperature and that this phosphorylation was responsible for its aberrant nuclear retention.     

Membranes,viruses, detergents,and endosomes

The fluid mosaic model for biological membranes was formulated 40 years ago. Ten years later endosomes were discovered as important prelysosomal organelles. At the outset of my research career, I was fortunate to witness both these turning points in biochemistry and cell biology from close up, and to participate in some of the studies. In this short essay, I will describe how this came about, and also try to provide some background as to the general starting situation in those not so distant pioneering years of membrane biology.     

Seasonal variability in the grazing potential of the invasive amphipod Gammarus tigrinus and the native amphipod Gammarus salinus (Amphipoda: Crustacea) in the northern Baltic Sea

Mesograzers are known to reduce the biomass of their host plant and modify the structure of the whole macrophyte community in many ecosystems. Thus, the introduction of an efficient mesograzer may destabilize macrophyte community and also affect the native grazers. We estimated how large proportion of macrophyte production are consumed by the alien gammarid G. tigrinus and the native gammarid G. salinus in the species poor ecosystem of the northern Baltic Sea. We analysed whether G. tigrinus consumes different diet as the native G. salinus and whether the effect of G. tigrinus on the survival of the native G. salinus is macrophyte species specific. Grazing experiments showed that there was a clear difference in the grazing rates of gammarids among the studied macrophyte species in summer and autumn but not in spring. The grazing rates were significantly higher in the prevailing macrophyte Pilayella littoralis as compared to other macrophytes. The grazing was inversely related to the diurnal net photosynthetic values of macrophytes. The gammarid amphipods potentially removed only a minor part of plant primary production except for summer and autumn when grazing of a few perennial species exceeded macrophyte production. Macrophyte species and presence of G. salinus had no effect on the survival of G. tigrinus. The presence of G. tigrinus, however, reduced the survival of the native gammarids within P. littoralis in summer. To conclude it is likely that both native and alien gammarid amphipods do not exert significant pressure on the macroalgal communities in the northern Baltic Sea. Competitive interactions between G. tigrinus and G. salinus within the prevailing macrophyte P. littoralis is the likely explanation of the decline of the native gammarid amphipods after the establishment of G. tigrinus in the northern Baltic Sea.