Biomedical subjects

Ari Helenius

Publications and source records attributed to Ari Helenius.

At least 19 recordsLinked to original sources

PubMed2006

Virus entry: open sesame.

Detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. Cell biology studies, live-cell imaging, and systems biology have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. These insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. In addition, such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication.

Animals↗

PubMed2005

Simulations of (an)isotropic diffusion on curved biological surfaces.

We present a computational particle method for the simulation of isotropic and anisotropic diffusion on curved biological surfaces that have been reconstructed from image data. The method is capable of handling surfaces of high curvature and complex shape, which are often encountered in biology. The method is validated on simple benchmark problems and is shown to be second-order accurate in space and time and of high parallel efficiency. It is applied to simulations of diffusion on the membrane of endoplasmic reticula (ER) in live cells. Diffusion simulations are conducted on geometries reconstructed from real ER samples and are compared to fluorescence recovery after photobleaching experiments in the same ER samples using the transmembrane protein tsO45-VSV-G, C-terminally tagged with green fluorescent protein. Such comparisons allow derivation of geometry-corrected molecular diffusion constants for membrane components from fluorescence recovery after photobleaching data. The results of the simulations indicate that the diffusion behavior of molecules in the ER membrane differs significantly from the volumetric diffusion of soluble molecules in the lumen of the same ER. The apparent speed of recovery differs by a factor of approximately 4, even when the molecular diffusion constants of the two molecules are identical. In addition, the specific shape of the membrane affects the recovery half-time, which is found to vary by a factor of approximately 2 in different ER samples.

Animals↗

PubMed2005

Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes.

The lateral mobility of individual murine polyoma virus-like particles (VLPs) bound to live cells and artificial lipid bilayers was studied by single fluorescent particle tracking using total internal reflection fluorescence microscopy. The particle trajectories were analyzed in terms of diffusion rates and modes of motion as described by the moment scaling spectrum. Although VLPs bound to their ganglioside receptor in lipid bilayers exhibited only free diffusion, analysis of trajectories on live 3T6 mouse fibroblasts revealed three distinct modes of mobility: rapid random motion, confined movement in small zones (30-60 nm in diameter), and confined movement in zones with a slow drift. After binding to the cell surface, particles typically underwent free diffusion for 5-10 s, and then they were confined in an actin filament-dependent manner without involvement of clathrin-coated pits or caveolae. Depletion of cholesterol dramatically reduced mobility of VLPs independently of actin, whereas inhibition of tyrosine kinases had no effect on confinement. The results suggested that clustering of ganglioside molecules by the multivalent VLPs induced transmembrane coupling that led to confinement of the virus/receptor complex by cortical actin filaments.

Actins↗

PubMed2005

Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters.

Using total internal reflection fluorescence microscopy (TIR-FM), fluorescence recovery after photobleaching (FRAP), and other light microscopy techniques, we analyzed the dynamics, the activation, and the assembly of caveolae labeled with fluorescently tagged caveolin-1 (Cav1). We found that when activated by simian virus 40 (SV40), a non-enveloped DNA virus that uses caveolae for cell entry, the fraction of mobile caveolae was dramatically enhanced both in the plasma membrane (PM) and in the caveosome, an intracellular organelle that functions as an intermediate station in caveolar endocytosis. Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles. We generated heterokaryons that contained GFP- and RFP-tagged caveolae by fusing cells expressing Cav1-GFP and -RFP, respectively, and showed that even when activated, individual caveolar domains underwent little exchange of Cav1. Only when the cells were subjected to transient cholesterol depletion, did the caveolae domain exchange Cav1. Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic. Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first.

Caveolae↗

PubMed2005

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.

Animals↗

PubMed2005

Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes.

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.

ADP-Ribosylation Factor 1↗

PubMed2005

Effects of organelle shape on fluorescence recovery after photobleaching.

The determination of diffusion coefficients from fluorescence recovery data is often complicated by geometric constraints imposed by the complex shapes of intracellular compartments. To address this issue, diffusion of proteins in the lumen of the endoplasmic reticulum (ER) is studied using cell biological and computational methods. Fluorescence recovery after photobleaching (FRAP) experiments are performed in tissue culture cells expressing GFP-KDEL, a soluble, fluorescent protein, in the ER lumen. The three-dimensional (3D) shape of the ER is determined by confocal microscopy and computationally reconstructed. Within these ER geometries diffusion of solutes is simulated using the method of particle strength exchange. The simulations are compared to experimental FRAP curves of GFP-KDEL in the same ER region. Comparisons of simulations in the 3D ER shapes to simulations in open 3D space show that the constraints imposed by the spatial confinement result in two- to fourfold underestimation of the molecular diffusion constant in the ER if the geometry is not taken into account. Using the same molecular diffusion constant in different simulations, the observed speed of fluorescence recovery varies by a factor of 2.5, depending on the particular ER geometry and the location of the bleached area. Organelle shape considerably influences diffusive transport and must be taken into account when relating experimental photobleaching data to molecular diffusion coefficients. This novel methodology combines experimental FRAP curves with high accuracy computer simulations of diffusion in the same ER geometry to determine the molecular diffusion constant of the solute in the particular ER lumen.

Animals↗

PubMed2005

Minor folding defects trigger local modification of glycoproteins by the ER folding sensor GT.

UDP-glucose:glycoprotein glucosyltransferase (GT) is a key component of the glycoprotein-specific folding and quality control system in the endoplasmic reticulum. By exclusively reglucosylating incompletely folded and assembled glycoproteins, it serves as a folding sensor that prolongs the association of newly synthesized glycoproteins with the chaperone-like lectins calnexin and calreticulin. Here, we address the mechanism by which GT recognizes and labels its substrates. Using an improved inhibitor assay based on soluble conformers of pancreatic ribonuclease in its glycosylated (RNase B) and unglycosylated (RNase A) forms, we found that the protein moiety of a misfolded conformer alone is sufficient for specific recognition by GT in vitro. To investigate the relationship between recognition and glucosylation, we tested a variety of glycosylation mutants of RNase S-Protein and an RNase mutant with a local folding defect [RNase C65S, C72S], as well as a series of loop insertion mutants. The results indicated that local folding defects in an otherwise correctly folded domain could be recognized by GT. Only glycans attached to the polypeptide within the misfolded sites were glucosylated.

Endoplasmic Reticulum↗

PubMed2005

Folding and dimerization of hepatitis C virus E1 and E2 glycoproteins in stably transfected CHO cells.

The recombinant E1E2 heterodimer of the hepatitis C virus is a candidate for a subunit vaccine. Folding analysis of E1 and E2 glycoproteins, stably expressed in CHO cells, showed that E1 folding was faster and more efficient than E2. The oxidized DTT-resistant conformation of E1 was completed within 2 h post-synthesis, while E2 not only required up to 6 h but also generated non-native species. Calnexin was found to assist E1 folding, whereas no chaperone association was found with E2. The assembly of E1 and E2 was assessed by co-immunoprecipitation and sedimentation velocity analysis. We found that the formation of native E1E2 heterodimers paralleled E2 oxidation kinetics, suggesting that E2 completed its folding process after association with E1. Once formed, sedimentation of the native E1E2 heterodimers was consistent with the absence of additional associated factors. Taken together, our data strongly suggest that productive folding of the major HCV spike protein E2 is assisted by E1.

Animals↗

PubMed2005

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae.

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

ADP-Ribosylation Factor 6↗

PubMed2004

Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic.

Endocytosis comprises several routes of internalization. An outstanding question is whether the caveolar and endosomal pathways intersect. Following transport of the caveolar protein Caveolin-1 and two cargo complexes, Simian Virus 40 and Cholera toxin, in live cells, we uncovered a Rab5-dependent pathway in which caveolar vesicles are targeted to early endosomes and form distinct and stable membrane domains. In endosomes, the low pH selectively allowed the toxin to diffuse out of the caveolar domains into the surrounding membrane, while the virus remained trapped. Thus, we conclude that, unlike cyclic assembly and disassembly of coat proteins in vesicular transport, oligomeric complexes of caveolin-1 confer permanent structural stability to caveolar vesicles that transiently interact with endosomes to form subdomains and release cargo selectively by compartment-specific cues.

Caveolae↗

PubMed2004

Echovirus 1 endocytosis into caveosomes requires lipid rafts, dynamin II, and signaling events.

Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the alpha2beta1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.

Animals↗

PubMed2004

How viruses enter animal cells.

Viruses replicate within living cells and use the cellular machinery for the synthesis of their genome and other components. To gain access, they have evolved a variety of elegant mechanisms to deliver their genes and accessory proteins into the host cell. Many animal viruses take advantage of endocytic pathways and rely on the cell to guide them through a complex entry and uncoating program. In the dialogue between the cell and the intruder, the cell provides critical cues that allow the virus to undergo molecular transformations that lead to successful internalization, intra-cellular transport, and uncoating.

Active Transport, Cell Nucleus↗

PubMed2004

ERp57 is a multifunctional thiol-disulfide oxidoreductase.

The thiol-disulfide oxidoreductase ERp57 is a soluble protein of the endoplasmic reticulum and the closest known homologue of protein disulfide isomerase. The protein interacts with the two lectin chaperones calnexin and calreticulin and thereby promotes the oxidative folding of newly synthesized glycoproteins. Here we have characterized several fundamental structural and functional properties of ERp57 in vitro, such as the domain organization, shape, redox potential, and the ability to catalyze different thiol-disulfide exchange reactions. Like protein disulfide isomerase, we find ERp57 to be comprised of four structural domains. The protein has an elongated shape of 3.4 +/- 0.1 nm in diameter and 16.8 +/- 0.5 nm in length. The two redox-active a and a' domains were determined to have redox potentials of -0.167 and -0.156 V, respectively. Furthermore, ERp57 was shown to efficiently catalyze disulfide reduction, disulfide isomerization, and dithiol oxidation in substrate proteins. The implications of these findings for the function of the protein in vivo are discussed.

Catalysis↗

PubMed2004

Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.

Animals↗

PubMed2004

Mutational analysis provides molecular insight into the carbohydrate-binding region of calreticulin: pivotal roles of tyrosine-109 and aspartate-135 in carbohydrate recognition.

Calreticulin (CRT) is a lectin chaperone present in the lumen of the endoplasmic reticulum. It interacts with various glycoproteins by binding via their attached Glc(1)Man(9)GlcNAc(2) moiety. To provide further insight into these lectin-glycan interactions, we are investigating the interaction of CRT with various sugars. We have earlier modeled the complex between CRT and the Glc(1)Man(3) tetrasaccharide, a derivative of the native Glc(1)Man(9)GlcNAc(2) sugar moiety. Here, we have systematically mutated the residues implicated by the model in the interaction of CRT to its sugar substrates and categorized the role played by each of the subsites of calreticulin toward the glycan binding. The CRT mutants Y109F and D135L did not show any binding to the sugar substrates interacting with the wild-type protein, demonstrating the great importance of these residues in the carbohydrate-binding site of CRT. Also, D317L and M131A showed weak affinity toward the trisaccharide. The mutation of residues from the primary binding site of CRT, i.e., those interacting with glucose, appears to be far less tolerated as compared to mutations in residues that interact with the mannose residues of the glycan. Also, methyl-2-deoxy-glucopyranosyl-alpha(1-->3)-mannopyranoside failed to bind, asserting to the significance of the interactions between the primary binding site of CRT and the 2'-OH of the glucose residue of the oligosaccharide substrate in generating specificity for this recognition. These studies provide detailed molecular insight into the sugar binding specificity of CRT.

Amino Acid Sequence↗

PubMed2004

Roles of N-linked glycans in the endoplasmic reticulum.

From a process involved in cell wall synthesis in archaea and some bacteria, N-linked glycosylation has evolved into the most common covalent protein modification in eukaryotic cells. The sugars are added to nascent proteins as a core oligosaccharide unit, which is then extensively modified by removal and addition of sugar residues in the endoplasmic reticulum (ER) and the Golgi complex. It has become evident that the modifications that take place in the ER reflect a spectrum of functions related to glycoprotein folding, quality control, sorting, degradation, and secretion. The glycans not only promote folding directly by stabilizing polypeptide structures but also indirectly by serving as recognition "tags" that allow glycoproteins to interact with a variety of lectins, glycosidases, and glycosyltranferases. Some of these (such as glucosidases I and II, calnexin, and calreticulin) have a central role in folding and retention, while others (such as alpha-mannosidases and EDEM) target unsalvageable glycoproteins for ER-associated degradation. Each residue in the core oligosaccharide and each step in the modification program have significance for the fate of newly synthesized glycoproteins.

Carbohydrate Sequence↗

PubMed2003

Ganglioside-dependent cell attachment and endocytosis of murine polyomavirus-like particles.

For murine polyomavirus (Py), previous studies suggest the cellular target is a terminal alpha2,3-linked sialic acid. Here, we investigate the binding and uptake of mouse polyomavirus-like particles (PyVLP) derived from bacterially expressed VP1. We find that in fibroblast 3T6 cells, binding of PyVLP was substantially reduced by sialidase treatment, but only moderately affected by protease treatment, suggesting glycolipids such as the sialic acid-containing gangliosides mediate cell attachment. We further tested the entry requirement of PyVLP using the ganglioside-deficient GM95 murine cell line, and find PyVLP binding and entry were reduced in these cells. Finally, we find that addition of gangliosides G(M1), G(D1a), and G(T1b) to GM95 cells restored cellular PyVLP binding and uptake. Taken together, results indicate that gangliosides function in PyVLP cell attachment and endocytosis.

Animals↗