bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure
Authors: Mironov Alexander , Colanzi Antonino , Silletta Maria Giuseppina , Fiucci Giusy , Flati Silvio , Fusella Aurora , Polishchuk Roman , Mironov Alexander , Tullio Giuseppe Di , Weigert Roberto , Malhotra Vivek , Corda Daniela , Matteis Maria Antonietta De , Luini Alberto
Published: July 2002

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.
Export of Cellubrevin from the Endoplasmic Reticulum Is Controlled by BAP31
Authors: Annaert Wim G. , Becker Bernd , Kistner Ute , Reth Michael , Jahn Reinhard
Published: July 2002

Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.
Muscle β1D Integrin Reinforces the Cytoskeleton–Matrix Link: Modulation of Integrin Adhesive Function by Alternative Splicing
Authors: Belkin Alexey M. , Retta S. Francesco , Pletjushkina Olga Y. , Balzac Fiorella , Silengo Lorenzo , Fassler Reinhard , Koteliansky Victor E. , Burridge Keith , Tarone Guido
Published: July 2002

Expression of muscle-specific β1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, β1 integrin-minus cells leads to their phenotypic conversion. β1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their β1A-expressing counterparts. The transfected β1D is targeted to focal adhesions and efficiently displaces the endogenous β1A and αvβ3 integrins from the sites of cell–matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in β1D transfectants. Whereas a significant part of cellular β1A integrin is extractable in digitonin, the majority of the transfected β1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of β1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, β1A interacts more strongly with α-actinin, than β1D. Inside-out driven activation of the β1D ectodomain increases ligand binding and fibronectin matrix assembly by β1D transfectants. Phenotypic effects of β1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of β1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton– matrix link in muscle cells. This reflects the major role for β1D integrin in muscle, where extremely stable association is required for contraction.
A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris
Authors: Elgersma Ype , Elgersma-Hooisma Minetta , Wenzel Thibaut , McCaffery J. Michael , Farquhar Marilyn G. , Subramani Suresh
Published: July 2002

Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence.
Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains
Authors: Hocking Denise C. , Sottile Jane , McKeown-Longo Paula J.
Published: July 2002

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α5β1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α5β1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α5β1 integrin, suggesting that these two domains of fibronectin cannot bind to the α5β1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α5β1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.
Localization, Dynamics, and Protein Interactions Reveal Distinct Roles for ER and Golgi SNAREs
Authors: Hay Jesse C. , Klumperman Judith , Oorschot Viola , Steegmaier Martin , Kuo Christin S. , Scheller Richard H.
Published: July 2002

ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.
A-kinase Anchoring Protein 100 (AKAP100) is Localized in Multiple Subcellular Compartments in the Adult Rat Heart
Authors: Yang Jiacheng , Drazba Judith A. , Ferguson Donald G. , Bond Meredith
Published: July 2002

Stimulation of β-adrenergic receptors activates type I and II cyclic AMP–dependent protein kinase A, resulting in phosphorylation of various proteins in the heart. It has been proposed that PKA II compartmentalization by A-kinase–anchoring proteins (AKAPs) regulates cyclic AMP–dependent signaling in the cell. We investigated the expression and localization of AKAP100 in adult hearts. By immunoblotting, we identified AKAP100 in adult rat and human hearts, and showed that type I and II regulatory (RI and II) subunits of PKA are present in the rat heart. By immunofluorescence and confocal microscopy of rat cardiac myocytes and cryostat sections of rat left ventricle papillary muscles, we localized AKAP100 to the nucleus, sarcolemma, intercalated disc, and at the level of the Z-line. After double immunostaining of transverse cross-sections of the papillary muscles with AKAP100 plus α-actinin–specific antibodies or AKAP100 plus ryanodine receptor–specific antibodies, confocal images showed AKAP100 localization at the region of the transverse tubule/junctional sarcoplasmic reticulum. RI is distributed differently from RII in the myocytes. RII, but not RI, was colocalized with AKAP100 in the rat heart. Our studies suggest that AKAP100 tethers PKA II to multiple subcellular compartments for phosphorylation of different pools of substrate proteins in the heart.
Rapid Movements of Vimentin on Microtubule Tracks: Kinesin-dependent Assembly of Intermediate Filament Networks
Authors: Prahlad Veena , Yoon Miri , Moir Robert D. , Vale Ronald D. , Goldman Robert D.
Published: July 2002

The assembly and maintenance of an extended intermediate filament (IF) network in fibroblasts requires microtubule (MT) integrity. Using a green fluorescent protein–vimentin construct, and spreading BHK-21 cells as a model system to study IF–MT interactions, we have discovered a novel mechanism involved in the assembly of the vimentin IF cytoskeleton. This entails the rapid, discontinuous, and MT-dependent movement of IF precursors towards the peripheral regions of the cytoplasm where they appear to assemble into short fibrils. These precursors, or vimentin dots, move at speeds averaging 0.55 ± 0.24 μm/s. The vimentin dots colocalize with MT and their motility is inhibited after treatment with nocodazole.

Our studies further implicate a conventional kinesin in the movement of the vimentin dots. The dots colocalize with conventional kinesin as shown by indirect immunofluorescence, and IF preparations from spreading cells are enriched in kinesin. Furthermore, microinjection of kinesin antibodies into spreading cells prevents the assembly of an extended IF network. These studies provide insights into the interactions between the IF and MT systems. They also suggest a role for conventional kinesin in the distribution of non-membranous protein cargo, and the local regulation of IF assembly.
DE-Cadherin Is Required for Intercellular Motility during Drosophila Oogenesis
Authors: Niewiadomska Paulina , Godt Dorothea , Tepass Ulrich
Published: July 2002

Cadherins are involved in a variety of morphogenetic movements during animal development. However, it has been difficult to pinpoint the precise function of cadherins in morphogenetic processes due to the multifunctional nature of cadherin requirement. The data presented here indicate that homophilic adhesion promoted by Drosophila E-cadherin (DE-cadherin) mediates two cell migration events during Drosophila oogenesis. In Drosophila follicles, two groups of follicle cells, the border cells and the centripetal cells migrate on the surface of germline cells. We show that the border cells migrate as an epithelial patch in which two centrally located cells retain epithelial polarity and peripheral cells are partially depolarized. Both follicle cells and germline cells express DE-cadherin, and border cells and centripetal cells strongly upregulate the expression of DE-cadherin shortly before and during their migration. Removing DE-cadherin from either the follicle cells or the germline cells blocks migration of border cells and centripetal cells on the surface of germline cells. The function of DE-cadherin in border cells appears to be specific for migration as the formation of the border cell cluster and the adhesion between border cells are not disrupted in the absence of DE-cadherin. The speed of migration depends on the level of DE-cadherin expression, as border cells migrate more slowly when DE-cadherin activity is reduced. Finally, we show that the upregulation of DE-cadherin expression in border cells depends on the activity of the Drosophila C/EBP transcription factor that is essential for border cell migration.
Recombinant Major Vault Protein Is Targeted to Neuritic Tips of PC12 Cells
Authors: Herrmann Christine , Golkaramnay Elaheh , Inman Elisabeth , Rome Leonard , Volknandt Walter
Published: July 2002

The major vault protein (MVP) is the predominant constituent of ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of unknown function. Vaults are multimeric protein complexes with several copies of an untranslated RNA. Double labeling employing laser-assisted confocal microscopy and indirect immunofluorescence demonstrates partial colocalization of vaults with cytoskeletal elements in Chinese hamster ovary (CHO) and nerve growth factor (NGF)-treated neuronlike PC12 cells. Transfection of CHO and PC12 cells with a cDNA encoding the rat major vault protein containing a vesicular stomatitis virus glycoprotein epitope tag demonstrates that the recombinant protein is sorted into vault particles and targeted like endogenous MVPs. In neuritic extensions of differentiated PC12 cells, there is an almost complete overlap of the distribution of microtubules and vaults. A pronounced colocalization of vaults with filamentous actin can be seen in the tips of neurites. Moreover, in NGF-treated PC12 cells the location of vaults partially coincides with vesicular markers. Within the terminal tips of neurites vaults are located near secretory organelles. Our observations suggest that the vault particles are transported along cytoskeletal-based cellular tracks.
Genetic Analysis of Collagen Q: Roles in Acetylcholinesterase and Butyrylcholinesterase Assembly and in Synaptic Structure and Function
Authors: Feng Guoping , Krejci Eric , Molgo Jordi , Cunningham Jeanette M. , Massoulié Jean , Sanes Joshua R.
Published: July 2002

Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ−/− mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ−/− muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ−/− neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.
wing blister, A New Drosophila Laminin α Chain Required for Cell Adhesion and Migration during Embryonic and Imaginal Development
Authors: Martin Doris , Zusman Susan , Li Xitong , Williams Erin L. , Khare Narmada , DaRocha Sol , Chiquet-Ehrismann Ruth , Baumgartner Stefan
Published: July 2002

We report the molecular and functional characterization of a new α chain of laminin in Drosophila. The new laminin chain appears to be the Drosophila counterpart of both vertebrate α2 (also called merosin) and α1 chains, with a slightly higher degree of homology to α2, suggesting that this chain is an ancestral version of both α1 and α2 chains. During embryogenesis, the protein is associated with basement membranes of the digestive system and muscle attachment sites, and during larval stage it is found in a specific pattern in wing and eye discs. The gene is assigned to a locus called wing blister (wb), which is essential for embryonic viability. Embryonic phenotypes include twisted germbands and fewer pericardial cells, resulting in gaps in the presumptive heart and tracheal trunks, and myotubes detached from their target muscle attachment sites. Most phenotypes are in common with those observed in Drosophila laminin α3, 5 mutant embryos and many are in common with those observed in integrin mutations. Adult phenotypes show blisters in the wings in viable allelic combinations, similar to phenotypes observed in integrin genes. Mutation analysis in the eye demonstrates a function in rhabdomere organization. In summary, this new laminin α chain is essential for embryonic viability and is involved in processes requiring cell migration and cell adhesion.
Characterization and Expression of the Laminin γ3 Chain: A Novel, Non-Basement Membrane–associated, Laminin Chain
Authors: Koch Manuel , Olson Pamela F. , Albus Anne , Jin William , Hunter Dale D. , Brunken William J. , Burgeson Robert E. , Champliaud Marie-France
Published: July 2002

Laminins are heterotrimeric molecules composed of an α, a β, and a γ chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known α and β chains but containing a novel γ chain, γ3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that γ3 contains all the expected domains of a γ chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that γ3-containing laminins are likely to exist in a stable matrix.

Studies of the tissue distribution of γ3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, γ3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The γ3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of γ3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.
Molecular Characterization of Caveolin Association with the Golgi Complex: Identification of a Cis-Golgi Targeting Domain in the Caveolin Molecule
Authors: Luetterforst Robert , Stang Espen , Zorzi Natasha , Carozzi Amanda , Way Michael , Parton Robert G.
Published: July 2002

Caveolins are integral membrane proteins which are a major component of caveolae. In addition, caveolins have been proposed to cycle between intracellular compartments and the cell surface but the exact trafficking route and targeting information in the caveolin molecule have not been defined. We show that antibodies against the caveolin scaffolding domain or against the COOH terminus of caveolin-1 show a striking specificity for the Golgi pool of caveolin and do not recognize surface caveolin by immunofluorescence. To analyze the Golgi targeting of caveolin in more detail, caveolin mutants were expressed in fibroblasts. Specific mutants lacking the NH2 terminus were targeted to the cis Golgi but were not detectable in surface caveolae. Moreover, a 32–amino acid segment of the putative COOH-terminal cytoplasmic domain of caveolin-3 was targeted specifically and exclusively to the Golgi complex and could target a soluble heterologous protein, green fluorescent protein, to this compartment. Palmitoylation-deficient COOH-terminal mutants showed negligible association with the Golgi complex. This study defines unique Golgi targeting information in the caveolin molecule and identifies the cis Golgi complex as an intermediate compartment on the caveolin cycling pathway.
Myosin VA Movements in Normal and Dilute-Lethal Axons Provide Support for a Dual Filament Motor Complex
Authors: Bridgman P.C.
Published: July 2002

To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va–associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein–myosin Va tail construct. Myosin Va–associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va null) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va–associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules.
Listeria monocytogenes Exploits Normal Host Cell Processes to Spread from Cell to Cell✪
Authors: Robbins Jennifer R. , Barth Angela I. , Marquis Hélène , de Hostos Eugenio L. , Nelson W. James , Theriot Julie A.
Published: July 2002

The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1–2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.
Gemin3
Authors: Charroux Bernard , Pellizzoni Livio , Perkinson Robert A. , Shevchenko Andrej , Mann Matthias , Dreyfuss Gideon
Published: July 2002

The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.
Inhibition of Cytokinesis by a Lipid Metabolite, Psychosine
Authors: Kanazawa Takayuki , Nakamura Sachiko , Momoi Michiko , Yamaji Toshiyuki , Takematsu Hiromu , Yano Hajime , Sabe Hisataka , Yamamoto Akitsugu , Kawasaki Toshisuke , Kozutsumi Yasunori
Published: July 2002

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.
A Functional Link between Dynamin and the Actin Cytoskeleton at Podosomes
Authors: Ochoa Gian-Carlo , Slepnev Vladimir I. , Neff Lynn , Ringstad Niels , Takei Kohji , Daniell Laurie , Kim Warren , Cao Hong , McNiven Mark , Baron Roland , De Camilli Pietro
Published: July 2002

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2–binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aaG273D abolished podosomes while GFP-dynaminK44A was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.
Calreticulin Is a Receptor for Nuclear Export
Authors: Holaska James M. , Black Ben E. , Love Dona C. , Hanover John A. , Leszyk John , Paschal Bryce M.
Published: July 2002

In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.
A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II
Authors: Pellizzoni Livio , Charroux Bernard , Rappsilber Juri , Mann Matthias , Dreyfuss Gideon
Published: July 2002

The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex–associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interaction is impaired in mutant SMNs found in SMA patients. Coimmunoprecipitation demonstrated that the SMN complex is associated with pol II, snRNPs, and RHA in vivo. In vitro experiments suggest that RHA mediates the association of SMN with the COOH-terminal domain of pol II. Moreover, transfection of cells with a dominant negative mutant of SMN, SMNΔN27, causes accumulation of pol II, snRNPs, and RHA in nuclear structures that contain the known markers of gems and coiled bodies, and inhibits RNA pol I and pol II transcription in vivo. These findings indicate a functional as well as physical association of the SMN complex with pol II and suggest a role for the SMN complex in the assembly of the pol II transcription/processing machinery.
Nascent Focal Adhesions Are Responsible for the Generation of Strong Propulsive Forces in Migrating Fibroblasts
Authors: Beningo Karen A. , Dembo Micah , Kaverina Irina , Small J. Victor , Wang Yu-li
Published: July 2002

Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In addition, detailed analysis reveals that the faint, small adhesions near the leading edge transmit strong propulsive tractions, whereas large, bright, mature focal adhesions exert weaker forces. This inverse relationship is unique to the leading edge of motile cells, and is not observed in the trailing edge or in stationary cells. Furthermore, time-lapse analysis indicates that traction forces decrease soon after the appearance of focal adhesions, whereas the size and zyxin concentration increase. As focal adhesions mature, changes in structure, protein content, or phosphorylation may cause the focal adhesion to change its function from the transmission of strong propulsive forces, to a passive anchorage device for maintaining a spread cell morphology.
α-Toxin is a mediator of Staphylococcus aureus–induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling
Authors: Bantel Heike , Sinha Bhanu , Domschke Wolfram , Peters Georg , Schulze-Osthoff Klaus , Jänicke Reiner U.
Published: July 2002

Infections with Staphylococcus aureus, a common inducer of septic and toxic shock, often result in tissue damage and death of various cell types. Although S. aureus was suggested to induce apoptosis, the underlying signal transduction pathways remained elusive. We show that caspase activation and DNA fragmentation were induced not only when Jurkat T cells were infected with intact bacteria, but also after treatment with supernatants of various S. aureus strains. We also demonstrate that S. aureus–induced cell death and caspase activation were mediated by α-toxin, a major cytotoxin of S. aureus, since both events were abrogated by two different anti–α-toxin antibodies and could not be induced with supernatants of an α-toxin–deficient S. aureus strain. Furthermore, α-toxin–induced caspase activation in CD95-resistant Jurkat sublines lacking CD95, Fas-activated death domain, or caspase-8 but not in cells stably expressing the antiapoptotic protein Bcl-2. Together with our finding that α-toxin induces cytochrome c release in intact cells and, interestingly, also from isolated mitochondria in a Bcl-2-controlled manner, our results demonstrate that S. aureus α-toxin triggers caspase activation via the intrinsic death pathway independently of death receptors. Hence, our findings clearly define a signaling pathway used in S. aureus–induced cytotoxicity and may provide a molecular rationale for future therapeutic interventions in bacterial infections.
Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments
Authors: Jenkins Scott M. , Bennett Vann
Published: July 2002

The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.
Specific SHP-2 partitioning in raft domains triggers integrin-mediated signaling via Rho activation
Authors: Lacalle Rosa Ana , Mira Emilia , Gómez-Moutón Concepción , Jiménez-Baranda Sonia , Martínez-A. Carlos , Mañes Santos
Published: July 2002

Cell signaling does not occur randomly over the cell surface, but is integrated within cholesterol-enriched membrane domains, termed rafts. By targeting SHP-2 to raft domains or to a non-raft plasma membrane fraction, we studied the functional role of rafts in signaling. Serum-depleted, nonattached cells expressing the raft SHP-2 form, but not non-raft SHP-2, display signaling events resembling those observed after fibronectin attachment, such as β1 integrin clustering, 397Y-FAK phosphorylation, and ERK activation, and also increases Rho-GTP levels. Expression of the dominant negative N19Rho abrogates raft-SHP-2–induced signaling, suggesting that Rho activation is a downstream event in SHP-2 signaling. Expression of a catalytic inactive SHP-2 mutant abrogates the adhesion-induced feedback inhibition of Rho activity, suggesting that SHP-2 contributes to adhesion-induced suppression of Rho activity. Because raft recruitment of SHP-2 occurs physiologically after cell attachment, these results provide a mechanism by which SHP-2 may influence cell adhesion and migration by spatially regulating Rho activity.
Regulatory T Cells Specific for the Same Framework 3 Region of the Vβ8.2 Chain Are Involved in the Control of Collagen II–induced Arthritis and Experimental Autoimmune Encephalomyelitis
Authors: Kumar Vipin , Aziz Fatema , Sercarz Eli , Miller Alexander
Published: July 2002

Recent evidence indicates that chronic autoimmune disease can result from breakdown of regulation and subsequent activation of self-reactive T cells. In many murine autoimmune disease systems and in the Lewis rat, antigen-specific T cells utilizing the T cell receptor (TCR) Vβ8.2 gene segment play a major role. In the myelin basic protein–induced experimental autoimmune encephalomyelitis (EAE) model in H-2u mice, we had shown that T cells recognizing a peptide determinant within the framework 3 region of the Vβ8.2 chain have a critical role in influencing the course of the disease. Here, we report experiments in another disease system, collagen II (CII)–induced arthritis (CIA) in DBA/1LacJ (H-2q) mice, indicating a remarkably parallel control circuit to that found for EAE. A critical role is played by CII-specific Vβ8.2bearing T cells in the CIA system, which we have confirmed. Animals treated with the superantigen SEB before CII administration are significantly protected from CIA. Next, we tested the ability of peptides encompassing the entire Vβ8.2 chain to induce proliferative responses. Only TCR peptide B5 (amino acids 76–101), a regulatory peptide in EAE, induced proliferation. B5 was then used to vaccinate DBA/1LacJ mice and was shown to reduce greatly the severity and incidence of CIA as measured by joint inflammation or histology. Furthermore, similar protection was found when B5 was administered after CII immunization. It was shown that there is physiological induction of a proliferative response to B5 during CIA and that the determinant within B5 is produced from a single chain TCR construct containing the entire Vβ8.2 chain. Finally, the regulation of CIA is discussed in the context of other experimental autoimmune diseases, especially EAE, with emphasis on what appear to be strikingly common mechanisms.
A T Helper Cell 2 (Th2) Immune Response against Non-self Antigens Modifies the Cytokine Profile of Autoimmune T Cells and Protects against Experimental Allergic Encephalomyelitis
Authors: Falcone Marika , Bloom Barry R.
Published: July 2002

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS), and the most commonly used experimental model for multiple sclerosis. It is mediated by autoreactive T cell clones exhibiting a T helper cell (Th) 1 cytokine profile. Nonencephalitogenic T lymphocytes specific for self or exogenous antigens have been found to suppress encephalitogenic T cell responses and to protect against autoimmune disease. The mechanisms by which exogenous antigens modulate autoimmunity are not fully understood. In this study, we tested the hypothesis that a Th2-type immune response against an exogenous, nonself antigen, keyhole limpet hemocyanin (KLH), by releasing IL-4 in the microenvironment, could shift the cytokine profile of encephalitogenic T cells from an inflammatory Th1 to a protective Th2 type. SJL/J mice were preimmunized with the KLH in incomplete Freund's adjuvant to induce a population of Th2 memory cells that would be expected to release Th2 cytokines when activated by the specific antigen at the time of EAE induction. Four weeks later, mice received an encephalitogenic challenge containing guinea pig myelin in complete Freund's adjuvant with or without KLH. All KLH primed animals not receiving the exogenous antigen at the time of EAE induction developed a severe clinical disease indistinguishable from control mice not KLH primed. In contrast, animals preimmunized and challenged with the encephalitogenic inoculum containing KLH showed either no, or markedly reduced, clinical signs. Enzyme-linked immunospot analysis demonstrated that KLH-specific T cells in the primed mice were producing IL-4 characteristic of Th2 cells. In the KLH-primed and restimulated mice, the cytokine profile of the autoreactive, myelin basic protein–specific T cells was shifted from an inflammatory Th1 towards a protective Th2 type. We infer that the presence of IL-4 secreted by KLH-specific memory Th2 cells in the lymphoid system microenvironment in which the autoreactive T cells were engaged by the encephalitogenic stimulus were able to bias their cytokine profile towards a protective Th2 phenotype. This interpretation is supported by the observation that the protective effect of preimmunization with KLH was overcome by rm– IL-12, which inhibited the production of IL-4 by the Th1 cells and biased the autoimmune response to a predominantly Th1 type. Since IL-4 mRNA could not be detected by reverse transcriptase PCR in the CNS, the protective effect was inferred to be mediated by Th2 cells in the lymphoid system, and not the target organ. We conclude that exogenous, nonself antigens that can induce Th2 responses, can modify the cytokine environment sufficiently to alter the cytokine phenotype of inflammatory, autoreactive T cell clones, and ultimately, to provide significant protection against EAE and possibly other T cell–mediated autoimmune diseases.