bluuebunny/crossref_metadata_2025 huggingface dataset:
CRP1, a LIM Domain Protein Implicated in Muscle Differentiation, Interacts with α-Actinin
Authors: Pomiès Pascal , Louis Heather A. , Beckerle Mary C.
Published: July 2002

Members of the cysteine-rich protein (CRP) family are LIM domain proteins that have been implicated in muscle differentiation. One strategy for defining the mechanism by which CRPs potentiate myogenesis is to characterize the repertoire of CRP binding partners. In order to identify proteins that interact with CRP1, a prominent protein in fibroblasts and smooth muscle cells, we subjected an avian smooth muscle extract to affinity chromatography on a CRP1 column. A 100-kD protein bound to the CRP1 column and could be eluted with a high salt buffer; Western immunoblot analysis confirmed that the 100-kD protein is α-actinin. We have shown that the CRP1–α-actinin interaction is direct, specific, and saturable in both solution and solid-phase binding assays. The Kd for the CRP1–α-actinin interaction is 1.8 ± 0.3 μM. The results of the in vitro protein binding studies are supported by double-label indirect immunofluorescence experiments that demonstrate a colocalization of CRP1 and α-actinin along the actin stress fibers of CEF and smooth muscle cells. Moreover, we have shown that α-actinin coimmunoprecipitates with CRP1 from a detergent extract of smooth muscle cells. By in vitro domain mapping studies, we have determined that CRP1 associates with the 27-kD actin–binding domain of α-actinin. In reciprocal mapping studies, we showed that α-actinin interacts with CRP1-LIM1, a deletion fragment that contains the NH2-terminal 107 amino acids (aa) of CRP1. To determine whether the α-actinin binding domain of CRP1 would localize to the actin cytoskeleton in living cells, expression constructs encoding epitope-tagged full-length CRP1, CRP1-LIM1(aa 1-107), or CRP1-LIM2 (aa 108-192) were microinjected into cells. By indirect immunofluorescence, we have determined that full-length CRP1 and CRP1-LIM1 localize along the actin stress fibers whereas CRP1-LIM2 fails to associate with the cytoskeleton. Collectively these data demonstrate that the NH2-terminal part of CRP1 that contains the α-actinin–binding site is sufficient to localize CRP1 to the actin cytoskeleton. The association of CRP1 with α-actinin may be critical for its role in muscle differentiation.
The Microtubule-dependent Motor Centromere–associated Protein E (CENP-E) Is an Integral Component of Kinetochore Corona Fibers That Link Centromeres to Spindle Microtubules
Authors: Yao Xuebiao , Anderson Karen L. , Cleveland Don W.
Published: July 2002

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Using immunoelectron microscopy, CENP-E is shown to be an integral component of the kinetochore corona fibers that tether centromeres to the spindle. Immediately upon nuclear envelope fragmentation, an associated plus end motor trafficks cytoplasmic CENP-E toward chromosomes along astral microtubules that enter the nuclear volume. Before or concurrently with initial lateral attachment of spindle microtubules, CENP-E targets to the outermost region of the developing kinetochores. After stable attachment, throughout chromosome congression, at metaphase, and throughout anaphase A, CENP-E is a constituent of the corona fibers, extending at least 50 nm away from the kinetochore outer plate and intertwining with spindle microtubules. In congressing chromosomes, CENP-E is preferentially associated with (or accessible at) the stretched, leading kinetochore known to provide the primary power for chromosome movement. Taken together, this evidence strongly supports a model in which CENP-E functions in congression to tether kinetochores to the disassembling microtubule plus ends.
Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations That Leads to Stable Epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells
Authors: Lochter André , Galosy Sybille , Muschler John , Freedman Neal , Werb Zena , Bissell Mina J.
Published: July 2002

Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell–cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.
The Integrin α6β4 Functions in Carcinoma Cell Migration on Laminin-1 by Mediating the Formation and Stabilization of Actin-containing Motility Structures
Authors: Rabinovitz Isaac , Mercurio Arthur M.
Published: July 2002

Functional studies on the α6β4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the α6β4 integrin in clone A cells, a colon carcinoma cell line that expresses α6β4 but no α6β1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the α6β4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the α6β4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although β1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the α6β4 integrin and F-actin was seen. An association between α6β4 and F-actin is supported by the fact that α6β4 integrin and actin were released from clone A cells by treatment with the F-actin– severing protein gelsolin and that α6β4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, α6β4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited α6β4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an α6-specific antibody. Together, these results indicate that the α6β4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.
Vascular Endothelial Growth Factor Induces Endothelial Fenestrations In Vitro
Authors: Esser Sybille , Wolburg Karen , Wolburg Hartwig , Breier Georg , Kurzchalia Teymuras , Risau Werner
Published: July 2002

Abstract. Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1–specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.
CAS/Crk Coupling Serves as a “Molecular Switch” for Induction of Cell Migration
Authors: Klemke Richard L. , Leng Jie , Molander Rachel , Brooks Peter C. , Vuori Kristiina , Cheresh David A.
Published: July 2002

Abstract. Carcinoma cells selected for their ability to migrate in vitro showed enhanced invasive properties in vivo. Associated with this induction of migration was the anchorage-dependent phosphorylation of p130CAS (Crk-associated substrate), leading to its coupling to the adaptor protein c-CrkII (Crk). In fact, expression of CAS or its adaptor protein partner Crk was sufficient to promote cell migration, and this depended on CAS tyrosine phosphorylation facilitating an SH2-mediated complex with Crk. Cytokine-stimulated cell migration was blocked by CAS lacking the Crk binding site or Crk containing a mutant SH2 domain. This migration response was characterized by CAS/Crk localization to membrane ruffles and blocked by the dominant-negative GTPase, Rac, but not Ras. Thus, CAS/Crk assembly serves as a “molecular switch” for the induction of cell migration and appears to contribute to the invasive property of tumors.
FH3, A Domain Found in Formins, Targets the Fission Yeast Formin Fus1 to the Projection Tip During Conjugation
Authors: Petersen Janni , Nielsen Olaf , Egel Richard , Hagan Iain M.
Published: July 2002

Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1+ with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain–GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.
Differential Nuclear Translocation and Transactivation Potential of β-Catenin and Plakoglobin
Authors: Simcha Inbal , Shtutman Michael , Salomon Daniela , Zhurinsky Jacob , Sadot Einat , Geiger Benjamin , Ben-Ze'ev Avri
Published: July 2002

β-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together with lymphoid-enhancing binding/T cell (LEF/TCF) transcription factors. Here we compared the nuclear translocation and transactivation abilities of β-catenin and plakoglobin in mammalian cells. Overexpression of each of the two proteins in MDCK cells resulted in nuclear translocation and formation of nuclear aggregates. The β-catenin-containing nuclear structures also contained LEF-1 and vinculin, while plakoglobin was inefficient in recruiting these molecules, suggesting that its interaction with LEF-1 and vinculin is significantly weaker. Moreover, transfection of LEF-1 translocated endogenous β-catenin, but not plakoglobin to the nucleus. Chimeras consisting of Gal4 DNA-binding domain and the transactivation domains of either plakoglobin or β-catenin were equally potent in transactivating a Gal4-responsive reporter, whereas activation of LEF-1– responsive transcription was significantly higher with β-catenin. Overexpression of wild-type plakoglobin or mutant β-catenin lacking the transactivation domain induced accumulation of the endogenous β-catenin in the nucleus and LEF-1–responsive transactivation. It is further shown that the constitutive β-catenin–dependent transactivation in SW480 colon carcinoma cells and its nuclear localization can be inhibited by overexpressing N-cadherin or α-catenin. The results indicate that (a) plakoglobin and β-catenin differ in their nuclear translocation and complexing with LEF-1 and vinculin; (b) LEF-1–dependent transactivation is preferentially driven by β-catenin; and (c) the cytoplasmic partners of β-catenin, cadherin and α-catenin, can sequester it to the cytoplasm and inhibit its transcriptional activity.
Cyclic Nucleotide-gated Channels on the Flagellum Control Ca2+ Entry into Sperm
Authors: Wiesner Burkhard , Weiner Jocelyn , Middendorff Ralf , Hagen Volker , Kaupp U. Benjamin , Weyand Ingo
Published: July 2002

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (α and β). The α subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of β subunits in testis. The α and β subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the α subunit is observed along the entire flagellum, whereas the short β subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of α and β subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.
The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1
Authors: Serini Guido , Bochaton-Piallat Marie-Luce , Ropraz Patricia , Geinoz Antoine , Borsi Laura , Zardi Luciano , Gabbiani Giulio
Published: July 2002

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.
The Perinucleolar Compartment and Transcription
Authors: Huang Sui , Deerinck Thomas J. , Ellisman Mark H. , Spector David L.
Published: July 2002

The perinucleolar compartment (PNC) is a unique nuclear structure localized at the periphery of the nucleolus. Several small RNAs transcribed by RNA polymerase III and two hnRNP proteins have been localized in the PNC (Ghetti, A., S. Piñol-Roma, W.M. Michael, C. Morandi, and G. Dreyfuss. 1992. Nucleic Acids Res. 20:3671–3678; Matera, A.G., M.R. Frey, K. Margelot, and S.L. Wolin. 1995. J. Cell Biol. 129:1181– 1193; Timchenko, L.T., J.W. Miller, N.A. Timchenko, D.R. DeVore, K.V. Datar, L. Lin, R. Roberts, C.T. Caskey, and M.S. Swanson. 1996. Nucleic Acids Res. 24: 4407–4414; Huang, S., T. Deerinck, M.H. Ellisman, and D.L. Spector. 1997. J. Cell Biol. 137:965–974). In this report, we show that the PNC incorporates Br-UTP and FITC-conjugated CTP within 5 min of pulse labeling. Selective inhibition of RNA polymerase I does not appreciably affect the nucleotide incorporation in the PNC. Inhibition of all RNA polymerases by actinomycin D blocks the incorporation completely, suggesting that Br-UTP incorporation in the PNC is due to transcription by RNA polymerases II and/or III. Treatment of cells with an RNA polymerase II and III inhibitor induces a significant reorganization of the PNC. In addition, double labeling experiments showed that poly(A) RNA and some of the factors required for pre-mRNA processing were localized in the PNC in addition to being distributed in their previously characterized nucleoplasmic domains. Fluorescence recovery after photobleaching (FRAP) analysis revealed a rapid turnover of polypyrimidine tract binding protein within the PNC, demonstrating the dynamic nature of the structure. Together, these findings suggest that the PNC is a functional compartment involved in RNA metabolism in the cell nucleus.
When Overexpressed, a Novel Centrosomal Protein, RanBPM, Causes Ectopic Microtubule Nucleation Similar to γ-Tubulin
Authors: Nakamura Masafumi , Masuda Hirohisa , Horii Johji , Kuma Kei-ichi , Yokoyama Nobuhiko , Ohba Tomoyuki , Nishitani Hideo , Miyata Takashi , Tanaka Masao , Nishimoto Takeharu
Published: July 2002

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with γ-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to γ-tubulin was faded by the addition of GTPγS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.
In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis
Authors: Obermann Wolfgang M.J. , Sondermann Holger , Russo Alicia A. , Pavletich Nikola P. , Hartl F. Ulrich
Published: July 2002

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.
Differences in the Localization and Morphology of Chromosomes in the Human Nucleus
Authors: Croft Jenny A. , Bridger Joanna M. , Boyle Shelagh , Perry Paul , Teague Peter , Bickmore Wendy A.
Published: July 2002

Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.
Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2
Authors: Gijón Miguel A. , Spencer Diane M. , Kaiser Alan L. , Leslie Christina C.
Published: July 2002

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.
I-Band Titin in Cardiac Muscle Is a Three-Element Molecular Spring and Is Critical for Maintaining Thin Filament Structure
Authors: Linke Wolfgang A. , Rudy Diane E. , Centner Thomas , Gautel Mathias , Witt Christian , Labeit Siegfried , Gregorio Carol C.
Published: July 2002

In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment.

We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends ∼60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH2 terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH2-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B–titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.
Crystal Structure of the Cytosolic C2a-C2b Domains of Synaptotagmin III
Authors: Sutton R. Bryan , Ernst James A. , Brunger Axel T.
Published: July 2002

Synaptotagmins are synaptic vesicle-associated, phospholipid-binding proteins most commonly associated with Ca+2-dependent exocytotic and Ca+2- independent endocytotic events. Synaptotagmin III is a 63.2-kD member of the synaptotagmin homology group; one of its characteristic properties is the ability to bind divalent cations and accessory proteins promiscuously. In the cytosolic portion of this protein, a flexible seven–amino acid linker joins two homologous C2 domains. The C2A domain binds to phospholipid membranes and other accessory proteins in a divalent cation-dependent fashion. The C2B domain promotes binding to other C2B domains, as well as accessory proteins independent of divalent cations. The 3.2 Å crystal structure of synaptotagmin III, residues 295–566, which includes the C2A and C2B domains, exhibits differences in the shape of the Ca+2-binding pocket, the electrostatic surface potential, and the stoichiometry of bound divalent cations for the two domains. These observations may explain the disparate binding properties of the two domains. The C2A and the C2B domains do not interact; synaptotagmin, therefore, covalently links two independent C2 domains, each with potentially different binding partners. A model of synaptotagmin's involvement in Ca+2-dependent regulation of membrane fusion through its interaction with the SNARE complex is presented.
The Cysteine-Rich Domain of Human Adam 12 Supports Cell Adhesion through Syndecans and Triggers Signaling Events That Lead to β1 Integrin–Dependent Cell Spreading
Authors: Iba Kousuke , Albrechtsen Reidar , Gilpin Brent , Fröhlich Camilla , Loechel Frosty , Zolkiewska Anna , Ishiguro Kazuhiro , Kojima Tetsuhito , Liu Wei , Langford J. Kevin , Sanderson Ralph D. , Brakebusch Cord , Fässler Reinhard , Wewer Ulla M.
Published: July 2002

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin β1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking β1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain–mediated cell adhesion, and then the β1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn2+ or the β1 integrin–activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12–syndecan complex fails to modulate the function of β1 integrin.
Sphingomyelin Hydrolysis to Ceramide during the Execution Phase of Apoptosis Results from Phospholipid Scrambling and Alters Cell-Surface Morphology
Authors: Tepper Annemiek D. , Ruurs Paula , Wiedmer Therese , Sims Peter J. , Borst Jannie , van Blitterswijk Wim J.
Published: July 2002

Apoptosis is generally accompanied by a late phase of ceramide (Cer) production, the significance of which is unknown. This study describes a previously unrecognized link between Cer accumulation and phosphatidylserine (PS) exposure at the cell surface, a characteristic of the execution phase of apoptosis resulting from a loss of plasma membrane phospholipid asymmetry. Using a fluorescent sphingomyelin (SM) analogue, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]–sphingosylphosphorylcholine (C6-NBD-SM), we show that Cer is derived from SM, initially located in the outer leaflet of the plasma membrane, which gains access to a cytosolic SMase by flipping to the inner leaflet in a process of lipid scrambling paralleling PS externalization. Lipid scrambling is both necessary and sufficient for SM conversion: Ca2+ ionophore induces both PS exposure and SM hydrolysis, whereas scrambling-deficient Raji cells do not show PS exposure or Cer formation. Cer is not required for mitochondrial or nuclear apoptotic features since these are still observed in Raji cells. SM hydrolysis facilitates cholesterol efflux to methyl-β-cyclodextrin, which is indicative of a loss of tight SM–cholesterol interaction in the plasma membrane. We provide evidence that these biophysical alterations in the lipid bilayer are essential for apoptotic membrane blebbing/vesiculation at the cell surface: Raji cells show aberrant apoptotic morphology, whereas replenishment of hydrolyzed SM by C6- NBD-SM inhibits blebbing in Jurkat cells. Thus, SM hydrolysis, during the execution phase of apoptosis, results from a loss of phospholipid asymmetry and contributes to structural changes at the plasma membrane.
HIV-1 Rev Depolymerizes Microtubules to Form Stable Bilayered Rings
Authors: Watts Norman R. , Sackett Dan L. , Ward Rita D. , Miller Mill W. , Wingfield Paul T. , Stahl Stephen S. , Steven Alasdair C.
Published: July 2002

We describe a novel interaction between HIV-1 Rev and microtubules (MTs) that results in the formation of bilayered rings that are 44–49 nm in external diameter, 3.4–4.2 MD (megadaltons) in mass, and have 28-, 30-, or 32-fold symmetry. Ring formation is not sensitive to taxol, colchicine, or microtubule-associated proteins, but requires Mg2+ and is inhibited by maytansine. The interaction involves the NH2-terminal domain of Rev and the face of tubulin exposed on the exterior of the MTs. The NH2-terminal half of Rev has unexpected sequence similarity to the tubulin-binding portion of the catalytic/motor domains of the microtubule-destabilizing Kin I kinesins. We propose a model wherein binding of Rev dimers to MTs at their ends causes segments of two neighboring protofilaments to peel off and close into rings, circumferentially containing 14, 15, or 16 tubulin heterodimers, with Rev bound on the inside. Rev has a strong inhibitory effect on aster formation in Xenopus egg extracts, demonstrating that it can interact with tubulin in the presence of normal levels of cellular constituents. These results suggest that Rev may interact with MTs to induce their destabilization, a proposition consistent with the previously described disruption of MTs after HIV-1 infection.
Profilin Enhances Cdc42-Induced Nucleation of Actin Polymerization
Authors: Yang Changsong , Huang Minzhou , DeBiasio John , Pring Martin , Joyce Michael , Miki Hiroaki , Takenawa Tadaomi , Zigmond Sally H.
Published: July 2002

We find that profilin contributes in several ways to Cdc42-induced nucleation of actin filaments in high speed supernatant of lysed neutrophils. Depletion of profilin inhibited Cdc42-induced nucleation; re-addition of profilin restored much of the activity. Mutant profilins with a decreased affinity for either actin or poly-l-proline were less effective at restoring activity. Whereas Cdc42 must activate Wiskott-Aldrich Syndrome protein (WASP) to stimulate nucleation by the Arp2/3 complex, VCA (verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASP) constitutively activates the Arp2/3 complex. Nucleation by VCA was not inhibited by profilin depletion. With purified N-WASP and Arp2/3 complex, Cdc42-induced nucleation did not require profilin but was enhanced by profilin, wild-type profilin being more effective than mutant profilin with reduced affinity for poly-l-proline.

Nucleation by the Arp2/3 complex is a function of the free G-actin concentration. Thus, when profilin addition decreased the free G-actin concentration, it inhibited Cdc42- and VCA-induced nucleation. However, when profilin was added with G-actin in a ratio that maintained the initial free G-actin concentration, it increased the rate of both Cdc42- and VCA-induced nucleation. This enhancement, also seen with purified proteins, was greatest when the free G-actin concentration was low. These data suggest that under conditions present in intact cells, profilin enhances nucleation by activated Arp2/3 complex.
Requirement for Matrix Metalloproteinase Stromelysin-3 in Cell Migration and Apoptosis during Tissue Remodeling in Xenopus laevis
Authors: Ishizuya-Oka Atsuko , Li Qing , Amano Tosikazu , Damjanovski Sashko , Ueda Shuichi , Shi Yun-Bo
Published: July 2002

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) was originally discovered as a gene whose expression was associated with human breast cancer carcinomas and with apoptosis during organogenesis and tissue remodeling. It has been shown previously, in our studies as well as those by others, that ST3 mRNA is highly upregulated during apoptotic tissue remodeling during Xenopus laevis metamorphosis. Using a function-blocking antibody against the catalytic domain of Xenopus ST3, we demonstrate here that ST3 protein is specifically expressed in the cells adjacent to the remodeling extracellular matrix (ECM) that lies beneath the apoptotic larval intestinal epithelium in X. laevis in vivo, and during thyroid hormone–induced intestinal remodeling in organ cultures. More importantly, addition of this antibody, but not the preimmune antiserum or unrelated antibodies, to the medium of intestinal organ cultures leads to an inhibition of thyroid hormone–induced ECM remodeling, apoptosis of the larval epithelium, and the invasion of the adult intestinal primodia into the connective tissue, a process critical for adult epithelial morphogenesis. On the other hand, the antibody has little effect on adult epithelial cell proliferation. Furthermore, a known MMP inhibitor can also inhibit epithelial transformation in vitro. These results indicate that ST3 is required for cell fate determination and cell migration during morphogenesis, most likely through ECM remodeling.
The Dynamin-Related Gtpase, Mgm1p, Is an Intermembrane Space Protein Required for Maintenance of Fusion Competent Mitochondria
Authors: Wong Edith D. , Wagner Jennifer A. , Gorsich Steven W. , McCaffery J. Michael , Shaw Janet M. , Nunnari Jodi
Published: July 2002

Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear. To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles. Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments. This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion. Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells. However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating. This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion. Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space. Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events. In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion.
βiv Spectrin, a New Spectrin Localized at Axon Initial Segments and Nodes of Ranvier in the Central and Peripheral Nervous System
Authors: Berghs Stanny , Aggujaro Diego , Dirkx Ronald , Maksimova Elena , Stabach Paul , Hermel Jean-Michel , Zhang Jian-Ping , Philbrick William , Slepnev Vladimir , Ort Tatiana , Solimena Michele
Published: July 2002

We report the identification of βIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The βIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of βIV spectrin generates at least four distinct isoforms, numbered βIVΣ1–βIVΣ4 spectrin. The longest isoform (βIVΣ1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. βIVΣ2 and βIVΣ3 spectrin encompass the NH2- and COOH-terminal halves of βIVΣ1 spectrin, respectively, while βIVΣ4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of βIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, βIVΣ1 spectrin is recognized as a protein of 250 kD. Anti–βIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from βIVΣ1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for βIV spectrin is more prominent in α than in β cells. In brain, βIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrinG 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, βIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, βIVΣ1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that βIVΣ1 spectrin interacts with ankyrinG 480/270-kD and participates in the clustering of voltage-gated Na+ channels and cell-adhesion molecules at initial segments and nodes of Ranvier.
Intraarterial Injection of Muscle-Derived Cd34+Sca-1+ Stem Cells Restores Dystrophin in mdx Mice
Authors: Torrente Yuan , Tremblay Jacques-P , Pisati Federica , Belicchi Marzia , Rossi Barbara , Sironi Manuela , Fortunato Franco , El Fahime Mostafa , D'Angelo Maria Grazia , Caron Nicolas J. , Constantin Gabriela , Paulin Denise , Scarlato Guglielmo , Bresolin Nereo
Published: July 2002

Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% β-galactosidase–positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.
The Dystroglycan Complex Is Necessary for Stabilization of Acetylcholine Receptor Clusters at Neuromuscular Junctions and Formation of the Synaptic Basement Membrane
Authors: Jacobson Christian , Côté Patrice D. , Rossi Susana G. , Rotundo Richard L. , Carbonetto Salvatore
Published: July 2002

The dystrophin-associated protein (DAP) complex spans the sarcolemmal membrane linking the cytoskeleton to the basement membrane surrounding each myofiber. Defects in the DAP complex have been linked previously to a variety of muscular dystrophies. Other evidence points to a role for the DAP complex in formation of nerve–muscle synapses. We show that myotubes differentiated from dystroglycan−/− embryonic stem cells are responsive to agrin, but produce acetylcholine receptor (AChR) clusters which are two to three times larger in area, about half as dense, and significantly less stable than those on dystroglycan+/+ myotubes. AChRs at neuromuscular junctions are similarly affected in dystroglycan-deficient chimeric mice and there is a coordinate increase in nerve terminal size at these junctions. In culture and in vivo the absence of dystroglycan disrupts the localization to AChR clusters of laminin, perlecan, and acetylcholinesterase (AChE), but not rapsyn or agrin. Treatment of myotubes in culture with laminin induces AChR clusters on dystroglycan+/+, but not −/− myotubes. These results suggest that dystroglycan is essential for the assembly of a synaptic basement membrane, most notably by localizing AChE through its binding to perlecan. In addition, they suggest that dystroglycan functions in the organization and stabilization of AChR clusters, which appear to be mediated through its binding of laminin.
The Transcription Coactivator Cbp Is a Dynamic Component of the Promyelocytic Leukemia Nuclear Body
Authors: Boisvert François-Michel , Kruhlak Michael J. , Box Alan K. , Hendzel Michael J. , Bazett-Jones David P.
Published: July 2002

The transcription coactivator and histone acetyltransferase CAMP response element–binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.