Biomedical subjects

B M Jockusch

Publications and source records attributed to B M Jockusch.

At least 19 recordsLinked to original sources

PubMed2005

Conformation-specific antibodies reveal distinct actin structures in the nucleus and the cytoplasm.

For many years the existence of actin in the nucleus has been doubted because of the lack of phalloidin staining as well as the failure to document nuclear actin filaments by electron microscopy. More recent findings reveal actin to be a component of chromatin remodeling complexes and of the machinery involved in RNA synthesis and transport. With distinct functions for nuclear actin emerging, the quest for its conformation and oligomeric/polymeric structure in the nucleus has resumed importance. We used chemically cross-linked 'lower dimer' (LD) to generate mouse monoclonal antibodies specific for different actin conformations. One of the resulting antibodies, termed 1C7, recognizes an epitope that is buried in the F-actin filament, but is surface-exposed in G-actin as well as in the LD. In immunofluorescence studies with different cell lines, 1C7 selectively reacts with non-filamentous actin in the cytoplasm. In addition, it detects a discrete form of actin in the nucleus, which is different from the nuclear actin revealed by the previously described 2G2 [Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., Hinssen, H., 1999. J. Cell Sci. 112, 797]. Upon latrunculin-induced disassembly of the filamentous cytoskeleton in Rat2 fibroblasts, we observed a perinuclear accumulation of the 1C7-reactive actin conformation. In addition, latrunculin treatment led to the assembly of phalloidin-staining actin structures in chromatin-free regions of the nucleus in these cells. Our results indicate that distinct actin conformations and/or structures are present in the nucleus and the cytoplasm of different cell types and that their distribution varies in response to external signals.

Actin Cytoskeleton↗

PubMed2001

Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins.

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and alpha-actinin and colocalizes with vinculin/metavinculin and alpha-actinin at microfilament attachment sites, such as cell-cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

Actin Cytoskeleton↗

PubMed2001

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin.

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).

Actins↗

PubMed2001

3-Dimensional organization of the N-terminal vinculin head fragment.

The cytoskeleton-associated protein vinculin is composed of a globular head and an elongated tail domain. The protein can be cleaved by V8 protease treatment into two fragments with apparent molecular masses of 90 and 29/27 kDa, respectively. So far, no high-resolution data on the tertiary structure of the N-terminal 90-kDa fragment are available. We analyzed the 90-kDa fragment in detail, using electron spectroscopic imaging in conjunction with modelling experiments. The front view projection of this fragment appears roughly rhomboidal, with 4 intensity maxima arranged at the vertices and a stain-filled region in the center. Based on a detailed examination of different particle projections, a 3-dimensional model was constructed which appears as a flattened tetrahedron. A comparison of the 90-kDa fragment with the intact protein allows for a correlation between the subdomain organization of the vinculin head and the biochemically defined V8 protease cleavage sites (aa 851 and 857).

Animals↗

PubMed2001

Characterization of functional domains of mDia1, a link between the small GTPase Rho and the actin cytoskeleton.

The widely expressed diaphanous proteins, a subclass of formins, comprise links between the Rho GTPases and the actin-based cytoskeleton. They contain several functional domains that are thought to be responsible for interaction with different ligands: the FH1 domain for binding the actin-associated protein profilin; the RBD for targeting activated Rho; and the C-terminal CIID module for autoregulation of the overall diaphanous activity. Using deletion constructs of the murine mDia1, we have analyzed the functional properties of these three domains separately in in vitro assays and in transiently and stably transfected cell lines. We show that the proline-rich FH1 domain effectively binds to profilins in vitro as well as in cells, that the RBD complexes with the CIID in a species-restricted manner and that overexpression of RBD causes spontaneous ruffling and loss of stress fibers, together with loss of directional motility. Supertransfection of cells stably expressing the RBD with dominant negative Rac effectively suppresses ruffling. Our data contribute to the understanding of the function of these domains in linking the actin cytoskeleton with the Rho-signaling cascade. Furthermore, they suggest that inactivation of Rho by exogenous RBD causes upregulation of Rac activity in the transfected cells.

3T3 Cells↗

PubMed2000

Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin.

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.

Actins↗

PubMed2000

Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1.

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells.

Animals↗

PubMed2000

Functional characterization of green fluorescent protein-profilin fusion proteins.

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.

3T3 Cells↗

PubMed2000

The armadillo repeat region targets ARVCF to cadherin-based cellular junctions.

The cytoplasmic domain of the transmembrane protein M-cadherin is involved in anchoring cytoskeletal elements to the plasma membrane at cell-cell contact sites. Several members of the armadillo repeat protein family mediate this linkage. We show here that ARVCF, a member of the p120 (ctn) subfamily, is a ligand for the cytoplasmic domain of M-cadherin, and characterize the regions involved in this interaction in detail. Complex formation in an in vivo environment was demonstrated in (1) yeast two-hybrid screens, using a cDNA library from differentiating skeletal muscle and part of the cytoplasmic M-cadherin tail as a bait, and (2) mammalian cells, using a novel experimental system, the MOM recruitment assay. Immunoprecipitation and in vitro binding assays confirmed this interaction. Ectopically expressed EGFP-ARVCF-C11, an N-terminal truncated fragment, targets to junctional structures in epithelial MCF7 cells and cardiomyocytes, where it colocalizes with the respective cadherins, beta-catenin and p120 (ctn). Hence, the N terminus of ARVCF is not required for junctional localization. In contrast, deletion of the four N-terminal armadillo repeats abolishes this ability in cardiomyocytes. Detailed mutational analysis revealed the armadillo repeat region of ARVCF as sufficient and necessary for interaction with the 55 membrane-proximal amino acids of the M-cadherin tail.

Amino Acid Sequence↗

PubMed2000

Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed.

The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role. We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells. Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria. The in vitro ligand-binding properties of this construct, e.g. the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP. Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells. In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria. In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria. Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria. Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility.

Actins↗

PubMed1999

A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the loss of alpha-motoneurons in the spinal cord followed by atrophy of skeletal muscles. SMA-determining candidate genes, SMN1 and SMN2, have been identified on human chromosome 5q. The corresponding SMN protein is expressed ubiquitously. It is coded by seven exons and contains conspicuous proline-rich motifs in its COOH-terminal third (exons 4, 5, and 6). Such motifs are known to bind to profilins (PFNs), small proteins engaged in the control of actin dynamics. We tested whether profilins interact with SMN via its polyproline stretches. Using the yeast two-hybrid system we show that profilins bind to SMN and that this binding depends on its proline-rich motifs. These results were confirmed by coimmunoprecipitation and by in vitro binding studies. Two PFN isoforms, I and II, are known, of which II is characteristic for central nervous system tissue. We show by in situ hybridization that both PFNs are highly expressed in mouse spinal cord and that PFN II is expressed predominantly in neurons. In motoneurons, the primary target of neurodegeneration in SMA, profilins are highly concentrated and colocalize with SMN in the cytoplasm of the cell body and in nuclear gems. Likewise, SMN and PFN I colocalize in gems of HeLa cells. Although SMN interacts with both profilin isoforms, binding of PFN II was stronger than of PFN I in all assays employed. Because the SMN genes are expressed ubiquitously, our findings suggest that the interaction of PFN II with SMN may be involved in neuron-specific effects of SMN mutations.

Amino Acid Motifs↗

PubMed1999

Binding of the Shigella protein IpaA to vinculin induces F-actin depolymerization.

Shigella flexneri, the causative agent of bacillary dysentery, enters into epithelial cells by a macropinocytic process. IpaA, a Shigella protein secreted upon cell contact, binds to the focal adhesion protein vinculin and is required for efficient bacterial uptake. IpaA was shown here to bind with high affinity to the N-terminal residues 1-265 of vinculin. Using co-sedimentation and solid-phase assays, we demonstrated that binding of IpaA to vinculin strongly increases the association of vinculin with F-actin. We also characterized a depolymerizing activity on actin filaments associated with the vinculin-IpaA complex both in vitro and in microinjected cells. We propose that the conformational change of vinculin induced by IpaA binding allows interaction of the vinculin-IpaA complex with F-actin and subsequent depolymerization of actin filaments.

Actin Cytoskeleton↗

PubMed1999

Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP.

The vasodilator-stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cell-cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C-terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.

3T3 Cells↗

PubMed1999

Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes.

Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.

Actins↗

PubMed1999

Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.

Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent. In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins. More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin. However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes. In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles.

Actins↗

PubMed1998

Differential actin organization by vinculin isoforms: implications for cell type-specific microfilament anchorage.

Vinculin is found in all adherens junctions, while metavinculin, a larger splice variant, is coexpressed with vinculin only in smooth and cardiac muscle. To understand the significance of metavinculin expression, we compared ligand binding between turkey vinculin and metavinculin. Residues 1-258 were found essential for head-tail interactions in both proteins. The tail domains (VT and MVT, respectively) both bind to F-actin. However, while VT bundles F-actin, MVT generates highly viscous F-actin webs. In transfected PtK2 cells, VT causes F-actin needles or coils, while MVT-expressing cells display a diffuse F-actin distribution. Thus, the MVT-specific insert induces an F-actin supraorganization different from the VT-based form, suggesting that metavinculin has a specific role in muscle.

Actin Cytoskeleton↗

PubMed1998

Vinculin is part of the cadherin-catenin junctional complex: complex formation between alpha-catenin and vinculin.

In epithelial cells, alpha-, beta-, and gamma-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. alpha-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell-matrix and cell-cell contacts, alpha-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell-cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and alpha-catenin. We show that alpha-catenin colocalizes at cell-cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to alpha-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The Kd of the complex was determined to 2-4 x 10(-7) M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of alpha-catenin is involved in this interaction. Complex formation of vinculin and alpha-catenin was challenged in transfected cells. In PtK2 cells, intact alpha-catenin and alpha-catenin1-670, harboring the beta-catenin- binding site, were directed to cell-cell contacts. In contrast, alpha-catenin697-906 fragments were recruited to cell-cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo alpha-catenin, like vinculin, is tightly regulated in its ligand binding activity.

Animals↗

PubMed1998

The interaction of the cell-contact proteins VASP and vinculin is regulated by phosphatidylinositol-4,5-bisphosphate.

BACKGROUND: Focal adhesion sites are cell-matrix contacts that are regulated by phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent pathways. Vinculin is a major structural component of these sites and is thought to be engaged in multiple ligand interactions at the cytoplasmic face of these contacts. Cytoplasmic vinculin is considered to be inactive due to its closed conformation involving intramolecular head-tail interactions. Recently, the vasodilator-stimulated phosphoprotein (VASP), a substrate of cyclic AMP-dependent or cyclic GMP-dependent kinases and a component of focal adhesion sites, was shown to bind to vinculin. RESULTS: VASP-vinculin complexes could be immunoprecipitated from cell lysates and, using immunofluorescence, both proteins were found to colocalize in nascent focal adhesions. Consistent with the view that vinculin must be activated at these sites, we found that PIP2, levels of which are elevated during the early stages of adhesion, bound to two discrete regions in the vinculin tail, disrupting the intramolecular head-tail interaction and inducing vinculin oligomerization. Vinculin-VASP complex formation was greatly enhanced by PIP2 and both the EVH1 and EVH2 domains of VASP participated in vinculin binding. CONCLUSIONS: Focal contact assembly involves interaction between VASP and vinculin, which is enhanced by PIP2-induced vinculin activation and oligomerization. Given that vinculin and VASP both bind to F-actin, vinculin-VASP complexes might bundle the distal ends of actin filaments in focal contacts. We propose that PIP2-dependent signalling modulates microfilament organization at cellular adhesion sites by regulating vinculin-VASP complexes.

Actins↗