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D Geerts

Publications and source records attributed to D Geerts.

16 recordsLinked to original sources
PubMed2001

The interaction of plectin with actin: evidence for cross-linking of actin filaments by dimerization of the actin-binding domain of plectin.

Plectin is a major component of the cytoskeleton and is expressed in a wide variety of cell types. It plays an important role in the integrity of the cytoskeleton by cross-linking the three filamentous networks and stabilizing cell-matrix and cell-cell contacts. Sequence analysis showed that plectin contains a highly conserved actin-binding domain, consisting of a pair of calponin-like subdomains. Using yeast two-hybrid assays in combination with in vitro binding experiments, we demonstrate that the actin-binding domain of plectin is fully functional and preferentially binds to polymeric actin. The sequences required for actin binding were identified at the C-terminal end of the first calponin homology domain within the actin-binding domain of plectin. We found that the actin-binding domain of plectin is able to bundle actin filaments and we present evidence that this is mediated by the dimerization of this domain. In addition we also show that plectin and another member of the plakin family, dystonin, can heterodimerize by their actin-binding domains. We propose a new mechanism by which plectin and possibly also other actin-binding proteins can regulate the organization of the F-actin network in the cell.

Actins↗
PubMed2000

The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes.

LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.

3T3 Cells↗
PubMed2000

Cytoplasmic domain mutants of beta1 integrin, expressed in beta 1-knockout lymphoma cells, have distinct effects on adhesion, invasion and metastasis.

Structural requirements for beta 1 integrin cytoplasmic domain functions in adhesion, migration and signaling have been studied mainly for fibroblasts in vitro. The relevance for beta 1-dependent in vivo migration of lymphoid cells has not been assessed. To study this, we transfected beta 1 mutants into beta 1-deficient double knockout (DKO) ESb lymphoma cells, and tested the capacity of the cells to metastasize to liver and spleen. This was compared to alpha 4 beta 1-dependent invasion into cell monolayers in vitro and Mn2+-induced adhesion to fibronectin. Deletion of the five C-terminal residues or mutation of both threonines T788 and T789 to alanines blocked invasion and metastasis and greatly reduced adhesion, in line with known in vitro effects. However, mutations of the NPXY motif tyrosines had unexpected consequences. A Y783F mutation had no effect at all, but a Y783,795F double mutation strongly reduced Mn2+-induced adhesion, whereas it had limited effects on invasion and metastasis. Furthermore, cells expressing a beta 1 beta 2 chimeric subunit, which contains phenylalanines in the NPXY/F motifs, adhered poorly but invasion and metastasis was fully restored to the same levels as for cells expressing wild-type beta 1. We conclude that part of the functions of the beta 1 cytoplasmic domain that are required for adhesion are not essential for beta 1-dependent invasion and metastasis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Formation of hemidesmosome-like structures in the absence of ligand binding by the (alpha)6(beta)4 integrin requires binding of HD1/plectin to the cytoplasmic domain of the (beta)4 integrin subunit.

Hemidesmosomes are adhesion structures that mediate anchorage of epithelial cells to the underlying basement membrane. We have previously shown that the (alpha)6(beta)4 integrin can induce the assembly of these multi-protein structures independent of binding to its ligand laminin-5 (ligand-independent formation of hemidesmosomes). Our results suggested a role for HD1/plectin, which binds to the cytoplasmic domain of the (beta)4 integrin subunit, in controlling the clustering of hemidesmosomal components at the basal side of the cell. Using keratinocytes derived from patients lacking HD1/plectin, we now show that ligand-independent formation of hemidesmosomal clusters indeed requires HD1/plectin, in contrast to the ligand-dependent assembly of hemidesmosomes. No clustering of the (alpha)6(beta)4 integrin, or of the bullous pemphigoid antigens BP180 and BP230, was seen when HD1/plectin-deficient keratinocytes were plated on fibronectin or type IV collagen. In (&bgr;)4-deficient keratinocytes, expression of an interleukin 2 receptor (IL2R) transmembrane chimera containing the (beta)4 cytoplasmic tail with the mutation R1281W, which abrogates HD1/plectin binding, resulted in a diffuse distribution of the chimeric receptor. In contrast, a (beta)4(R1281W) mutant that can associate with (alpha)6 and bind ligand, was found to be directed to the basal surface of the cells, at sites where laminin-5 was deposited. In addition, this mutant induced clustering of BP180 and BP230 at these sites. Together, these results show that the formation of hemidesmosomes requires binding of either ligand or HD1/plectin to the (beta)4 integrin subunit. Intriguingly, we found that IL2R/(beta)4 chimeras become localized in pre-existing hemidesmosomes of HD1/plectin-deficient keratinocytes, and that this localization requires a domain in the (beta)4 cytoplasmic tail that is also required for HD1/plectin binding (residues 1115-1356). Because this part of (beta)4 lacks the BP180 binding site, and since we show in this study that it is unable to interact with the same part on another (beta)4 molecule, we suggest that the chimera becomes incorporated into hemidesmosomes of HD1/plectin-deficient keratinocytes by interacting with an as yet unidentified hemidesmosomal component.

Antigens, CD↗
PubMed1999

Binding of integrin alpha6beta4 to plectin prevents plectin association with F-actin but does not interfere with intermediate filament binding.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

Actins↗
PubMed1999

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains.

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.

Amino Acid Sequence↗
PubMed1998

Hemidesmosome formation is initiated by the beta4 integrin subunit, requires complex formation of beta4 and HD1/plectin, and involves a direct interaction between beta4 and the bullous pemphigoid antigen 180.

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the beta4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in beta4 expression. We found that the expression of wild-type beta4 restored the ability of the beta4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the beta4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the beta4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between beta4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for alpha6 and beta4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of beta4 with BP180. Nevertheless, beta4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of beta4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of beta4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of beta4 with other hemidesmosomal components, e.g., BP180, is regulated.

Animals↗
PubMed1998

Ligand-independent role of the beta 4 integrin subunit in the formation of hemidesmosomes.

Recently, we have shown that a region within the beta4 cytoplasmic domain, encompassing the second fibronectin type III (FNIII) repeat and the first 27 amino acids of the connecting segment, is critical for the localization of alpha6 beta4 in hemidesmosomes. In addition, this region was shown to regulate the distribution of HD1/plectin in transfected cells. In order to investigate the function of the beta4 extracellular and cytoplasmic domains in the assembly and integrity of hemidesmosomes, we have constructed chimeric receptors consisting of the extracellular and transmembrane domains of the interleukin 2 receptor (IL2R), fused to different parts of the beta4 cytoplasmic domain. These chimeras are expressed as single subunits at the plasma membrane. The results show that the first and the second FNIII repeat, together with the first part of the connecting segment (in total a stretch of 241 amino acids spanning amino acids 1,115 to 1,356) are both essential and sufficient for the localization of beta4 in pre-existing hemidesmosomes. Moreover, expression of the IL2R/beta4 chimeric constructs in COS-7 and CHO cells, which do not express alpha6 beta4 or the bullous pemphigoid (BP) antigens but do express HD1/plectin, revealed that the stretch of 241 amino acids is sufficient for inducing the formation of type II hemidesmosomes. Expression of the IL2R/beta4 chimeras in a keratinocyte cell line derived from a patient lacking beta4 expression, showed that amino acids 1,115 to 1,356 can also induce the formation of type I hemidesmosomes. We further demonstrate that type I and II hemidesmosomes can also be formed upon adhesion of alpha6 beta4-expressing cells to fibronectin. These findings establish that the beta4 extracellular domain is not essential for the induction of hemidesmosome assembly. Moreover, they demonstrate that binding of alpha6 beta4 to ligand, and heterodimerization of alpha6 with beta4, are not required for hemidesmosome formation. This indicates that the assembly of hemidesmosomes can be regulated from within the cell.

Animals↗
PubMed1995

Inducible expression of heterologous genes targeted to a chromosomal platform in the cyanobacterium Synechococcus sp. PCC 7942.

High-level, inducible expression of heterologous genes in the cyanobacterium Synechococcus sp. strain PCC 7942 was obtained using the Escherichia coli trc promoter and lacI repressor. The petE gene of Anabaena sp. strain PCC 7937 encoding plastocyanin precursor protein and the E. coli uidA gene encoding beta-glucuronidase were initially placed under the control of the trc promoter and lacI repressor by cloning into the E. coli pTrc99C expression vector and were introduced into the chromosomal platform for integration in metF (PIM) of the Synechococcus R2-PIM9 recipient strain. These pTrc99C-derived constructs often gave rise to transformants that did not contain a complete insert gene, probably because of gene conversion events. Selection of the desired Synechococcus R2-PIM9 transformants was vastly improved using the new pTrcIS vector that contains the aadA gene encoding streptomycin resistance as an extra antibiotic resistance marker. The influence of IPTG concentration and induction time on gene expression with the E. coli trc/lacI system in Synechococcus was determined using beta-glucuronidase as a reporter. The Anabaena PCC 7937 petE gene in Synechococcus was expressed to a high level upon induction with IPTG as shown by RNA and immunoblot analysis. The general usability of pTrcIS as a cloning vector for inducible heterologous gene expression in Synechococcus was confirmed by the introduction of several more genes.

Amino Acid Sequence↗
PubMed1994

Expression of Anabaena PCC 7937 plastocyanin in Synechococcus PCC 7942 enhances photosynthetic electron transfer and alters the electron distribution between photosystem I and cytochrome-c oxidase.

The petE gene encoding plastocyanin precursor protein from the cyanobacterium Anabaena PCC 7937 was introduced in the cyanobacterial host strain Synechococcus PCC 7942. The host normally only uses cytochrome c553 as Photosystem I (PS I) donor. The heterologous gene was efficiently expressed using the inducible Escherichia coli trc promoter. Accumulation of plastocyanin protein depended on the presence of Cu2+. The protein was accurately targeted to the thylakoid lumen, from which it could be isolated in the mature form. Redox difference spectroscopy proved the presence of a Cu2+ ion in the holoenzyme. Isolated heterologous plastocyanin was functional in reconstitution of in vitro electron transfer to PS I. The presence of Anabaena plastocyanin in Synechococcus thylakoid membranes increased PS I electron transfer rate 2.5 times. Analysis of P700 redox and PS II fluorescence transients in vivo showed a faster electron transfer through PS I because of enhanced electron supply in the presence of plastocyanin. In addition, the distribution of electrons between photosynthetic and respiratory electron transfer changed. Plastocyanin preferentially donates electrons to PS I rather than to the respiratory cytochrome-c oxidase complex and is not functionally equivalent to cytochrome c553.

Anabaena↗
PubMed1989

Amino acid sequence of a conserved neutralizing epitope of murine coronaviruses.

We identified the binding site of monoclonal antibody 19.2, which cross-neutralizes several mouse hepatitis virus (MHV) strains, inhibits fusion of MHV-infected cells, and protects against lethal infection (P. J. Talbot and M. J. Buchmeier, Virus Res. 2:317-328, 1985). We used fusion proteins, generated by expression of fragments of the MHV A59 E2 gene in pEX plasmids, and synthetic peptides in a PEPSCAN.

Amino Acid Sequence↗
PubMed1989

[Axial length and radial keratotomy].

The axial length of 68 eyes has been measured before radial keratotomy. This measurement can influence the decision of a procedure. In 49 cases, a postoperative measurement was done, and a mean variation of the length of the eyeball of -0.1 mm is demonstrated.

Biometry↗