References of "Golsteyn, R. M"
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See detailActA and human zyxin harbour Arp2/3-independent actin-polymerization activity.
Fradelizi, J.; Noireaux, V.; Plastino, J. et al

in Nature cell biology (2001), 3(8), 699-707

The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin ... [more ▼]

The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization. [less ▲]

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See detailLPP, an actin cytoskeleton protein related to zyxin, harbors a nuclear export signal and transcriptional activation capacity.
Petit, M. M.; Fradelizi, J.; Golsteyn, R. M. et al

in Molecular biology of the cell (2000), 11(1), 117-29

The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of ... [more ▼]

The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus. [less ▲]

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See detailGrowing an actin gel on spherical surfaces.
Noireaux, V.; Golsteyn, R. M.; Friederich, Evelyne UL et al

in Biophysical journal (2000), 78(3), 1643-54

Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of ... [more ▼]

Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails. [less ▲]

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See detailCharacterization of the interaction between zyxin and members of the Ena/vasodilator-stimulated phosphoprotein family of proteins.
Drees, B.; Friederich, Evelyne UL; Fradelizi, J. et al

in The Journal of biological chemistry (2000), 275(29), 22503-11

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human ... [more ▼]

Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading. [less ▲]

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See detailQuantitative measurement of proteins by western blotting with Cy5-coupled secondary antibodies.
Fradelizi, J.; Friederich, Evelyne UL; Beckerle, M. C. et al

in BioTechniques (1999), 26(3), 484-6488490

The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting ... [more ▼]

The concentration of proteins in cells is an important parameter that determines how a protein will interact with other proteins or pharmacological agents. Recent developments in Western blotting techniques have now made this a method of choice to measure protein concentration in complex solutions such as total cell extracts. We show that detection of Cy5-coupled secondary antibodies by PhosphorImager analysis produces signals that approach linearity with respect to protein concentration over a 20-fold range. We used this technique to estimate cellular levels of zyxin, which is an important protein component of the actin cytoskeleton in mammalian cells. By producing specific protein standards based on sequences that are available from public databases, it is now possible to estimate the concentration of almost any protein by this technique. [less ▲]

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See detailThe role of actin binding proteins in epithelial morphogenesis: models based upon Listeria movement.
Golsteyn, R. M.; Louvard, D.; Friederich, Evelyne UL

in Biophysical chemistry (1997), 68(1-3), 73-82

We summarize recent findings on the organization of the protein actin in eucaryotic cells. In particular we focus on how actin can be used to generate a vectorial force that is required for cell movement ... [more ▼]

We summarize recent findings on the organization of the protein actin in eucaryotic cells. In particular we focus on how actin can be used to generate a vectorial force that is required for cell movement. These forces arise from protein molecules that recruit actin to the plasma membrane in such a manner that actin filaments extend outward from the cell body. This type of actin dependent force generation has been described in a nucleation-release model, which is one of several models currently being tested to explain actin dependent cell movement. Data in support of this model has arisen unexpectedly from studies of an intracellular bacteria, Listeria monocytogenes. This bacteria uses actin to propel itself during infection of eucaryotic cells. By studying Listeria movement, the roles of several eucaryotic actin interacting proteins have been identified. One of these is zyxin, a human protein that shares important structural and possibly functional properties with ActA, an actin dependent force generating protein of Listeria. We intend to test the function of these and other actin interacting proteins in a simplified system that should facilitate precise measurement of their properties of force generation in vitro. [less ▲]

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See detailStructural and functional similarities between the human cytoskeletal protein zyxin and the ActA protein of Listeria monocytogenes.
Golsteyn, R. M.; Beckerle, M. C.; Koay, T. et al

in Journal of cell science (1997), 110 ( Pt 16)

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The ... [more ▼]

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells. Therefore we examined the possibility that an ActA-like protein is present in mammalian cells. By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types. We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box). Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences. A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells. Furthermore, zyxin and ActA exhibit common protein partners in vitro. On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells. [less ▲]

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