umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Effects of invasin and YopH of Yersinia pseudotuberculosis on host cell signaling
Umeå University, Faculty of Medicine, Molecular Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Effekter av proteinerna invasin och YopH från bakterien Yersinia pseudotuberculosis på värdcellen (Swedish)
Abstract [en]

Integrins are a large family of membrane-spanning heterodimeric (αβ) receptors that bind to ligands on other cells or to extracellular matrix (ECM) proteins. These receptors mediate bidirectional signaling over the cell membrane to induce signaling cascades mediating functions as cell adhesion, spreading and migration. This signaling takes place at cell-matrix adhesions, which are sites where clustered and ligand-bound integrins connect to and mediate stabilization of the actin cytoskeleton, and induce signaling cascades. Integrins have a short cytoplasmic tail that is crucial for the bidirectional signaling, and the β1-integrin subunit exists in five splice variants only differing in the membrane-distal part of the cytoplasmic tail. This region of the almost ubiquitously expressed β1-integrin, β1A, contains two protein tyrosine motifs (NPXYs) interspaced with a threonine-rich region, while this region of the β1B splice variant is completely different and lacks known motifs. In contrast to the β1A-integrin, the β1B variant cannot mediate cell-matrix adhesion formation following binding to ECM ligands.

The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1-integrins on the host cell with invasin, and this stimulates uptake of the bacterium. However, upon binding to the host cell, pathogenic Yersinia strains inject virulence effectors that block uptake. One effector responsible for the blocking is a tyrosine phosphatase, YopH. We identified the targets for this effector in the macrophage-like cell line J774A.1, which represent a professional phagocyte and thus is the likely target cell for the antiphagocytic effect of Yersinia. Two YopH target proteins were p130Cas and ADAP, of which the latter interestingly is an adapter protein specifically expressed in hematopoietic cells. ADAP has previously been implicated to participate in Fc-receptor-mediated phagocytosis and in communication between T-cell receptors and integrins.

We also studied the importance of the cytoplasmic tail of β1-integrin for uptake of Yersinia. The GD25 cell line, which is a fibroblast-like cell line that lacks endogenous β1-integrins, was used together with GD25 cells transfected with β1B, β1Α or cytoplasmic tail mutants of β1A. These studies revealed that β1B-integrins could bind to invasin but not mediate uptake of Yersinia, while β1A both bound to invasin and mediated uptake. The first NPXY motif (unphosphorylated) and the double-threonines of the unique part of β1A were important for the ability of integrin to mediate uptake of Yersinia. These studies lead to the interesting finding that, when these cells were allowed to spread on invasin, those that expressed β1A spread as normal fibroblasts while for β1B-integrin-expressing cells, only finger-like protrusions of filopodia were formed. This provided us with a tool to study formation of filopodia without interference of the tightly linked process of lamellipodia formation. Initially, proteins that localized to the tip complex of these filopodia were identified. These were talin, VASP and interestingly the p130Cas-Crk-DOCK180 scaffold, while FAK, paxillin and vinculin were absent. In addition, VASP, p130Cas and Crk were shown to be important for the filopodia formation in GD25β1B. Further, the role of the actin motor myosin X, which previously has been implicated in formation of filopodia, was studied in the GD25Β1B cells and it was shown that myosin X not was important for filopodia formation, but that it recruited FAK and vinculin to the tip complexes of filopodia.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet) , 2004. , 67 p.
Keyword [en]
Molecular biology, β1-integrin, Yersinia pseudotuberculosis, Invasin, YopH, Cell-Matrix Adhesion, Filopodia, Cell Spreading
Keyword [sv]
Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-183ISBN: 91-7305-588-3 (print)OAI: oai:DiVA.org:umu-183DiVA: diva2:142141
Public defence
2004-03-05, Major Groove, 6L, NUS, Inst f molekylärbiologi, Umeå Universitet, 901 87 Umeå, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2010-01-22Bibliographically approved
List of papers
1. YopH dephosphorylates Cas and Fyn-binding protein in macrophages
Open this publication in new window or tab >>YopH dephosphorylates Cas and Fyn-binding protein in macrophages
Show others...
1999 (English)In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 27, no 4, 231-242 p.Article in journal (Refereed) Published
Abstract [en]

The tyrosine phosphatase YopH is an essential virulence effector of pathogenic Yersinia spp. YopH, which is translocated from extracellularly located bacteria into interacting target cells, blocks phagocytosis by professional phagocytes. We show here that immunoprecipitation of YopH from lysates of J774 cells infected with Y. pseudotuberculosis expressing an inactive form of YopH resulted in co-precipitation of certain phosphotyrosine proteins. The association between the inactive YopH and phosphotyrosine proteins in the 120 kDa range was rapid and could be detected after 2 min of infection. The proteins were identified as the docking proteins Cas and Fyn-binding protein (FYB). Upon infection of J774 cells with Y. pseudotuberculosis lacking YopH expression both of these proteins became tyrosine phosphorylated. Moreover, this infection caused recruitment of Cas to peripheral focal complexes, and FYB was relocalized to areas surrounding these structures. Both Cas and FYB became dephosphorylated upon infection with Y. pseudotuberculosis expressing active YopH, and this was associated with disruption of focal complexes. With regard to the previous identification of Cas and focal complexes as targets of YopH in HeLa cells, the present study supports an important role for these targets in a general mechanism of bacterial uptake. Copyright 1999 Academic Press.

Keyword
Yersinia pseudotuberculosis, phagocytosis, Cas, FYB, focal complexes, PTPase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-3442 (URN)10.1006/mpat.1999.0301 (DOI)10502464 (PubMedID)
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2013-02-06Bibliographically approved
2. Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia
Open this publication in new window or tab >>Role of the β1-integrin cytoplasmic tail in mediating invasin-promoted internalization of Yersinia
Show others...
2002 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 115, no 13, 2669-2678 p.Article in journal (Refereed) Published
Abstract [en]

Invasin of Yersinia pseudotuberculosis binds to beta1-integrins on host cells and triggers internalization of the bacterium. To elucidate the mechanism behind the beta1-integrin-mediated internalization of Yersinia, a beta1-integrin-deficient cell line, GD25, transfected with wild-type beta1A, beta1B or different mutants of the beta1A subunit was used. Both beta1A and beta1B bound to invasin-expressing bacteria, but only beta1A was able to mediate internalization of the bacteria. The cytoplasmic region of beta1A, differing from beta1B, contains two NPXY motifs surrounding a double threonine site. Exchanging the tyrosines of the two NPXYs to phenylalanines did not inhibit the uptake, whereas a marked reduction was seen when the first tyrosine (Y783) was exchanged to alanine. A similar reduction was seen when the two nearby threonines (TT788-9) were exchanged with alanines. It was also noted that cells affected in bacterial internalization exhibited reduced spreading capability when seeded onto invasin, suggesting a correlation between the internalization of invasin-expressing bacteria and invasin-induced spreading. Likewise, integrins defective in forming peripheral focal complex structures was unable to mediate uptake of invasin-expressing bacteria.

Keyword
Invasin, b1-integrin, Yersinia pseudotuberculosis, Focal complexes, Bacterial internalization
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-3443 (URN)12077358 (PubMedID)
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2010-04-20Bibliographically approved
3. Temporal dissection of beta1-integrin signaling indicates a role for p130Cas-Crk in filopodia formation.
Open this publication in new window or tab >>Temporal dissection of beta1-integrin signaling indicates a role for p130Cas-Crk in filopodia formation.
2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 22, 22893-22901 p.Article in journal (Refereed) Published
Abstract [en]

Invasin-promoted spreading of beta1-integrin-deficient cells, transfected with the beta1A- or beta1B-integrin splice variants, were used to dissect early beta1-integrin signaling events. The beta1B isoform, which has a different membrane-distal part of the cytoplasmic tail from beta1A, is defective in signaling and function. When plated on surfaces coated with the high affinity ligand invasin, beta1B-integrin-expressing cells spread by forming filopodia with distinct adhesive phosphotyrosine complexes at the tips, without signs of lamellipodia. This suggested that the beta1B-integrin mediated a partial signaling sufficient for formation of filopodia but insufficient for lamellipodia formation. When screening for proteins present in the distal filopodial phosphotyrosine complexes of beta1B cells, p130Cas and the filopodia proteins vasodilator-stimulated phosphoprotein and talin were found, whereas the typical focal complex proteins focal adhesion kinase, paxillin, and vinculin were not. Invasin-promoted adhesion induced complex formation of p130Cas and the adapter Crk. Moreover, Crk together with Dock180 were present at the filopodial tips of beta1B-integrin-expressing cells, and there was a prominent Rac1 activation. Expression of dominant negative variants of p130Cas or CrkII blocked beta1B-integrin-mediated filopodia formation, indicating that this signaling scaffold is central in this process.

Keyword
Animals, Antigens; CD29/*physiology, Crk-Associated Substrate Protein, Phosphorylation, Proteins/*physiology, Proto-Oncogene Proteins/physiology, Proto-Oncogene Proteins c-crk, Pseudopodia/*physiology, Rabbits, Rats, Retinoblastoma-Like Protein p130, Signal Transduction
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-16654 (URN)10.1074/jbc.M309693200 (DOI)15044442 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2010-04-20Bibliographically approved
4. Myosin X recruits FAK and vinculin to the tip complexes of filopodia
Open this publication in new window or tab >>Myosin X recruits FAK and vinculin to the tip complexes of filopodia
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-3445 (URN)
Available from: 2004-01-30 Created: 2004-01-30 Last updated: 2010-01-13Bibliographically approved

Open Access in DiVA

fulltext(1494 kB)1383 downloads
File information
File name FULLTEXT01.pdfFile size 1494 kBChecksum SHA-1
c1772864dbcdeacb41628e37be0afaf3f052c0ba18266dbbeeeece27c413b361e9fa9379
Type fulltextMimetype application/pdf

By organisation
Molecular Biology
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1383 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 431 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf