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Profilin:actin in cell motility: A search for profilin:actin binding proteins
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The profilin:actin complex is a major source of actin for actin filament growth in vivo. A number of proteins regulating either profilin or actin has been described since profilin:actin was isolated during the 1970s. Since then, profilin and actin and their binding partners have been intensively studied. The ability of profilin:actin to interact with the fast polymerizing end of actin filaments focus the interest to components that regulates this interaction; this is the theme in this thesis.

A chemically cross-linked and therefore non-dissociable profilin:actin complex, called PxA, was used in these studies which led to development of a rapid screening method to search for proteins that bind to profilin:actin. The method allows a simultaneous detection of proteins that separately interact with profilin, actin and/or profilin:actin. Here the technique was used to screen cell and tissue extracts, before and after gel filtration, for components that showed a unique interaction with the profilin:actin complex. Mass spectrometry was then used for their identification. Furthermore it was demonstrated that profilin:actin binding components are present in RNA containing, large molecular weight complexes.

Two different PxA immunizations generated two separate populations of affinity purified profilin and actin antibodies. The actin antibodies from these two populations showed significant differences in the staining pattern when used for fluorescence microscopy of tissue cultured cells. One of these appeared to bind monomeric actin while the other bound to filamentous actin. Both of the profilin antibody preparations stained cells in a dotted pattern. The distribution of epitopes recognized by the different actin antibody preparations was determined using a combination of protease digestion, gel electrophoresis and mass spectrometry. The result demonstrated partially different epitope recognition.

The actin associated protein palladin contains sequence motifs typical for profilin-binding proteins suggesting that profilin may bind palladin. The potential profilin-palladin interaction was studied using a combination of biochemical and histochemical techniques. The interaction was observed in vitro, and the two proteins co-distributed in actin rich regions in tissue cultured cells. These results suggest that palladin recruits profilin and/or profilin:actin to sites of actin dynamics.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi , 2005. , 75 p.
Keyword [sv]
profilin, actin
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-727ISBN: 91-7155-153-0 (print)OAI: oai:DiVA.org:su-727DiVA: diva2:197956
Public defence
2005-12-19, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2005-11-28 Created: 2005-11-28 Last updated: 2012-01-12Bibliographically approved
List of papers
1. Detection of binding partners to the profilin:actin complex by far Western and mass spectrometry analyses
Open this publication in new window or tab >>Detection of binding partners to the profilin:actin complex by far Western and mass spectrometry analyses
2004 In: Anal Biochem, Vol. 335, no 2, 228-234 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24625 (URN)
Note
Part of urn:nbn:se:su:diva-727Available from: 2005-11-28 Created: 2005-11-28Bibliographically approved
2. Profilin:actin binding proteins are present in cell extracts in high molecular weight RNA-containing complexes
Open this publication in new window or tab >>Profilin:actin binding proteins are present in cell extracts in high molecular weight RNA-containing complexes
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24626 (URN)
Note
Part of urn:nbn:se:su:diva-727Available from: 2005-11-28 Created: 2005-11-28 Last updated: 2010-01-13Bibliographically approved
3. Anti-actin antibodies generated against profilin: actin distinguish between non-filamentous and filamentous actin, and label cultured cells in a dotted pattern
Open this publication in new window or tab >>Anti-actin antibodies generated against profilin: actin distinguish between non-filamentous and filamentous actin, and label cultured cells in a dotted pattern
2004 (English)In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 83, no 8, 413-423 p.Article in journal (Refereed) Published
Abstract [en]

Actin polymerization is a prominent feature of migrating cells, where it powers the protrusion of the leading edge. Many studies have characterized the well-ordered and dynamic arrangement of filamentous actin in this submembraneous space. However, less is known about the organization of unpolymerized actin. Previously, we reported on the use of covalently coupled profilin:actin to study actin dynamics and presented evidence that profilin-bound actin is a major source of actin for filament growth. To locate profilin:actin in the cell we have now used this non-dissociable complex for antibody generation, and obtained monospecific anti-actin and antiprofilin antibodies from two separate immunizations. Fluorescence microscopy revealed drastic differences in the staining pattern generated by the anti-actin antibody preparations. With one, distinct puncta appeared at the actin-rich leading edge and sometimes aligned with microtubules in the interior of the lamella, while the other displayed typical actin filament staining. Labelling experiments in vitro demonstrated failure of the first antibody to recognize filamentous actin and none of the two bound microtubules. The two anti-profilin antibodies purified in parallel generated a punctated pattern similar to that seen with the first anti-actin antibody. All antibody preparations labelled the nuclei.

Keyword
profilin-actin; antibodies; fluorescence microscopy; immunohistochemistry; microfilament dynamics
National Category
Natural Sciences
Identifiers
urn:nbn:se:su:diva-24627 (URN)10.1078/0171-9335-00400 (DOI)
Available from: 2005-11-28 Created: 2005-11-28 Last updated: 2012-01-12Bibliographically approved
4. The proline-rich protein palladin is a binding partner for profilin
Open this publication in new window or tab >>The proline-rich protein palladin is a binding partner for profilin
Show others...
2006 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 1, 26-33 p.Article in journal (Refereed) Published
Abstract [en]

Palladin is an actin-associated protein that has been suggested to play critical roles in establishing cell morphology and maintaining cytoskeletal organization in a wide variety of cell types. Palladin has been shown previously to bind directly to three different actin-binding proteins vasodilator-stimulated phosphoprotein (VASP), α-actinin and ezrin, suggesting that it functions as an organizing unit that recruits actin-regulatory proteins to specific subcellular sites. Palladin contains sequences resembling a motif known to bind profilin. Here, we demonstrate that palladin is a binding partner for profilin, interacting with profilin via a poly proline-containing sequence in the amino-terminal half of palladin. Double-label immunofluorescence staining shows that palladin and profilin partially colocalize in actin-rich structures in cultured astrocytes. Our results suggest that palladin may play an important role in recruiting profilin to sites of actin dynamics.

Keyword
Actin assembly Ena/Mena/VASP lamellipodium migration
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-24628 (URN)10.1111/j.1742-4658.2005.05036.x (DOI)
Note
Part of urn:nbn:se:su:diva-727Available from: 2005-11-28 Created: 2005-11-28 Last updated: 2010-07-08Bibliographically approved

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