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Anti-actin antibodies generated against profilin: actin distinguish between non-filamentous and filamentous actin, and label cultured cells in a dotted pattern
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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.

Place, publisher, year, edition, pages
2004. Vol. 83, no 8, 413-423 p.
Keyword [en]
profilin-actin; antibodies; fluorescence microscopy; immunohistochemistry; microfilament dynamics
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-24627DOI: 10.1078/0171-9335-00400OAI: oai:DiVA.org:su-24627DiVA: diva2:197954
Available from: 2005-11-28 Created: 2005-11-28 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Profilin:actin in cell motility: A search for profilin:actin binding proteins
Open this publication in new window or tab >>Profilin:actin in cell motility: A search for profilin:actin binding proteins
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
profilin, actin
National Category
Cell Biology
Identifiers
urn:nbn:se:su:diva-727 (URN)91-7155-153-0 (ISBN)
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
2. Cross-linked Profilin:actin - A tool to study actin dynamics in non-muscle cells
Open this publication in new window or tab >>Cross-linked Profilin:actin - A tool to study actin dynamics in non-muscle cells
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The microfilament system, consisting of actin and a number of auxiliary proteins, is fundamental for cell motility. Its dynamic organization depends on receptor-mediated signals, leading to rapid polymerizations and depolymerizations of actin. Profilin binds to non-filamentous actin, inhibits spontaneous filament formation, and functions as a regulator of actin polymerization. The profilin:actin complex, is thought to be the principal source of actin for filament formation although the role of profilin is not fully elucidated.

In this thesis, a cross-linked profilin:actin complex (PxA), that retains the properties of ordinary profilin:actin, except for being non-dissociable, has been used to characterize the role of profilin and profilin:actin in non-muscle cells. A rapid screening method, employing PxA and based on the far western technique and mass-spectrometry, was designed to identify cellular components that specifically bind profilin:actin. Microinjection of PxA into cells infected with the bacteria Listeria monocytogenes impaired bacterial motility but a mutant PxA, unable to bind proline-rich sequences had no effect, demonstrating that profilin:actin is vital for the activity of the actin polymer-forming complex that the pathogen recruits to its surface upon infection.

Fluorescence microscopy using two distinct sets of affinity-purified actin and profilin antibodies generated against PxA enabled localization of monomeric actin in cells. One of the actin and both profilin antibodies resulted in a dotted pattern of fluorescence partially aligning with microtubules whereas the other actin antibody detected filamentous actin. The result demonstrates extensive variability in epitope recognition, and indicates that unpolymerized actin, i.e. profilin:actin and maybe other complex-bound forms of actin, distributes in small packages that might be transported along microtubules. Microinjection of PxA into lamprey axons demonstrated the involvement of actin polymerization during synaptic signaling.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi, 2004. 64 p.
Keyword
actin dynamics, profilin, actin, Profilin-actin complex, Cell motility, synaptic vesicle trafficing, Far western, fluorescence microscopy
National Category
Cell Biology
Identifiers
urn:nbn:se:su:diva-168 (URN)91-7265-854-1 (ISBN)
Public defence
2004-06-04, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2012-01-12Bibliographically approved
3. Elements in regulation of the microfilament system
Open this publication in new window or tab >>Elements in regulation of the microfilament system
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis deals with cell motility. The process of rapid actin polymerization in the lamellipodium of a migrating cell is responsible for its protrusion. Studies have been made on some of the elements behind this event and special interest has been focused on the protein tropomyosin. Muscle tropomyosin and its function in regulating muscle contraction, have been studied for decades, but there are also multiple tropomyosin isoforms in non-muscle cells, whose detailed function has not been revealed. Previous work at this department has shown an involvement of tropomyosin in the assembly of actin in vitro in the presence of gelsolin, and initial studies located tropomyosin to the lamellipodium of stimulated human fibroblasts. However, the general view is that tropomyosin is depleted from the advancing cell edge, observations noted in support of the current model of Arp2/3 dependent formation of actin filaments in lamellipodia. We have demonstrated the presence of tropomyosins in lamellae of migrating cells using different antibodies against non-muscle tropomyosin in indirect immunofluorescence. Also, the distribution of tagged non-muscle tropomyosin isoforms was analyzed in transfected cells. We conclude that tropomyosin is present in lamellipodia, all the way to their advancing edges. The presence of tropomyosin in the leading edge urges for tropomyosin to be taken into account when modelling cell motility. Furthermore, the nature of cytosolic tropomyosin was investigated by gel filtration chromatography and the conclusion was that in fibroblasts, approximately 10% of the tropomyosin is present in the cytosol, while the remaining 90% is associated with actin microfilaments in the cytomatrix. Interestingly, the soluble tropomyosin was found to exist mostly in a multimeric form of high molecular weight. Surprisingly, skeletal muscle tropomyosin and recombinant TM1 expressed in Escherichia coli forms multimers, a phenomenon not observed previously.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi, 2008. 46 p.
Keyword
cell motility, microfilament system, actin, tropomyosin
National Category
Biochemistry and Molecular Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-7633 (URN)978-91-7155-667-7 (ISBN)
Public defence
2008-06-02, De Geersalen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-05-12 Created: 2008-05-02 Last updated: 2012-01-12Bibliographically approved

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