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Elements in regulation of the microfilament system
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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 [en]
cell motility, microfilament system, actin, tropomyosin
National Category
Biochemistry and Molecular Biology
Research subject
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-7633ISBN: 978-91-7155-667-7 (print)OAI: oai:DiVA.org:su-7633DiVA: diva2:198734
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
List of papers
1. 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: 2017-12-13Bibliographically approved
2. Tropomyosins are present in lamellipodia of motile cells
Open this publication in new window or tab >>Tropomyosins are present in lamellipodia of motile cells
Show others...
2006 (English)In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 85, no 5, 399-409 p.Article in journal (Refereed) Published
Abstract [en]

This paper shows that high-molecular-weight tropomyosins (TMs), as well as shorter isoforms of this protein, are present in significant amounts in lamellipodia and filopodia of spreading normal and transformed cells. The presence of TM in these locales was ascertained by staining of cells with antibodies reacting with endogenous TMs and through the expression of hemaglutinin- and green fluorescent protein-tagged TM isoforms. The observations are contrary to recent reports suggesting the absence of TMs in regions,where polymerization of actin takes place, and indicate that the view of the role of TM in the formation of actin filaments needs to be significantly revised.

Keyword
Arp2/3, Actin, Lamellipodia, Tropomyosin, VASP
Identifiers
urn:nbn:se:su:diva-25020 (URN)10.1016/j.ejcb.2005.12.005 (DOI)
Note
Part of urn:nbn:se:su:diva-7633Available from: 2008-05-12 Created: 2008-05-02 Last updated: 2017-12-13Bibliographically approved
3. Tropomyosin assembly intermediates in the control of MF-system turnover
Open this publication in new window or tab >>Tropomyosin assembly intermediates in the control of MF-system turnover
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2008 (English)In: European Journal of Cell Biology, ISSN 0171-9335, E-ISSN 1618-1298, Vol. 87, no 11, 905-920 p.Article in journal (Refereed) Published
Abstract [en]

Tropomyosin is a coiled-coil α-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MWapp 180,000–250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.

Keyword
Actin, Tropomyosin, Tropomyosin multimers, Lamellipodia, Filopodia, Tumorigenesis
Identifiers
urn:nbn:se:su:diva-25021 (URN)10.1016/j.ejcb.2008.06.006 (DOI)000260600800004 ()
Note
Part of urn:nbn:se:su:diva-7633Available from: 2008-05-12 Created: 2008-05-02 Last updated: 2017-12-13Bibliographically approved

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