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Cross-linked Profilin:actin - A tool to study actin dynamics in non-muscle cells
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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 [en]
actin dynamics, profilin, actin, Profilin-actin complex, Cell motility, synaptic vesicle trafficing, Far western, fluorescence microscopy
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-168ISBN: 91-7265-854-1 (print)OAI: oai:DiVA.org:su-168DiVA: diva2:190415
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
List of papers
1. Detection of Binding Partners to the Profilin:actin Complex by Far western and MS-analyses
Open this publication in new window or tab >>Detection of Binding Partners to the Profilin:actin Complex by Far western and MS-analyses
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23149 (URN)
Note
Part of urn:nbn:se:su:diva-168Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2010-01-13Bibliographically approved
2. A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes
Open this publication in new window or tab >>A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes
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2003 (English)In: EMBO reports, ISSN 1469-221, Vol. 4, no 5, 523-529 p.Article in journal (Refereed) Published
Abstract [en]

We have examined the effect of covalently crosslinked profilin–actin (PxA), which closely matches the biochemical properties of ordinary profilin–actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin–actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin–actin is important for actin-based processes, and show that it has a key function in Listeria motility.

Place, publisher, year, edition, pages
London: Nature Publishing Group, 2003
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-23150 (URN)10.1038/sj.embor.embor823 (DOI)
Note
Part of urn:nbn:se:su:diva-168Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2009-12-22Bibliographically 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: 2017-12-13Bibliographically approved
4. Omega-shaped structures in the active zone caused by non-dissociable profilin:actin
Open this publication in new window or tab >>Omega-shaped structures in the active zone caused by non-dissociable profilin:actin
Show others...
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-23152 (URN)
Note
Part of urn:nbn:se:su:diva-168Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2010-01-13Bibliographically approved

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