The actin filament system

The Actin Filament System
Its Involvement in Cell Migration and Neurotransmitter Release

Anna-Karin Johnsson
Department of Cell Biology, The Wenner-Gren Institute, Stockholm University, 2011

©Anna-Karin Johnsson, Stockholm 2011 ISBN 978-91-7447-292-9 US-AB, 2011

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SUMMARY
The microfilament system consists of actin filaments as the major component and is regulated by a numberof actin binding proteins. It is juxtaposed to the plasma membrane where it forms a dense cortical weave from where it pervades into the cell interior. This filament system has multiple roles and participates in virtually all motile processes where its dynamic activities depend on receptor mediated signaling leading to constant polymerizations and depolymerizations. These activities are now alsoknown to affect gene regulation. This thesis discusses these dynamic reorganizations of the microfilament system and how components are supplied to support these motile processes. The focus is on profilin/profilin:actin, actin polymerization and the localization of the transcripts of these proteins. The localization of profilin mRNA was examined in relation to the distribution of β-actin mRNAusing fluorescent in situ hybridization. It was concluded that both these mRNAs localize to sites of massive actin polymerization called dorsal ruffles albeit the data obtained suggests that this localization must be dependent on distinct mechanisms. Additionally signal transduction and cell motility was studied after depleting the two profilin isoforms 1 and 2. The activity of thetranscription factor SRF is known to be coupled to microfilament system dynamics via the cofactor MAL which binds to actin monomers and is released upon receptor mediated actin polymerization. Depletion of profilin was seen to influence SRF dependent signaling, most likely because the lack of profilin enables more MAL to bind actin monomers thereby preventing SRF dependent transcription. Finally, it wasalso investigated how the synaptic vesicle protein synaptotagmin 1 which is involved in exocytosis, has a role in actin polymerization. This protein has previously been described to cause filopodia formation when ectopically expressed. A polybasic sequence motif was identified as the effector sequence for this activity and it was established that this sequence interacts with anionic lipids. It isalso discussed how this sequence could have a role in neurotransmitter release and actin polymerization in the nerve synapse.

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LIST OF PUBLICATIONS
Paper I:
Microtubule-dependent Localization of Profilin mRNA to Actin Polymerization Sites in Serumstimulated Cells Johnsson, A-K., Karlsson, R. (2010) European J of Cell Biology 89:394-401.

Paper II:

Profilin I and II are BothInfluencing SRF-dependent Signaling in B16 Melanoma Cells and Loss of Profilin I Interferes with Cell Migration Sadi, S., Johnsson, A-K., Karlsson, R. (2011) Manuscript.

Paper III: Synaptotagmin 1 causes Phosphatidyl-(4,5)-

bisphosphate-dependent Actin Remodeling in Cultured Non-neuronal and Neuronal cells Johnsson, A-K., Karlsson, R. (2011) Submitted, under review.

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TABLE OF CONTENTSIntroduction................................................................................... 7 Cell motility and microfilament system organization ...................... 8 Intracellular trafficking .................................................................... 13 Actin structure ................................................................................ 14 Profilin............................................................................................. 19 PIP2 and microfilament dynamics .................................................. 27 Rho GTPases .................................................................................... 29 Actin regulatory proteins ................................................................ 32 Actin depolymerizing factors...