Profilins : The Control of the Microfilament and Microtubule systems
Sammanfattning: The microtubule and actin filament systems are important for many cellular processes such as intracellular organelle trafficking, cell adhesion, migration and mitosis. The two filament systems are closely connected, operate in parallel and are essential for proper cell function. This thesis demonstrates that profilin, an actin monomer binding protein that enhances actin nucleotide exchange, is central for actin polymerization control and binds poly-proline sequences present in many actin nucleation and elongation promoting factors,also contributes to the coordination of the actin and microtubule systems.The results show that profilin is linked to microtubules via members of the formin family of proteins. This was realized by combining different microscopy techniques with biochemical analyses. Exposure of the cells to the formin inhibitor SMIFH2 led to a significant decrease of the profilin-microtubule co-distribution, and this was also observed by simultaneously decreasing expression of two formins, Diaphaneous 1 and 2, by siRNA-transfection. Together this provided strong evidence for the formins as major components associating profilin to the microtubules. With the use of actin-targeted drugs, I further showed that the turn-over of the actin microfilament system balances the extent of profilin-microtubule association, and, moreover, down-regulation of profilin expression resulted in increased levels of acetylated tubulin and faster microtubule elongation. All in congruence with profilin being central in coordinating the activities of the microtubule and actin microfilament systems.In some of the experiments above, I used a specifically developed fluorescent variant of profilin that carries the fluorescent protein citrine internally in a loop extending away from the actin and proline-binding surfaces. In a separate study it was shown to bind poly-proline, phosphatidylinositol lipids and actin, and also to distribute properly in cells after expression. Furthermore, this fusion-construct largely rescued the phenotypes displayed by a Crispr/Cas 9 profilin I gene knock-out, and therefore opens for detailed future analyses of profilin-dependent processes by imaging of living cells.In a third study, encouraged by the result with citrine-profilin I, a similar approach was taken to generate a fluorescent variant of the profilin isoform IIa with the mKate2 protein as the fused fluorophore. The cellular localisation of this profilin-constuct revealed an association with focal adhesions. Further analysis after introduction of a mutation in the profilin-moiety that distorts poly-proline binding, clearly showed that the distribution to focal adhesions depends on the binding to poly-proline, pointing to a small group of candidate proteins as recruiting molecules. It was not possible to detect a localization to microtubules suggesting that this interaction is unique to profilin I. This means that the data in my thesis also point to distinct functions for the two major mammalian profilin isoforms; profilin I being connected to microtubule organization in addition to its classical role as an actin partner, and profilin IIa-function may be more closely linked to focal adhesion and cell-substratum attachments.
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