No description available.

Internal and external signaling dependent changes in cell behavior are directly linked to force-generating remodeling of the actin microfilament system which is juxtaposed to the inside of the plasma membrane. This dynamic filament system is involved in many processes in the cytoplasm and the nucleus of eukaryotic cells.   This thesis studies profilin, a regulator of actin filament dynamics which functions during incorporation of new actin molecules at growing filament ends at the cell periphery. Profilin is also present in the nucleus but its function is less well understood in this compartment. Here I present results concerning profilin and the activity of the transcription factor SRF, which is known to control the expression of actin and many actin-binding proteins in a process requiring the MRTF-A co-factor. MRTF-A binds monomeric actin and is released upon receptor mediated actin polymerization. Depletion of the two profilin isoforms I and IIa reduced MRTF-A/SRF-dependent transcription, most likely since the lack of profilin enable more MRTF-A to bind actin monomers and thereby prevent SRF-transcription. Interestingly profilin depletion also seemed to affect general transcription in the two cell lines investigated. In a separate study, a close connection between profilin, and possibly also profilin:actin, with microtubules was revealed. Microtubules are important for intracellular trafficking of vesicles as well as directional cell migration and the observation made here suggests the existence of a microtubule-associated platform for actin filaments formation. In congruence, the microtubule-associated actin nucleation promoting factor WHAMM was found to interact with profilin. Finally, the intracellular distribution of profilin was investigated by fluorescence microscopy using different peptide specific antibodies. Since these antibodies showed unique but varying results our work emphasizes common problems connected with this technique. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper1: Manuscript; Paper 2: Manuscript; Paper 3: Manuscript</p>

Das Cyanobakterium Microcystis aeruginosa PCC 7806 enthält zwei Proteine unbekannter Funktion, welche eine hohe Sequenzähnlichkeit mit Bausteinen des eukaryotischen Aktinzytoskeletts haben. Eines dieser Proteine ist Aktin selbst, das andere ist das Aktinbindeprotein Profilin. Die vorliegende Arbeit enthält eine detaillierte Charakterisierung beider Proteine sowie Vergleiche mit ihren eukaryotischen Verwandten. So inhibiert, im Gegensatz zu Eukaryoten, cyanobakterielles Aktin nicht das Enzym DNaseI. Es bildet jedoch Polymere, die hier mit Phalloidin visualisiert wurden. Konfokale Mikroskopie offenbart klare Unterschiede in den Polymeren, da die cyanobakteriellen eine Länge von 10 µm nicht überschreiten und breiter sind als die zylindrischen, ca. 100 µm langen Filamente eukaryotischen Aktins. Röntgen-Kleinwinkelstreuungsdaten zeigen, dass cyanobakterielle Aktinpolymere in ihrer Form am ehesten einem Band ähneln. Es bestehen auch Unterschiede hinsichtlich des Profilins: während es in Eukaryoten ausschließlich Aktinmonomere bindet, assoziiert cyanobakterielles Profilin mit Aktinfilamenten und vermittelt die Entstehung flächiger Heteropolymere. GFP-Fusionsstudien zeigen, dass die Koexpression von Aktin und Profilin die Bildung eines Hohlraumkompartiments in E.coli nach sich zieht. Ähnliche Gebilde wurden bereits in Microcystis gezeigt und könnten auf die beobachteten Heteropolymere zurückzuführen sein. Diese Arbeit verdeutlicht, dass beide Proteine in einer natürlichen Bakterienpopulation etabliert sind und dort Merkmale tragen, die ihre eukaryotischen Vorläufer nicht zeigen. Folglich könnte die Anpassung an die räumlichen Begrenzungen einer Bakterienzelle, welcher die für die Regulierung der Polymerisation notwendigen Aktinbindeproteine fehlen, die Triebkraft für eine Koevolution von cyanobakteriellem Aktin und Profilin gewesen sein. Dieser Prozess gipfelte möglicherweise in der Entstehung eines neuartigen intrazellulären Gebildes von potentiell struktureller Bedeutung. / The cyanobacterium Microcystis aeruginosa PCC 7806 harbors two proteins with unknown functions that were transferred horizontally from eukaryotes and show a high degree of sequence identity with key components of the eukaryotic actin cytoskeleton. One is actin itself; the other is profilin, an actin binding protein. This work presents the detailed characterization of both proteins and comparisons with the eukaryotic archetype. In contrast to bona fide actin, its cyanobacterial counterpart does not inhibit DNaseI. It forms polymers that can be visualized with labeled phalloidin, resembling eukaryotic actin in that respect. However, confocal microscopy reveals key differences between polymers of eukaryotic and cyanobacterial actin. Whereas the former appear as cylindrical filaments about 100 µm in length, the latter are shorter and wider arresting polymerization at 5-10 µm. Structural elucidation by Small-angle X-ray scattering shows that cyanobacterial actin polymers are ribbon-shaped. This work also shows fundamental differences between cyanobacterial and eukaryotic profilin. Most importantly, cyanobacterial profilin binds actin filaments and mediates their assembly into heteropolymeric sheets. GFP labeling experiments show that the co-expression of cyanobacterial profilin and actin results in the formation of large hollow enclosures in E.coli. These structures resemble the shell-like distribution of actin in Microcystis aeruginosa and may be based on the actin/profilin heteropolymers observed in vitro. This work shows that both cyanobacterial proteins are established in a natural bacterial community where they have gained properties unknown from their eukaryotic ancestors. Consequently, the adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes may have driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity of potential structural relevance.

University, Diss., 2000--München.

No description available.

No description available.

The spatio-temporal organization and dynamic behavior of microtubules accurately react to cellular needs during intracellular transport, signal transduction, growth, division, and differentiation. The cell generates centrosomal microtubules de novo with the help of γ-tubulin complexes (γTuRCs). The post-translational modifications fine-tune microtubule nucleation by targeting the proteins, interacting with γTuRCs. However, the exact signaling pathways, regulating centrosomal microtubule nucleation, remain mostly unknown. In the presented thesis, we functionally characterized protein tyrosine phosphatase SHP-1 and E3 UFM-protein ligase 1 (UFL1) with its interacting protein CDK5RAP3 (C53) in the regulation of centrosomal microtubule nucleation. We also elucidated the role of actin regulatory protein profilin 1 in this process. We found that SHP-1 formed complexes with γTuRC proteins and negatively regulated microtubule nucleation by modulating the amount of γ-tubulin/γTuRC at the centrosomes in bone marrow-derived mast cells (BMMCs). We suggested a novel mechanism with centrosomal tyrosine-phosphorylated Syk kinase, targeted by SHP-1 during Ag-induced BMMCs activation, regulating microtubules. We showed for the first time that UFL1/C53 protein complex is involved in the regulation of microtubule...

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.