Rho-GTPases and Muscle Differentiation: Spatio-Temporal Control of Rac1-Activity and Specific Signaling Pathways
Final Report Abstract
Muscles develop from undifferentiated myoblasts by a complex differentiation process to multinucleated and contractile muscle fibers. Proteins of the family of RhoGTPases have been implicated in regulating many differentiation steps. RhoGTPases are molecular switches which can shuttle between an active and an inactive state. Guanine-nucleotide exchange factors (GEFs) activate RhoGTPases, whereas GTPase activating proteins (GAPs) inactivate them. However, the exact involvement of one the RhoGTPase Rac1 is controversial and especially very little is known about the regulation of this RhoGTPase. This project intended to understand further details about the regulation of Rac1 during myoblast differentiation, especially with respect to the spatio-temporal aspect. For that it was first attempted to use RhoGTPase biosensors, which allow to detect active RhoGTPases in living cells. However, this approach turned out not to be appropriate for myoblast differentiation, as this process is too long-termed. The involvement of the GEF Def-6 in myoblast differentiation was discovered in detail. Def-6 was found to bind to the intracellular domain of the integrin alpha7A chain. By cell free binding assays with purified proteins, this binding could be proven to occur directly without any other cellular proteins mediating this interaction. The alpha7A integrin chain is a splice variant, which is exclusively expressed during skeletal muscle development and also in terminally differentiated skeletal muscles. Particular functions of the alpha7A chain were never described before and Def-6 is the first protein which was shown to bind exclusively to the alpha7A splice isoform. Overexpression of Def-6 in the myoblasts cell line C2C12 under growth conditions did not have a significant impact, as the cells showed a normal morphology and did not have increased Rac1.GTP levels. In contrast, when Def-6 overexpressing C2C12 cells were induced to differentiate to myotubes, differences compared to control cells were observed. Although general differentiation markers were upregulated in these cells after induction of differentiation, the formation of resulting myotubes was strongly impaired, as excessive cell fusions occurred (measured by the fusion index) and the resulting myotubes had an altered morphology (strongly branched myotubes with increased diameter). Interestingly, Rac1 activity was found to be increased in Def-6 overexpressing cells during the first days of differentiation, before Rac1 activity reached the same level as the control cells. These results showed that Def-6 has to be tightly regulated during myogenesis and that the protein is involved in controlling coordinated membrane fusions during myoblast differentiation, a process for which the involvement of Rac1 was suggested before. By the interaction with the alpha7A chain it seemed likely that Def-6 GEF activity is guided to certain areas in the differentiating cell. Indeed, by confocal microscopy I found Def-6 to colocalize with the apha7 chain in myotubes. The regulation of Def-6 was previously suggested to occur via phosphorylation of a particular tyrosine residue, and I found a fraction of the endogenous Def-6 to be tyrosine phosphorylated in myoblasts and myotubes. Further, the binding of Def-6 to the alpha7A chain was found to modulate GEF activity of Def-6. In another part of this research project I performed a screen to identify other novel Racl GEFs involved in myoblast differentiation. By this screen I found that expression of the Rac1/cdc42 GEF ßPIX is upregulated early during differentiation of myoblasts. According to known functions of ßPIX, it has to be assumed that this protein regulates early migration and spreading events during myogenesis and first experiments using ßPIX depleted myoblasts confirmed this. Taken together, my results indicate functions of different GEFs for RhoGTPases during different steps of myoblasts differentiation, which is an essential process for the development and regeneration of muscles.
Publications
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Def-6, a guanine nucleotide exchange factor for Rac1, interacts with the skeletal muscle integrin chain alpha7A and influences myoblast differentiation. J Biol Chem. 2007 May 25;282(21):15730-42
Samson T, Will C, Knoblauch A, Sharek L, von der Mark K, Burridge K, Wixler V
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RhoG regulates endothelial apical cup assembly downstream from ICAM1 engagement and is involved in leukocyte transendothelial migration. J Cell Biol. 2007 Sep 24;178(7):1279-93
van Buul JD, Allingham MJ, Samson T, Meller J, Boulter E, Garcia-Mata R, Burridge K.
