Cellular Functions and Dynamics of Reggie Proteins

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LANGHORST, Matthias F., 2006. Cellular Functions and Dynamics of Reggie Proteins [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Langhorst2006Cellu-8837, title={Cellular Functions and Dynamics of Reggie Proteins}, year={2006}, author={Langhorst, Matthias F.}, address={Konstanz}, school={Universität Konstanz} }

2011-03-24T17:47:13Z Langhorst, Matthias F. 2011-03-24T17:47:13Z application/pdf Cellular Functions and Dynamics of Reggie Proteins Langhorst, Matthias F. The reggies/flotillins are two proteins of 47 kDa, which are associated with the detergent-resistant membrane fraction. They are evolutionarily highly conserved and expressed in most tissues, with particularly high expression in the developing and regenerating nervous system. The reggies localize predominantly to the plasma membrane, where they form clusters of<br />50 100 nm formed by homo- and hetero-oligomerization. These clusters scaffold specific membrane microdomains and facilitate the regulated assembly of multiprotein complexes.<br />In this study, the cellular dynamics and functions of the reggies in neurons and lymphocytes were investigated. Analysis of the biosynthetic pathways of the reggies showed vesicular, Golgi- and microtubule-dependent trafficking of the reggies. At the plasma membrane, rapid vesicular cycling was observed by TIRF microscopy, which could be stimulated by serum and protein phosphatase inhibitors. During docking, reggie-fluorescence did not diffuse into the adjacent plasma membrane, suggesting that the reggies are trafficked as stable entities. In FRAP experiments, reggie-1 proved to be highly mobile at the bulk plasma membrane in both neurons and lymphocytes. But at the basis of membrane protrusions in PC12 cells and within the polarized accumulation in a preformed cap in lymphocytes, reggie-1 was largely immobilized. Immobilization at these specific structures and lateral mobility of reggie-1 in general was shown to be regulated by the SPFH domain. This domain interacted with the actin cytoskeleton both in vitro and in vivo. Interestingly, vesicular docking at the plasma membrane was also dependent on the SPFH domain. Thus, both vesicular and lateral dynamics of the reggies can be tightly regulated for the formation of stable reggie microdomains at specific sites of the cell.<br />The results of this study show that reggie microdomains are of particular importance for signalling complexes regulating the remodelling of the actin cytoskeleton. Expression of a trans-negative reggie-1 deletion mutant, which interferes with oligomerization of the endogenous reggies, selectively inhibits cytoskeleton remodelling, both in lymphocytes and in neurons. In lymphocytes, the inhibition of actin remodelling impaired the formation of a stable signalling macrodomain after activation. In neurons, differentiation and neurite outgrowth were impaired, both in neuroblastoma cells and in primary neurons. The massive defects in actin cytoskeleton remodelling were caused by an impaired Rho-GTPase signalling. In lymphocytes, the guanine-nucleotide exchange factor Vav did not localize to sites of active actin remodelling in cells expressing the trans-negative reggie-1 mutant. In neurons, the recruitment of the adaptor protein CAP/ponsin, which in turn interacts with several Rho-GTPase regulators, to the plasma membrane failed upon expression of the trans-negative mutant. Thus, microdomains scaffolded by oligomeric reggie clusters are platforms for the assembly of Rho-GTPase signalling complexes and are thus crucial for cytoskeletal remodelling. This function of the reggies is in excellent agreement with their discovery as proteins upregulated during axon regeneration.<br />Thus, this study expanded the view on reggie localization from static subcellular distributions to a dynamic, regulated equilibrium of vesicular pathways and lateral mobility in the plasma membrane. The formation of remarkably stable microdomains at specific sites of the cell, which are important regulators of the actin cytoskeleton, was shown to be the most prominent function of the reggies in neurons and lymphocytes. Zelluläre Funktion und Dynamik der Reggie-Proteine deposit-license 2006 eng

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