Laboratory Management

The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs): two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP' part) of the B1 subunit of the cyclic GMP-gated channel.

In rod outer segments, GARP proteins have been proposed to control subcellular propagation of Ca2+ signals from the Ca2+-entry site, the cyclic nucleotide-gated channel, to the cytosol. GARP1 and GARP2 can be regarded as loose coils with low-affinity, high-capacity Ca2+ binding. Thus, GARPs might be involved in photoreceptor adaptation, which is controlled by Ca2+. Importantly, the GARP' part of the CNG channel may function as a molecular wire that is "channeling" Ca2+ from the exit site of the channel onto the surface of the disk and thereby compartmentalises Ca2+ microdomains.

GARPs have been shown to interact with proteins at the rim of the disc membrane. Amino acid sequence analysis of GARPs revealed structural features typical of "natively unfolded" proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder.

Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.

We aim to analyse the protein interactions at the rim region on the single-molecule level using biochemical and biophysical techniques including cryo-electron microscopy, scanning electron microscopy (SEM), and high-resolution transmission electron microscopy (TEM).

Publications

Raju, D.N., Hansen, J.N., Rassmann, S., Stüven, B., Jikeli, J.F., Strünker, T., Körschen, H.G., Möglich, A., Wachten, D. (2019) Cyclic Nucleotide-Specific Optogenetics Highlights Compartmentalization of the Sperm Flagellum into cAMP Microdomains. Cells 8, 648. Link to MPG Publication Repository


Stabel, R., Stüven, B., Hansen, J.N., Körschen, H.G., Wachten, D., Möglich, A. (2019) Revisiting and Redesigning Light-Activated Cyclic-Mononucleotide Phosphodiesterases. J Mol Biol, [Epub ahead of print 07/2019: In Press, Corrected Proof]. Link to MPG Publication Repository

Woeste, M.A., Stern, S., Raju, D.N., Grahn, E., Dittmann, D., Gutbrod, K., Doermann, P., Hansen, J.N., Schonauer, S., Marx, C.E., et al. (2019). Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations. J Biol Chem 294, 3853-387 Link to MPG Publication Repository

Windler, F., Bönigk, W., Körschen, H.G., Grahn, E., Strünker, T., Seifert, R. & Kaupp, U.B. (2018) "The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclic-nucleotide binding" Nature Communications 9, 2809 Link to MPG Publication Repository

Schonauer, S., Körschen, H.G., Penno, A., Rennhack, A., Breiden, B., Sandhoff, K., Gutbrod, K., Dörmann, P., Raju, D.N., Haberkant, P., Gerl, M.J., Brügger, B., Zigdon, H., Vardi, A., Futerman, A.H., Wachten, D. (2017) Identification of a feedback loop involving beta-glucosidase 2 and its product sphingosine sheds light on the molecular mechanisms in Gaucher disease" J. Biol. Chem., M116.762831 Link to MPG Publication Repository

Schumacher, C.H., Körschen, H.G., Nicol, C., Gasser, C., Seifert, R., Schwarzel, M. & Moglich, A. (2016) "A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators" Methods Mol. Biol. 1408, 93-105

Raju, D., Schonauer, S., Hamzeh, H., Flynn, K.C., Bradke, F., vom Dorp, K., Dörmann, P., Yildiz, Y., Trötschel, C., Poetsch, A., Breiden, B., Sandhoff, K., Körschen, H.G. & Wachten, D. (2015) "Accumulation of glucosylceramide in the absence of the Beta-Glucosidase GBA2 alters cytoskeletal dynamics" PLoS Genet 11, e1005063

Scheib, U., Stehfest, K., Gee, C.E., Körschen, H.G., Fudim, R., Oertner, T.G. & Hegemann, P. (2015) "The rhodopsin-guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling" Sci. Sinal. 8, rs8

Pichlo, M., Bungert-Plümke, S., Weyand, I., Seifert, R., Bönigk, W., Strünker, T., Kashikar, N.D., Goodwin, N., Müller, A., Pelzer, P., Van, Q., Enderlein, J., Klemm, C., Krause, E., Trotschel, C., Poetsch, A., Kremmer, E. & Kaupp, U.B. (2014) "High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor" J. Cell. Biol. 206, 541-557

Körschen, H.G., Yildiz, Y., Raju, D.N., Schonauer, S., Bönigk, W., Jansen, V., Kremmer, E., Kaupp, U.B. & Wachten, D. (2013) "The non-lysosomal beta-glucosidase GBA2 is a non-integral membrane-associated protein at the ER and Golgi" J. Biol. Chem. 288, 3381-3393

Haber-Pohlmeier, S., Abarca-Heidemann, K., Körschen, H.G., Dhiman, H.K., Heberle, J., Schwalbe, H., Klein-Seetharaman, J., Kaupp, U.B. & Pohlmeier, A. (2007) "Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment" Biophys. J.92, 3207-3214

Batra-Safferling, R., Abarca-Heidemann, K., Körschen, H.G., Tziatzios, C., Stoldt, M., Budyak, I., Willbold, D., Schwalbe, H., Klein-Seetharaman, J. & Kaupp, U.B. (2006)"Glutamic acid-rich proteins of rod photoreceptors are natively unfolded" J. Biol. Chem. 281,1449-1460

Saenz de Tejada, I., Angulo, J., Cuevas, P., Fernández, A., Moncada, I., Allona, A., Lledó, E., Körschen, H.G., Niewöhner, U., Haning, H., Pages, E. & Bischoff, E (2001)"The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new PDE5 inhibitor vardenafil" Int. J. Impot. Res. 13, 282-290

Körschen, H.G., Beyermann, M., Müller, F., Heck, M., Vantler, M., Koch, K.W., Kellner, R., Wolfrum, U., Bode, C., Hofmann, K.P. & Kaupp, U.B. (1999) "Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors" Nature 400, 761-766

Weitz, D., Zoche, M., Müller, F., Beyermann, M., Körschen, H.G., Kaupp, U.B. & Koch, K.W. (1998) "Calmodulin controls the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the b-subunit" EMBO J. 17, 2273-2284

Körschen, H.G., Illing, M., Seifert, R., Sesti, F., Williams, A., Gotzes, S., Colville, C., Müller, F., Dosé, A., Godde, M., Molday, L.,Kaupp, U.B. & Molday R.S. (1995) "A 240 kDa protein represents the complete beta subunit of the cyclic nucleotide-gated channel from rod photoreceptor" Neuron 15, 627-636