Functional analysis of cell signaling events following inhibition of clathrin/ AP2-mediated endocytosis
Final Report Abstract
Clathrin-mediated endocytosis (CME) regulates internalization, recycling, and degradation of a large variety of signaling receptors, channels, transporters and provides an entry point for bacteria or viruses. During the funding period we have developed and further refined the first known inhibitors of clathrin function that due to their ability to acutely and reversibly perturb clathrin-coated pit (CCP) dynamics have been termed PitstopsTM. Acute treatment of mammalian cells with Pitstop 2, a cell-permeable clathrin inhibitor, which acts by competing with endogenous ligands for access to the clathrin terminal domain (TD), reversibly inhibits CME of transferrin and of other receptors while secretory traffic of VSVG, retrograde traffic of COPI vesicles between the Golgi and the ER, or endosomal recycling proceed unperturbed. Application of Pitstop 2 furthermore causes the dispersion of mannose 6-phosphate receptors known to shuttle between the trans-Golgi network and endosomes by clathrin/ AP-1 and clathrin/ GGA-mediated transport processes to deliver lysosomal enzymes. During the funding period we have also probed potential applications of Pitstop compounds as antiviral or antimitotic agents. For example, we could show that Pitstop 2 inhibits the entry and replication of viruses including HIV and crimean-Congo hemorrhagic fever virus (CCHFV) suggesting that Pitstop compounds may serve as a starting point for the development of a novel class of antiviral drugs. Moreover, together with Megan Chircop and Phil Robinson we were able to demonstrate that application of Pitstop 2 in a variety of cell lines slows mitosis by interfering with clathrin function at the mitotic spindle. Additional work has resulted in the functional and biochemical dissection of select AP-2-endocytic ligand interactions and in the generation of the first conditional knockout mice lacking the clathrin adaptor AP-2(µ). These serve as an important starting point for future studies that will address the role of clathrin/ AP-2 in neuronal signaling.
Publications
- (2010) -p90 with clathrin adaptor AP-2, J. Biol. Chem., 285, 2734-2749
Kahlfeldt, N., Vahedi-Faridi, A., Schäfer, J.G., Krainer, G., Keller, S., Saenger, W., Krauss, M., Haucke V.
(See online at https://doi.org/10.1074/jbc.M109.074906) - (2010) Regulation of synaptic vesicle recycling by complex formation between intersectin 1 and the clathrin adaptor complex AP2. Proc. Natl. Acad. Sci. USA, 107, 4206-4211
Pechstein, A., Bacetic, J., Vahedi-Faridi, A., Gromova, K., Sundborger, A., Tomlin, N., Krainer, N., Vorontsova, O., Schäfer, J.G., Owe, S.G., Cousin, M.A., Saenger, W., Shupliakov, O., Haucke, V.
(See online at https://doi.org/10.1073/pnas.0911073107) - (2011) Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell, 146, 471-484
von Kleist, L., Stahlschmidt, W., Bulut, H., Gromova, K., Puchkov, D., Robertson, M., MacGregor, K.A., Tomlin, N., Pechstein, A., Chau, N., Chircop, M., Sakoff, J., von Kries, J., Saenger, W., Kräusslich, H.-G., Shupliakov, O., Robinson, P., McCluskey, A., Haucke, V.
(See online at https://doi.org/10.1016/j.cell.2011.06.025) - (2012) At the crossroads of chemistry and cell biology: inhibiting membrane traffic by small molecules Traffic, 13, 495-504
von Kleist, L. and Haucke, V.
(See online at https://doi.org/10.1111/j.1600-0854.2011.01292.x) - (2013) Crimean-Congo hemorrhagic fever virus utilizes a clathrin- and early endosome-dependent entry pathway. Virology, 444, 45-54
Garrison, A.R., Radoshitzky, S.R., Kota, K.P., Pegoraro, G., Ruthel, G., Kuhn, J.H., Altamura, L.A., Kwilas, S.A., Bavari, S., Haucke, V., Schmaljohn, C.S.
(See online at https://doi.org/10.1016/j.virol.2013.05.030) - (2013) Development of 1,8-Naphthalimides as clathrin inhibitors. J. Med. Chem. 57, 131-43
Macgregor, K.A., Robertson, M.J., Young, K.A., von Kleist, L., Stahlschmidt, W., Whiting, A., Chau, N., Robinson, P.J., Haucke V., McCluskey, A.
(See online at https://doi.org/10.1021/jm4015263) - (2013) Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells. Mol. Cancer, 12:4
Smith, C.M., Haucke, V., McCluskey, A., Robinson, P.J., Chircop, M.
(See online at https://doi.org/10.1186/1476-4598-12-4) - (2014) Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors. J Biol. Chem. 289, 4906-4918
Stahlschmidt, W., Robertson, M.J., Robinson, P.J., McCluskey, A., Haucke, V.
(See online at https://doi.org/10.1074/jbc.M113.535211)
