Contact

Prof. Dr. Volker Haucke

phone +49 30 94793 100
haucke(at)fmp-berlin.de

Research Areas of the Haucke Lab

Neurotransmission and presynaptic membrane turnover

Regulation of membrane homeostasis in endocytosis & the endolysosomal system by phosphoinositides and phosphoinositide metabolizing enzymes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Research Department Molecular Pharmacology and Cell Biology (Volker Haucke)

Regulation of cell signalling and membrane homeostasis in endocytosis and the endolysosomal system by phosphoinositides and phosphoinositide metabolizing enzymes

Membrane lipids, in particular phosphoinositides, are implicated in the regulation of intracellular membrane traffic and in cell signalling. Dysfunction of phosphoinositide metabolizing enzymes is implicated in a variety of diseases ranging from developmental defects and inherited rare disease such as myotubular myopathies and Charcot Marie Tooth disease to renal and brain disorders and to cancer.

Research from our laboratory has unravelled key roles for distinct phosphoinositide species in the regulation of defined steps of clathrin-mediated endocytosis (CME) (Figure 1).

Figure 1: Stages of CME visualized by electron microscopy.

CME is initiated by PI(4,5)P2 synthesized by type PIPK (Krauß et al., J. Cell Biol. 2003; Krauß et al., Proc. Natl. Acad. Sci. USA 2006; Krauß & Haucke, EMBO Rep. 2007). Combined biochemical and structural studies from our lab have uncovered that multiple binding sites within PIPK type Ig, the major PI(4,5)P2-synthesizing enzyme at synapses, orchestrate AP-2-PIPKIg complex formation and hence, PI(4,5)P2-synthesis during endocytosis (Kahlfeldt et al., 2010). Moreover, we and others have discovered that many proteins involved in CME, such as the a and m2 subunits of the heterotetrameric clathrin adaptor complex AP-2 (Rohde et al., J Cell Biol. 2002; Höning et al., Mol. Cell 2005), the A/ENTH domains of AP180 or CALM, epsins, the PX domains of SNXs, or the pleckstrin homology (PH) domain of the large GTPase dynamin (Faelber et al., Nature 2011; Reubold et al., Nature 2015) can interact directly with PI(4,5)P2. Depletion of PI(4,5)P2 leads to loss of plasmalemmal clathrin/ AP-2 coated pits and complete inhibition of clathrin-dependent receptor internalization. Together with the laboratory of Prof. Oliver Daumke (MDC Berlin) we have also dissected the mechanisms of membrane fission by the large GTPase dynamin and the way dynamin assembly at membranes is regulated (Faelber et al., Nature 2011; Reubold et al., Nature 2015).

         More recently, we have identified phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) as a a novel key lipid in endocytosis. We were able to show that formation of PI(3,4)P2 by class II phosphatidylinositol-3-kinase C2α (PI(3)K C2α) spatiotemporally controls clathrin-mediated endocytosis. Depletion of PI(3,4)P2 or PI(3)K C2α impaired the maturation of late-stage clathrin-coated pits before fission (Figure 2). We could further show that timed formation of PI(3,4)P2 by PI(3)K C2α was required for selective enrichment of the BAR domain protein SNX9 at late-stage endocytic intermediates.

Figure 2: Phosphatidylinositol (3,4)-bisphosphate synthesis by PI3KC2α spatiotemporally controls clathrin-mediated endocytosis and coated pit dynamics. (A) Colocalization of phosphatidylinositol (3,4)-bisphosphate with clathrin heavy chain. (B) Depletion of plasma membrane phosphatidylinositol (3,4)-bisphosphate inhibits clathrin-mediated transferrin endocytosis. (C) Depletion of the phosphatidylinositol (3,4)-bisphosphate-synthesizing enzyme PI3KC2α stalls clathrin-coated pit dynamics. (D) Recruitment profile of endocytic proteins including PI3KC2α determined by TIRF imaging. Adapted from: Posor et al., Nature 2013.

These findings have provided a mechanistic framework for the role of PI(3,4)P2 in endocytosis (Posor et al., Nature 2013) and suggest a pathway for phosphoinositide conversion at the plasma membrane en route to endosomes (highlighted by News & Views articles in Nature and Nature Cell Biology).
We hypothesize that PI(3,4)P2 is required for conformational activation of its effector protein SNX9 (Daumke et al., Cell 2014) akin to the dynamin/lipid-based activation of the BAR domain protein syndapin demonstrated earlier by us (using protein X-ray crystallography; Rao et al., Proc. Natl. Acad. Sci. USA 2010). These and other findings have led us to propose a model how the dynamic assembly and disassembly of BAR domain protein scaffolds constrains the membrane into distinct shapes (Figure 3) as the pathway progresses towards fission by the GTPase dynamin.

Figure 3: Domain organization, structure, and assembly of BAR domain proteins and dynamin. At the left, the domain organization of human FCHO I , FBP17, SNX9, endophilin A1, amphiphysin 1 and dynamin-1 are shown. Dimeric building blocks are boxed in green, cylinders indicate the approximate diameter of the encircled lipid tubule, the outer diameter of the protein-coated oligomer is indicated. Taken from: Daumke et al., 2014.

Another major focus of our more recent work has been the elucidation of the mechanism of phosphosinositide conversion as a key step in the turnover of membrane identity during membrane traffic and signalling. We have discovered that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans link to Latest Thinking video. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2a (PI4K2a-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane (Figure 4) and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.

Figure 4: Model of PI conversion during endosomal exocytosis. SE: sorting endosome; RE: recycling endosome; 2α: PI4K2α.

Other work has focussed on the function of phosphatidylinositol 4-kinase complexes in endosomal sorting and Wnt signaling during development (Mössinger et al., EMBO Rep. 2012; Wieffer et al., Curr. Biol. 2013). These studies are of fundamental importance for our understanding of the mechanisms by which phosphoinositides control endocytosis, endosomal membrane traffic, and development.

At present, we are investigating how metabolism of various phosphoinositides regulates intracellular membrane transport and cell signalling and vice versa (Marat and Haucke, EMBO J., 2016). We also study how phosphoinositides spatiotemporally control the assembly of membrane scaffolds (i.e. BAR domain protein assemblies) at endocytic sites and on compartments of the endo-lysosomal system.

Selected references

  • Ketel, K., Krauss, M., Nicot, A.S., Puchkov, D., Wieffer, M., Müller, R., Subramanian, D., Schultz, C., Laporte, J., Haucke, V. (2016) A phosphoinositide conversion mechanism for exit from endosomes. Nature, 529, 408-412
  • Marat, A.L., Haucke, V. (2016) Phosphatidylinositol 3-phosphates - at the interface between cell signalling and membrane traffic. EMBO J., 35, 561-579
  • Haucke, V. (2015) Cell biology: On the endocytosis rollercoaster. Nature, 517, 446-447
  • Reubold, T.F., Faelber, K., Plattner, N., Posor, Y., Ketel, K., Curth, U., Schlegel, J., Anand, R., Manstein, D.J., Noé, F., Haucke, V., Daumke, O., Eschenburg, S. (2015) Crystal structure of the dynamin tetramer. Nature, 525, 404-408
  • Daumke, O., Roux, A., Haucke, V. (2014) BAR domain scaffolds in dynamin-mediated membrane fission. Cell 156, 882-892
  • Franco, I., Gulluni, F., Campa, C.C., Costa, C., Margaria, P., Ciraolo, E., Martini, M., Monteyne, D., De Luca, D., Germena, G.,  Posor, Y., Maffucci, T., Marengo, S., Haucke, V., Falasca, M., Perez-Morga, D., Boletta, A., Merlo, G.R.,  Hirsch, E. (2014) PI3K class II α controls spatially restricted endosomal PtdIns3P and Rab11 activation to promote primary cilium function. Dev. Cell, 28, 647-58
  • Posor, Y., Eichhorn-Grünig, M., Puchkov, D., Schöneberg, J., Ullrich, A., Lampe, A., Müller, R., Zarbakhsh, Gulluni, F., Hirsch, E., Krauss, M., Schultz, C., Noe, F., Haucke, V. (2013) Spatiotemporal Control of Endocytosis by Phosphatidylinositol 3,4-Bisphosphate. Nature,499, 233-237
  • Wieffer, M., Cibrián Uhalte, E., Posor, Y., Otten, C., Branz, K., Schütz, I., Mössinger,      J., Schu, P., Abdelilah-Seyfried, S., Krauß, M., Haucke, V. (2013) PI4K2/ AP-1-based TGN-endosomal sorting regulates Wnt signaling. Curr Biol., 23, 2185-90
  • Puchkov, D. and Haucke, V. (2013) Greasing the synaptic vesicle cycle by membrane lipids. Trends Cell Biol., 23, 493–503
  • Mössinger, J., Wieffer, M., Krause, E., Freund, C., Gerth, F., Krauss, M., Haucke, V. (2012) Phosphatidylinositol 4-kinase IIα function at endosomes is regulated by the ubiquitin ligase ltch. EMBO Rep., 13, 1087-94
  • Rao, Y., Ma, Q., Vahedi-Faridi, A., Sundborger, A., Pechstein, A., Puchkov, D., Luo, L., Shupliakov, O., Saenger, W., Haucke, V. (2010) Molecular basis for SH3-domain regulation of F-BAR-mediated membrane deformation. Proc. Natl. Acad. Sci. USA, 107, 8213-8218.
  • Kahlfeldt, N., Vahedi-Faridi, A., Schäfer, J.G., Krainer, G., Keller, S., Saenger, W., Krauss, M., Haucke V. (2010) Molecular basis for the association of PIPKIg-p90 with the clathrin adaptor AP-2, J. Biol. Chem., 285, 2734-2749
  • Krauss, M. and Haucke, V. (2007) Phosphoinositide modifying enzymes at the interface between membrane traffic and cell signaling. EMBO Rep., 3, 241-246
  • Krauss M., Kukhtina, V., Pechstein, A., and Haucke V. (2006) Stimulation of PIPK type I-mediated PI(4,5)P2 synthesis by endocytic AP-2mu adaptor cargo complexes. Proc. Natl. Acad. Sci. USA 103, 11934-9

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)
info(at)fmp-berlin.de

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