Lipid Signaling & Membrane Traffic

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

Membrane lipids, in particular phosphoinositides (PIs), are key players in the control of intracellular membrane traffic and in cell signalling. Dysfunction of PI-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 (Marat and Haucke, EMBO J., 2016).

Research from our laboratory has unraveled key roles for distinct PI species in the regulation of defined steps of clathrin-mediated endocytosis (CME) (Figure 1), during endosomal sorting and recycling, in nutrient signaling at lysosomes, and during cell signaling downstream of receptor activation.

Work in the laboratory thus focuses on three fundamental questions:

1. How do phosphoinositide-metabolizing enzymes and their products define membrane identity during endocytosis and endolysosomal sorting, and how can their mutation lead to the development of diseases, such as peripheral myopathies (Marat and Haucke, EMBO J., 2016; Ketel et al., Nature 2016; Ebner et al., Biochem. Soc. Trans. 2019)?

2. Which molecular mechanisms control the activities of the enzymes involved in phosphoinositide conversion and their downstream effector proteins (Wang et al., Mol. Cell 2018; Lo et al., Dev. Cell 2017)?

3. How do PI-metabolizing enzymes control organelle position and function, e.g. during regulation of nutrient signalling (Marat et al., Science 2017; Wallroth et al., Nature Cell Biol., 2019)? We combine quantitative live and super-resolution imaging, electron microscopy and tomography with genome engineering (e.g. via CRISPR), structural biochemical and proteomic approaches to address these questions, often in collaboration with theoreticians.

PI conversion systems during clathrin-mediated endocytosis and endosomal sorting

Clathrin-coated pit nucleation is initiated by the local synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (see Further Contributions). We found that progression of clathrin-medated endocytosis is associated with a partial conversion of membrane lipid identity from PI(4,5)P2 to phoshatidylinositol 3,4-bisphosphate (PI(3,4)P2) and, finally to PI(3)P, the predominant PI at endosomes (Wang et al., Curr. Opin. Cell Biol. 2019). Synthesis of  PI(3,4)P2 by class II PI 3-kinase type C2α at late stages of endocytosis (Figure 2)  (Posor et al., Nature 2013; Schöneberg et al., Nat. Commun. 2017; Wang et al., J. Biol. Chem 2019) serves as  signal for the conformational activation and assembly of the BAR domain protein SNX9 to direct membrane constriction prior to dynamin-mediated fission (Daumke et al., Cell 2014; Lo et al., Dev. Cell 2017). A similar mechanism controls  activation of the BAR domain protein syndapin (using protein X-ray crystallography; Rao et al., Proc. Natl. Acad. Sci. USA 2010) during bulk endocytosis in neurons and other cell types.

Further on-going studies aim at defining the role of early-acting BAR-domain proteins and their partners in endocytosis. For instance, we found that FCHo2 assembles at the rim of early clathrin-coated pits to control their growth and lifetime by coupling the invagination of early endocytic intermediates to clathrin lattice assembly (Lehmann et al., Science Advances 2019).

PI conversion not only directs the endocytic internalization of membrane vesicles but is also a key controller of the surface delivery of molecules. We have discovered that recycling 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. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion (Figure 3). 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 3) and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans (Ketel et al., Nature 2016).

At present we aim to develop tools that can rescue this disease, e.g. by rebalancing phosphoinositide levels or integrin exo- and endocytic cycling.

Molecular mechanisms underlying the modulation of activity of PI-metabolizing enzymes

Using HDX-MS we have demonstrated that the PX-C2 module in PI(3)K C2α can fold back onto the kinase domain, and thereby inhibit its catalytic activity. Consistently, destabilization of this inhibitory conformation leads to an accumulation of PI(3,4)P2, increased recruitment of SNX9 to endocytic sites and facilitates endocytosis in fibroblasts. Binding of the PX-C2 module to PI(4,5)P2-enriched membranes relieves the kinase from autoinhibition and renders the enzyme fully active (Wang et al., Mol. Cell 2018) (Figure 4).

Current projects focus on structural studies of class II PI 3-kinases (Lo et al., Nat. Struct. Mol Biol. 2022) and their binding partners and on the development of small-molecule inhibitors that target these enzymes.

Control of lysosome function and nutrient signaling by PI-metabolizing enzymes

A number of phosphoinositide-metabolizing enzymes reside on lysosomes, where they mediate formation of diverse products (in particular PI(3)P, PI(4)P, PI(3,4)P2, PI(3,5)P2 and PI(4,5)P2). Of note, mutations of these enzymes cause lysosomal dysfunction and are associated with numerous diseases, ranging from neurodegeneration to cancer (Ebner et al., Biochem. Soc. Trans. 2019). However, little is known about potential effector proteins within the lysosomal membrane or at the lysosomal surface. Also, it is largely unclear how exactly these effectors regulate important aspects of lysosomal life cycle and function.
Work in our laboratory has recently elucidated a fundamental role of PI(3)K C2β in nutrient signaling (Marat et al., Science 2017; Wallroth et al., Nature Cell Biol., 2019) (Figure 5). Under growth factor deprivation the kinase is recruited to the surface of late endosomes/lysosomes by Rab7, where it synthesizes a pool of PI(3,4)P2 that negatively regulates mTorc1 by promoting the association of Raptor with inhibitory 14-3-3 proteins. Consistently, loss of PI(3)K C2β leads to hyperactivation of mTorc1. In presence of growth factor signaling the enzyme is kept in an inactive state by phosphorylation through protein kinase N, which mediates its association with 14-3-3 proteins and thereby sequesters PI(3)K C2β in the cytoplasm (Figure 5).