Lipid Signaling & Membrane Traffic

Regulation of cell physiology, membrane dynamics, and cell signaling by phosphoinositides 

Membrane lipids, in particular phosphoinositides (PIs), are key players in the control of intracellular membrane traffic and in cell signaling. 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 (e.g. epilepsy) and to cancer (Marat and Haucke, EMBO J., 2016; Posor, Jang, Haucke, Nat Rev Mol Cell Biol 2022).

Research from our laboratory has unraveled key roles for distinct PI species in the regulation of endocytosis, during endosomal sorting and recycling, in lysosome function, and during lysosomal nutrient signaling.

Work in the laboratory thus focuses on three fundamental questions:

1. How do phosphoinositide-metabolizing enzymes and their products define membrane identity and flux during endocytosis and within the endolysosomal system, 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; Jang et al., Science 2022)?

2. Which molecular mechanisms control the activities of the enzymes involved in phosphoinositide conversion and their downstream effector proteins (Lo et al., Nat Struct Mol Biol 2022; Wallroth et al., Nat Cell Biol 2019) and can these enzymes be targeted by small molecules to develop novel therapies for human disease (Lo et al., Nat Chem Biol 2023)?

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

PI conversion in the endocytic pathway and at ER-endosome membrane contacts

Endocytic pit formation is initiated by the local synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) (see Further Contributions). We found that progression of clathrin-mediated 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 1)  (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 studies have defined 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 (Figure 2).

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 2). Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane 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 (Samso, Koch et al., Proc Natl. Acad. Sci USA 2022).

In a more recent study, we uncovered an important function of MTM1 in the nutrient-controlled rewiring of organelle dynamics. Specifically, we found that endosomal signaling lipid turnover via MTM1 controls mitochondrial morphology and function by reshaping the endoplasmic reticulum (ER). Starvation-induced endosomal recruitment of MTM1 impairs PI(3)P-dependent contact formation between tubular ER membranes and early endosomes resulting in the conversion of ER tubules into sheets and the inhibition of mitochondrial fission to sustain oxidative metabolism (Figure 3). These results unravel an important role for early endosomal lipid signaling in controlling ER shape and, thereby, mitochondrial form and function to enable cells to adapt to altering nutrient environments (Jang et al., Science, 2022; Jang & Haucke, Trends Cell Biol, 2024).

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

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis (Posor et al. Nature 2013), angiogenesis, viral replication, platelet formation, and roles in cell division (Gulluni et al. Science 2022). Using a combination of protein X-ray crystallography, single particle cryo electron micrsocopy and cross-linking mass spectrometry we determined structures of PI3KC2α in its active and inactive conformations. We uncovered a coincident mechanism of lipid-induced activation of PI3KC2α at membranes that involves large-scale repositioning of its Ras-binding and lipid-binding distal PX and C-C2 domains and can serve as a paradigm for entire the class II PI3K family. Moreover, we describe a PI3KC2α-specific helical bundle domain that underlies its scaffolding function at the mitotic spindle (Lo et al., Nat Struct Mol Biol. 2022).

            The determination of the 3-dimensional structure of PI3KC2α has also enabled us to identify PITCOINs as potent and highly selective small molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity towards PI3KC2α due to their unique mode of interaction with the ATP binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α -mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodelling during platelet-dependent thrombus formation (Lo et al., Nat Chem Biol. 2023). To our knowledge, PITCOINs are potent and selective cell permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer. We are currently expanding these approaches to other members of the class II PI3K subfamily for the treatment of human diseases such as acute ischemic stroke.

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; Posor, Jang, Haucke, Nat Rev Mol Cell Biol 2022). 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 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)KC2β 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 4).

In most recent work, we identified a nutrient-regulated switch that controls lysosome function by local signaling lipid conversion. We discovered that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling lipid switch that triggers the conversion between peripheral motile mTOR complex 1 (mTORC1) signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center. Starvation-triggered relocalization of PI(4)P metabolizing enzymes reshapes the lysosomal surface proteome to facilitate lysosomal proteolysis and to repress mTORC1-signaling. Concomitantly, lysosomal PI(3)P, which marks motile signaling-active lysosomes in the cell periphery, is erased (Figure 5). Interference with this PI(3)P/ PI(4)P lipid switch module impairs the adaptive response of cells to altering nutrient supply. These data unravel a key function for lysosomal phosphoinositide metabolism in rewiring organellar membrane dynamics in response to cellular nutrient status (Ebner et al., Cell 2023).