Rainer Müller

A multifunctional phosphatidylethanolamine (PE) derivative to follow lipid transport and to determine the PE interactome in intact cells.

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3‐phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2‐mediated hyperactivation of Cdk5 downstream of receptor‐ and activity‐dependent calcium influx. Our results unravel an unexpected function for PI(3)P‐containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P‐producing VPS34 kinase in neurological disease and neurodegeneration.

Structural changes in HIV maturation: Nascent HIV particles assemble at the plasma membrane of an infected cell and bud into a membrane-enveloped, immature virion. Assembly and budding are driven by a polyprotein called Gag, which consists of a matrix domain (MA) that is recruited to the plasma membrane, a capsid domain (CA) responsible for self-assembly, and a nucleocapsid domain (NC) that recruits the viral RNA genome. Gag cleavage results in a structural rearrangement that produces the mature virion. Qu et al . imaged mature and immature HIV particles by electron tomography and focused in on the MA domain (see the Perspective by Hikichi and Freed). They found that MA rearranges between two distinct hexameric lattices, and mature MA modulates the viral membrane by binding to a membrane lipid. This finding suggests that MA may play functional roles in the mature virion. —VV

Phosphatidylinositol (PI) is the biosynthetic precursor for seven phosphoinositides, important signaling lipids in cells. A membrane‐permeant caged PI derivative featuring a photo‐removable coumarinyl group masking the negative charge of the phosphate, as well as two enzymatically removable butyrate esters for increased lipophilicity and for preventing phosphate migration, were synthesized. Rapid cell entry and cellular labeling in fixed cells was demonstrated by a photo‐cross‐linkable diazirine followed by attachment of a fluorophore through click chemistry. Using this technique, we found that the multifunctional caged PI derivative resided predominantly at internal membranes but rapidly changed to the plasma membrane after uncaging. Accordingly, a preliminary proteomic analysis of the lipid–protein conjugates revealed that the two major PI transport proteins PITPα and β were prime targets of the photo‐cross‐linked PI derivative.

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] is essential for exocytosis. Classical ways of manipulating PI(4,5)P₂ levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P₂ from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P₂, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P₂ levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P₂ uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca²⁺ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P₂ activation of exocytosis did not depend on the PI(4,5)P₂-binding CAPS-proteins, suggesting that PI(4,5)P₂ uncaging may bypass CAPS-function. Finally, PI(4,5)P₂ uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P₂ in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors.

Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂], synthesized by class II PI3K β (PI3KC2β) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2β hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2β with the Raptor subunit of mTORC1. Local PI(3,4)P₂ synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P₂ production in shutting off mTORC1.