Photoactivatable and Cell‐Membrane‐Permeable Phosphatidylinositol 3,4,5‐Trisphosphate

Mentel, Matthias; Laketa, Vibor; Subramanian, Devaraj; Gillandt, Hartmut; Schultz, Carsten

doi:10.1002/anie.201007796

Cited by 70 publications

(53 citation statements)

References 24 publications

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“…We first aimed to experimentally increase the intracellular PI3P concentration in cultured neurons by applying a membranepermeable PI3P-acetoxymethyl (AM) ester derivative, its photoactivatable "caged" coumarin-PI3P-AM variant, and, as a negative control, its regioisomer PI4P-AM, all of which had previously been validated in HeLa cells (21) and were recently used for studying vesicular trafficking in muscle cells of X-linked centronuclear myopathy patients (23). Previous studies indicate a distribution of both coumarin-caged and -uncaged PIP-AM probes into most cellular membranes (21,24). However, despite their wide subcellular distribution, these probes demonstrated high structural specificity, presumably through their specific interaction with endogenous PIP-binding proteins.…”

Section: Pi3p Promotes Postsynaptic Gfp Gephyring Clustering-tomentioning

confidence: 99%

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses

Papadopoulos

Rhee

Subramanian

et al. 2017

Journal of Biological Chemistry

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Edited by Thomas SöllnerThe formation of neuronal synapses and the dynamic regulation of their efficacy depend on the proper assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic postsynapses requires the scaffold protein gephyrin and the guanine nucleotide exchange factor collybistin (Cb). In vitro, the pleckstrin homology domain of Cb binds phosphoinositides, specifically phosphatidylinositol 3-phosphate (PI3P). However, whether PI3P is required for inhibitory postsynapse formation is currently unknown. Here, we investigated the role of PI3P at developing GABAergic postsynapses by using a membrane-permeant PI3P derivative, timelapse confocal imaging, electrophysiology, as well as knockdown and overexpression of PI3P-metabolizing enzymes. Our results provide the first in cellula evidence that PI3P located at early/ sorting endosomes regulates the postsynaptic clustering of gephyrin and GABA A receptors and the strength of inhibitory, but not excitatory, postsynapses in cultured hippocampal neurons. In human embryonic kidney 293 cells, stimulation of gephyrin cluster formation by PI3P depends on Cb. We therefore conclude that the endosomal pool of PI3P, generated by the class III phosphatidylinositol 3-kinase, is important for the Cbmediated recruitment of gephyrin and GABA A receptors to developing inhibitory postsynapses and thus the formation of postsynaptic membrane specializations.Phosphoinositides, phosphorylated derivatives of phosphatidylinositol, are critical regulators of intracellular signaling, membrane traffic, and cell compartmentalization (1-3). The functional roles of phosphoinositide metabolism have been studied in great detail at the presynaptic terminal, where phosphoinositide turnover is of critical importance for synaptic vesicle (SV) 3 recycling and synapse function (4). Little, however, is known about specific roles of phosphoinositides at postsynapses.Core components of most inhibitory GABAergic postsynapses are GABA A receptors (GABA A Rs), the cell adhesion protein neuroligin 2 (NL2), the scaffolding protein gephyrin, and the guanine nucleotide exchange factor collybistin (Cb) (5, 6). During the formation of many GABAergic synapses, most notably those at neuronal somata, NL2 is thought to activate Cb, which promotes Cb membrane association and the subsequent recruitment of gephyrin and GABA A Rs (7-9). In vitro binding studies indicate that the pleckstrin homology (PH) domain of Cb specifically binds phosphatidylinositol 3-phosphate (PI3P) (9 -13), and this interaction is thought to be essential for the anchoring of NL2-gephyrin-Cb complexes at the postsynaptic plasma membrane of synapses that depend on Cb (7, 9). The notion that PI3P binding by the PH domain is required for proper Cb function is supported by the observation that deletion of the PH domain (14), or the substitution therein of two arginine residues that are essential for PI3P binding (9, 11), causes a marked reduction in the density of postsynaptic gephyrin cluste...

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Section: Pi3p Promotes Postsynaptic Gfp Gephyring Clustering-tomentioning

confidence: 99%

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses

Papadopoulos

Rhee

Subramanian

et al. 2017

Journal of Biological Chemistry

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“…For example, the addition of aminophospholipid increases endocytosis in living cells (Farge et al 1999) and adding the biologically active isoform, but not other isoforms, of LBPA reduces cholesterol overload in NPC cells (Chevallier et al 2008). Finally, a promising approach has been elaborated by Carsten Schultz and colleagues that developed very elegant strategies to introduce membrane-permeant versions of phosphoinositides into cells so that the lipid is released upon cleavage by cytosolic esterases (Laketa et al 2009;Mentel et al 2011). Such strategies, together with the development of novel chemical biology tools, offer promising new ways to study the function of membrane lipids (Riezman and Johnsson 2011;Wymann and Schultz 2012).…”

Section: Concluding Remarks and Future Challengesmentioning

confidence: 99%

Lipid Sorting and Multivesicular Endosome Biogenesis

Bissig

Grüenberg

2013

Cold Spring Harbor Perspectives in Biology

133

107

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Intracellular organelles, including endosomes, show differences not only in protein but also in lipid composition. It is becoming clear from the work of many laboratories that the mechanisms necessary to achieve such lipid segregation can operate at very different levels, including the membrane biophysical properties, the interactions with other lipids and proteins, and the turnover rates or distribution of metabolic enzymes. In turn, lipids can directly influence the organelle membrane properties by changing biophysical parameters and by recruiting partner effector proteins involved in protein sorting and membrane dynamics. In this review, we will discuss how lipids are sorted in endosomal membranes and how they impact on endosome functions.

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“…A more general way to rapidly increase lipid concentration is the use of caged lipids. These are equipped with a photocleavable protecting group (caging group), which blocks biological activity and renders them resistant to metabolic turnover before the active lipid is released using a flash of light (2,(5)(6)(7). The sudden increase in target lipid concentration facilitates analysis of downstream lipid signaling events as well as lipid metabolism within living cells in pulse− chase experiments.…”

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confidence: 99%

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

Höglinger

Nadler

Haberkant

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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110

105

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Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo-cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.

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Photoactivatable and Cell‐Membrane‐Permeable Phosphatidylinositol 3,4,5‐Trisphosphate

Cited by 70 publications

References 24 publications

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses

Endosomal Phosphatidylinositol 3-Phosphate Promotes Gephyrin Clustering and GABAergic Neurotransmission at Inhibitory Postsynapses

Lipid Sorting and Multivesicular Endosome Biogenesis

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

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