- Award ID(s):
- 1749919
- NSF-PAR ID:
- 10155323
- Date Published:
- Journal Name:
- The Journal of Cell Biology
- Volume:
- 219
- Issue:
- 3
- ISSN:
- 0021-9525
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans -cyclooctene–containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD’s lipid substrate, phosphatidylcholine. The resultant trans -cyclooctene–containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels–Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.more » « less
-
Abstract Intracellular organelles are membrane‐bound and biochemically distinct compartments constructed to serve specialized functions in eukaryotic cells. Through extensive interactions, they form networks to coordinate and integrate their specialized functions for cell physiology. A fundamental property of these organelle networks is that they constantly undergo dynamic organization via membrane fusion and fission to remodel their internal connections and to mediate direct material exchange between compartments. The dynamic organization not only enables them to serve critical physiological functions adaptively but also differentiates them from many other biological networks such as gene regulatory networks and cell signaling networks. This review examines this fundamental property of the organelle networks from a systems point of view. The focus is exclusively on homotypic networks formed by mitochondria, lysosomes, endosomes, and the endoplasmic reticulum, respectively. First, key mechanisms that drive the dynamic organization of these networks are summarized. Then, several distinct organizational properties of these networks are highlighted. Next, spatial properties of the dynamic organization of these networks are emphasized, and their functional implications are examined. Finally, some representative molecular machineries that mediate the dynamic organization of these networks are surveyed. Overall, the dynamic organization of intracellular organelle networks is emerging as a fundamental and unifying paradigm in the internal organization of eukaryotic cells.
This article is categorized under:
Metabolic Diseases > Molecular and Cellular Physiology
-
Summary Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G‐protein complex. We have recently established that in Arabidopsis, the regulator of G‐protein signaling (
RGS 1) protein and a lipid‐hydrolyzing enzyme, phospholipase Dα1 (PLD α1), both act asGTP ase‐activity accelerating proteins (GAP s) for the Gα protein to attenuate its activity.RGS 1 andPLD α1 interact with each other, andRGS 1 inhibits the activity ofPLD α1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA ), a second messenger typically derived from the lipid‐hydrolyzing activity ofPLD α1, is a molecular target ofRGS 1.PA binds and inhibits theGAP activity ofRGS 1. A conserved lysine residue inRGS 1 (Lys259) is directly involved inRGS 1–PA binding. Introduction of thisRGS 1 protein variant in thergs1 mutant background makes plants hypersensitive to a subset of abscisic acid‐mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal–response output. -
Santos, AL (Ed.)Peroxisomes are key regulators of cellular and metabolic homeostasis. These organelles play important roles in redox metabolism, the oxidation of very-long-chain fatty acids (VLCFAs), and the biosynthesis of ether phospholipids. Given the essential role of peroxisomes in cellular homeostasis, peroxisomal dysfunction has been linked to various pathological conditions, tissue functional decline, and aging. In the past few decades, a variety of cellular signaling and metabolic changes have been reported to be associated with defective peroxisomes, suggesting that many cellular processes and functions depend on peroxisomes. Peroxisomes communicate with other subcellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum (ER), and lysosomes. These inter-organelle communications are highly linked to the key mechanisms by which cells surveil defective peroxisomes and mount adaptive responses to protect them from damages. In this review, we highlight the major cellular changes that accompany peroxisomal dysfunction and peroxisomal inter-organelle communication through membrane contact sites, metabolic signaling, and retrograde signaling. We also discuss the age-related decline of peroxisomal protein import and its role in animal aging and age-related diseases. Unlike other organelle stress response pathways, such as the unfolded protein response (UPR) in the ER and mitochondria, the cellular signaling pathways that mediate stress responses to malfunctioning peroxisomes have not been systematically studied and investigated. Here, we coin these signaling pathways as “peroxisomal stress response pathways”. Understanding peroxisomal stress response pathways and how peroxisomes communicate with other organelles are important and emerging areas of peroxisome research.more » « less
-
Abstract Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein‐protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light‐activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light‐tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC.
This article was corrected on 25 July 2022. See the end of the full text for details.
Basic Protocol 1 : Genetic engineering strategy for the generation of modular light‐activated protein dimerization unitsSupport Protocol 1 : Molecular cloningBasic Protocol 2 : Cell culture and transfectionSupport Protocol 2 : Production of dark containers for optogenetic samplesBasic Protocol 3 : Confocal microscopy and light‐dependent activation of the dimerization systemAlternate Protocol 1 : Protein recruitment to intracellular compartmentsAlternate Protocol 2 : Induction of organelles’ membrane tetheringAlternate Protocol 3 : Optogenetic reconstitution of protein functionBasic Protocol 4 : Image analysisSupport Protocol 3 : Analysis of apparent on‐ and off‐kineticsSupport Protocol 4 : Analysis of changes in organelle overlap over time