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Isozyme-specific interaction of protein kinase C? with mitochondria dissected using live cell fluorescence imaging.

ABSTRACT: PKC? signaling to mitochondria has been implicated in both mitochondrial apoptosis and metabolism. However, the mechanism by which PKC? interacts with mitochondria is not well understood. Using FRET-based imaging, we show that PKC? interacts with mitochondria by a novel and isozyme-specific mechanism distinct from its canonical recruitment to other membranes such as the plasma membrane or Golgi. Specifically, we show that PKC? interacts with mitochondria following stimulation with phorbol esters or, in L6 myocytes, with insulin via a mechanism that requires two steps. In the first step, PKC? translocates acutely to mitochondria by a mechanism that requires its C1A and C1B domains and a Leu-Asn sequence in its turn motif. In the second step, PKC? is retained at mitochondria by a mechanism that depends on its C2 domain, a unique Glu residue in its activation loop, intrinsic catalytic activity, and the mitochondrial membrane potential. In contrast, of these determinants, only the C1B domain is required for the phorbol ester-stimulated translocation of PKC? to other membranes. PKC? also basally localizes to mitochondria and increases mitochondrial respiration via many of the same determinants that promote its agonist-evoked interaction. PKC? localized to mitochondria has robust activity, as revealed by a FRET reporter of PKC?-specific activity (?CKAR). These data support a model in which multiple determinants unique to PKC? drive a specific interaction with mitochondria that promotes mitochondrial respiration.

SUBMITTER: Wu-Zhang AX

PROVIDER: S-EPMC3488061 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Isozyme-specific interaction of protein kinase Cδ with mitochondria dissected using live cell fluorescence imaging.

Wu-Zhang Alyssa X AX Murphy Anne N AN Bachman Mackenzie M Newton Alexandra C AC

The Journal of biological chemistry 20120917 45

PKCδ signaling to mitochondria has been implicated in both mitochondrial apoptosis and metabolism. However, the mechanism by which PKCδ interacts with mitochondria is not well understood. Using FRET-based imaging, we show that PKCδ interacts with mitochondria by a novel and isozyme-specific mechanism distinct from its canonical recruitment to other membranes such as the plasma membrane or Golgi. Specifically, we show that PKCδ interacts with mitochondria following stimulation with phorbol esters ...[more]

PMID: 22988234

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Project description:Thermally activated delayed fluorescence (TADF) molecules offer nowadays a powerful tool in the development of novel organic light emitting diodes due to their capability of harvesting energy from non-emissive triplet states without using heavy-metal complexes. TADF emitters have very small energy difference between the singlet and triplet excited states, which makes thermally activated reverse intersystem crossing from the triplet states back to the singlet manifold viable. This mechanism generates a long-lived delayed fluorescence component which can be explored in the sensing of oxygen concentration, local temperature, or used in time-gated optical cell-imaging, to suppress interference from autofluorescence and scattering. Despite this strong potential, until recently the application of TADF outside lighting devices has been hindered due to the low biocompatibility, low aqueous solubility and poor performance in polar media shown by the vast majority of TADF emitters. To achieve TADF luminescence in biological media, careful selection or design of emitters is required. Unfortunately, most TADF molecules are not emissive in polar media, thus complexation with biomolecules or the formation of emissive aggregate states is required, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein, we demonstrate a facile method with great generalization potential that maintains the photophysical properties of solvated dyes by combining luminescent molecules with polymeric nanoparticles. Using an established swelling procedure, two known TADF emitters are loaded onto polystyrene nanoparticles to prepare TADF emitting nanomaterials able to be used in live-cell imaging. The obtained particles were characterized by optical spectroscopy and exhibited the desired TADF emission in aqueous media, due to the polymeric matrix shielding the dye from solvent polarity effects. The prepared nanoparticles were incubated with live human cancer cells and showed very low cytotoxicity and good cellular uptake, thus making fluorescence microscopy imaging possible at low dye concentrations.

Dataset Information

Isozyme-specific interaction of protein kinase C? with mitochondria dissected using live cell fluorescence imaging.

Publications

Isozyme-specific interaction of protein kinase Cδ with mitochondria dissected using live cell fluorescence imaging.

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