Fast and precise protein tracking using repeated reversible photoactivation.

Dmitriy M Chudakov; Tatyana V Chepurnykh; Vsevolod V Belousov; Sergey Lukyanov; Konstantin A Lukyanov

doi:10.1111/j.1600-0854.2006.00468.x

Home/Archive/PubMed

PubMed · 16889652

Fast and precise protein tracking using repeated reversible photoactivation.

Dmitriy M Chudakov·Tatyana V Chepurnykh·Vsevolod V Belousov·Sergey Lukyanov·Konstantin A Lukyanov

View original source ↗Export metadata ↓

Abstract

Photoactivatable fluorescent proteins opened principally novel possibilities to study proteins' movement pathways. In particular, reversibly photoactivatable proteins enable multiple tracking experiments in a long-drawn work with a single cell. Here we report 'protein rivers tracking' technique based on repeated identical rounds of photoactivation and subsequent images averaging, which results in dramatic increase of imaging resolution for fast protein movement events.

Explore related subjects

BH3 Interacting Domain Death Agonist Protein Fluorescent Dyes Green Fluorescent Proteins HeLa Cells Humans Lasers Light Luminescent Proteins Microscopy, Fluorescence Recombinant Fusion Proteins Staining and Labeling Journal Article Research Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Dmitriy M Chudakov, Tatyana V Chepurnykh, Vsevolod V Belousov, Sergey Lukyanov, Konstantin A Lukyanov. 2006-08-02. Fast and precise protein tracking using repeated reversible photoactivation.. https://doi.org/10.1111/j.1600-0854.2006.00468.x

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Expressional analysis of anti-apoptotic phospho-BAD protein and mutational analysis of pro-apoptotic BAD gene in hepatocellular carcinomas.

BACKGROUND: It has become clear that, together with proliferation, deregulation of apoptosis plays a pivotal role in tumourigenesis. BAD is a pro-apoptotic Bcl-2 family protein and regulates the intrinsic apoptosis pathway. Phosphorylation of BAD inhibits the apoptosis function of BAD. AIMS: To investigate whether alteration of the phospho-BAD protein and somatic mutation of BAD gene are characteristics of human hepatocellular carcinoma. PATIENTS AND METHODS: We analysed the expression of phospho-BAD in 20 hepatocellular carcinomas by immunohistochemistry. Also, we analysed the BAD gene for the detection of somatic mutations by a single-strand conformation polymorphism assay in 69 hepatocellular carcinomas. RESULTS: Phospho-BAD expression in the non-tumour hepatocytes was seen in all of the hepatocellular carcinomas, while the expression in the cancer cells was observed in 15% (3 of the 20) of the hepatocellular carcinomas. There was no somatic mutation of BAD Bcl-2 homology 3 (BH3) domain in the 69 hepatocellular carcinomas. CONCLUSIONS: The data showed that loss of phospho-BAD expression, but not BAD gene mutation, is a feature of hepatocellular carcinomas. The decreased expression of phospho-BAD in the hepatocellular carcinoma cells compared to the non-tumour hepatocytes suggests that loss of phospho-BAD expression may play a role in hepatocellular tumourigenesis.

BH3 Interacting Domain Death Agonist Protein↗

PubMed2005

The interaction between tBid and cardiolipin or monolysocardiolipin.

Bid, a BH3-only pro-apoptotic member of the Bcl-2 family, is cleaved by caspase 8 in apoptosis induced by death domain receptors. The carboxyl terminus of the cleavage product, tBid, remains associated with the amino terminal fragment (nBid) after cleavage. Dissociation of tBid from nBid occurs during targeting of tBid to mitochondria. We use an in vitro system and demonstrate that cardiolipin is sufficient for the dissociation. Monolysocardiolipin, a metabolite of cardiolipin that increases in mitochondria during apoptosis, has the same affinity to tBid as cardiolipin and is also capable of inducing dissociation of tBid from nBid. In contrast, phosphatidylethanolamine could not induce dissociation of tBid from nBid. To determine the site of tBid that interacts with cardiolipin, we performed mutational analysis by eliminating the positive-charged residues in helices 4-6. None of the single mutations can abolish the ability of tBid to target to mitochondria and to induce cytochrome c release, suggesting that positive-charged residues in helices 4-6 may not be required for mitochondrial targeting of tBid.

BH3 Interacting Domain Death Agonist Protein↗

PubMed2005

tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1.

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of beta-oxidation flux by labeled palmitate demonstrates that tBid inhibits beta-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on beta-oxidation. The unexpected role of tBid in the regulation of lipid beta-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

BH3 Interacting Domain Death Agonist Protein↗