Study of molecular events in cells by fluorescence correlation spectroscopy

Vukojević, Vladana; Pramanik, Aladdin; Yakovleva, Tatiana; Rigler, Rudolf; Terenius, Lars; Bakalkin, Georgy

doi:10.1007/s00018-004-4305-7

Cited by 85 publications

(72 citation statements)

References 123 publications

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“…Next, we analyzed how the M1 mutation affects the association of eIF5 with the translational machinery by using FCS. This method measures fluctuation of fluorescent molecules in a subfemtoliter confocal volume at very high time resolution (25). By calculating the autocorrelation function of such fluctuating fluorescence signals, complex formation with relatively immobile molecules can be detected in living cells.…”

Section: Resultsmentioning

confidence: 99%

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Homma

Wada

Suzuki

et al. 2005

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

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Casein kinase 2 (CK2) is a ubiquitous eukaryotic Ser͞Thr protein kinase that plays an important role in cell cycle progression. Although its function in this process remains unclear, it is known to be required for the G 1 and G2͞M phase transitions in yeast. Here, we show that CK2 activity changes notably during cell cycle progression and is increased within 3 h of serum stimulation of quiescent cells. During the time period in which it exhibits high enzymatic activity, CK2 associates with and phosphorylates a key molecule for translation initiation, eukaryotic translation initiation factor (eIF) 5. Using MS, we show that Ser-389 and -390 of eIF5 are major sites of phosphorylation by CK2. This is confirmed using eIF5 mutants that lack CK2 sites; the phosphorylation levels of mutant eIF5 proteins are significantly reduced, relative to WT eIF5, both in vitro and in vivo. Expression of these mutants reveals that they have a dominant-negative effect on phosphorylation of endogenous eIF5, and that they perturb synchronous progression of cells through S to M phase, resulting in a significant reduction in growth rate. Furthermore, the formation of mature eIF5͞eIF2͞eIF3 complex is reduced in these cells, and, in fact, restricted diffusional motion of WT eIF5 was almost abolished in a GFP-tagged eIF5 mutant lacking CK2 phosphorylation sites, as measured by fluorescence correlation spectroscopy. These results suggest that CK2 may be involved in the regulation of cell cycle progression by associating with and phosphorylating a key molecule for translation initiation. C asein kinase 2 (CK2) (1-4) is composed of two subunits, ␣ or ␣Ј and ␤, which combine to form a native ␣ 2 ␤ 2 tetramer. Disruption of the catalytic subunits (␣ and ␣Ј) is lethal in Saccharomyces cerevisiae (5) and disruption of the regulatory ␤ subunit in mice leads to early embryonic lethality (6). CK2 phosphorylates a range of cellular targets in a variety of subcellular sites and appears to be highly pleiotropic; it is involved in many key biological functions, including growth and cell cycle control (7), signal transduction (3), circadian rhythms (8, 9), and gene expression (10, 11). CK2 is also a stress-activated kinase and might participate in the transduction of survival signals to avoid damage by mutagenic UV radiation (12, 13). An important role for CK2 in promoting cell proliferation and transformation has been indicated by several studies. In mammalian systems, its targeted overexpression in mice results in the development of T cell lymphoma and mammary tumorigenesis (5). Despite these findings, there is still much uncertainty regarding the activation of CK2 in response to stimuli (14). The mechanism by which it is regulated and its precise function in cell cycle progression and proliferation is still poorly understood.CK2 activity and stability are believed to be regulated in part by holoenzyme formation via a self-assembly mechanism and by phosphorylation. Phosphorylation by p34 cdc2 of the catalytic ␣ subunit at the C-terminal domain occurs in ...

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Section: Resultsmentioning

confidence: 99%

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Homma

Wada

Suzuki

et al. 2005

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

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“…FCS Study-FCS, a confocal technique for examining molecular events in real time and with single molecule detection sensitivity (40), was applied to investigate peptide translocation into live cells, including dependence of translocation on the peptide concentrations and kinetics of translocation. Interactions of Rh-Dyn A with live cells were compared with those of Rh-Dyn B, Rh-TP10, a prototypical CPP, and the fluorescent label alone.…”

Section: Streptavidin and Immunolabeling Of Dynorphinsmentioning

confidence: 99%

“…Evaluations obtained by fitting the experimental autocorrelation curves in the cytoplasm with a threecomponent model (40,42) suggest that about 50% of Rh-Dyn A formed complexes with intracellular components characterized by diffusion time diff Ϸ 1 s; about 15% of Rh-Dyn A was associated with smaller complexes characterized by diff Ϸ 6 ms, whereas about 25% of Rh-Dyn A was detected in the free form ( diff ϭ 0.13 ms). The determination of diffusion times was complicated because of the large size of the complexes; the values given should be considered as a first approximation.…”

Section: Streptavidin and Immunolabeling Of Dynorphinsmentioning

confidence: 99%

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

Marinova

Vukojević

Surcheva

et al. 2005

Journal of Biological Chemistry

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Several peptides, including penetratin and Tat, are known to translocate across the plasma membrane. Dynorphin opioid peptides are similar to cell-penetrating peptides in a high content of basic and hydrophobic amino acid residues. We demonstrate that dynorphin A and big dynorphin, consisting of dynorphins A and B, can penetrate into neurons and non-neuronal cells using confocal fluorescence microscopy/immunolabeling. The peptide distribution was characterized by cytoplasmic labeling with minimal signal in the cell nucleus and on the plasma membrane. Translocated peptides were associated with the endoplasmic reticulum but not with the Golgi apparatus or clathrin-coated endocytotic vesicles. Rapid entry of dynorphin A into the cytoplasm of live cells was revealed by fluorescence correlation spectroscopy. The translocation potential of dynorphin A was comparable with that of transportan-10, a prototypical cell-penetrating peptide. A central big dynorphin fragment, which retains all basic amino acids, and dynorphin B did not enter the cells. The latter two peptides interacted with negatively charged phospholipid vesicles similarly to big dynorphin and dynorphin A, suggesting that interactions of these peptides with phospholipids in the plasma membrane are not impaired. Translocation was not mediated via opioid receptors. The potential of dynorphins to penetrate into cells correlates with their ability to induce non-opioid effects in animals. Translocation across the plasma membrane may represent a previously unknown mechanism by which dynorphins can signal information to the cell interior.

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“…The stochastic character of the observed intensity fluctuations in FCS, however, requires statistic analysis of a multitude of fluctuations, while in principle a single FRAP observation of entire molecule populations is sufficient to determine transport or reaction constants (Petersen & Elson 1986). In FCS, the statistical analysis of autocorrelation of the random fluctuations will yield the diffusion coefficient and the concentration of independently diffusing molecules and their association status (Elson & Qian 1989;Vukojevic et al 2005). It can also be used to separate diffusion processes that occur on different timescales, such as the motion of small and large fluorescent molecules in a mixture, or free and bound states of the same molecule.…”

Section: Introductionmentioning

confidence: 99%

Dynamics of the CapG actin-binding protein in the cell nucleus studied by FRAP and FCS

Renz

Langowski

2008

Chromosome Res

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FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) are spectroscopic methods for monitoring the dynamic distribution of proteins inside the nucleus of living cells. As an example we report our studies on the intracellular mobility of the actin-binding protein CapG in live breast cancer cells. This Gelsolin-related protein is a putative oncogene. It appears to be overexpressed especially in metastasizing breast cancer. Furthermore, the CapG protein is known to be involved in the motility control of non-muscle benign cells. Its increased expression triggers an increase in cell motility of benign cells. Thus it can be expected that in cancer cells overexpressing the CapG protein, motility, invasiveness and metastasis might be particularly promoted. Since the nuclear CapG fraction seems to be pivotal to the increase in cell motility, we focused our studies on the CapG mobility in cell nuclei of live breast cancer cells. Using FCS and FRAP we showed that the eGFP-tagged CapG is monomeric and characterized its diffusional properties on the microsecond to minute timescale. This information about the mobility and compartmentalization of CapG might help to provide insight into its function within the cell nucleus and give clues about its altered cellular function in malignant dedifferentiation. Abbreviations ACF autocorrelation function CapGGelsolin-related actin binding capping protein CapG-eGFP CapG-eGFP fusion protein eGFP enhanced green fluorescent protein FCS fluorescence correlation spectroscopy FRAP fluorescence recovery after photobleaching NLS nuclear localization signal PI 3-kinase phosphatidylinositol 3-hydroxy kinase PIP 2 -binding phospatidylinositol 4,5-bisphosphate

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Study of molecular events in cells by fluorescence correlation spectroscopy

Cited by 85 publications

References 123 publications

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression

Translocation of Dynorphin Neuropeptides across the Plasma Membrane

Dynamics of the CapG actin-binding protein in the cell nucleus studied by FRAP and FCS

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