Dynamics of yeast prion aggregates in single living cells

Kawai-Noma, Shigeko; Ayano, Satoru; Pack, Chan Gi; Kinjo, Masataka; Yoshida, Masasuke; Yasuda, Kenji; Taguchi, Hideki

doi:10.1111/j.1365-2443.2006.01004.x

Cited by 46 publications

(109 citation statements)

References 48 publications

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“…Fig. 1) and the apparent viscosity of a yeast cell 51 . This fast component with a large D value was barely detected for FCFs of cross-correlation functions, which exclude independent photochemical effects (such as blinking) from the two fluorescent molecules ( Supplementary Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The cells were mounted on a glass-bottom dish (MatTek Corporation). FCS and FCCS measurements were all performed at 25°C with a ConfoCor2 (Carl Zeiss) microscope, as described 31,[51][52][53] . The ConfoCor2 consisted of a CW Ar þ and He-Ne lasers, a water-immersion objective (C-Apochromat, 40 Â , 1.2 numerical aperture; Carl Zeiss), and two channels of avalanche photodiodes (SPCM-200-PQ; EG&G).…”

Section: Methodsmentioning

confidence: 99%

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Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

et al. 2014

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The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-a mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome

et al. 2014

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“…To overcome the limitation of HILO in the observation of the smaller aggregates, we utilized FCS. FCS is a powerful method to measure the size of molecules at single-molecule sensitivity in solution or in living cells (40,41). The FCS measurement enabled us to investigate the smaller aggregates, which were further disaggregated by Hsp104/Hsp70.…”

Section: Establishment Of Experimental Systems Tomentioning

confidence: 99%

Single-molecule Analyses of the Dynamics of Heat Shock Protein 104 (Hsp104) and Protein Aggregates

Okuda

Niwa

Taguchi

2015

Journal of Biological Chemistry

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Background:The process of disaggregation by heat shock protein 104 (Hsp104) and heat shock protein 70/40 (Hsp70/40) has not been elucidated. Results: We developed several methods to investigate the dynamics of Hsp104 at single-molecule levels. Conclusion: Statistical analyses revealed that Hsp70/40 affected the dynamics of Hsp104. Significance: Single-molecule approaches are a unique way to unravel the functional mechanisms of disaggregases.

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“…101 and 102), direct proof of Hsp104-dependent fragmentation in vivo was gleaned from observations of prion protein dynamics in live cells. When new protein synthesis is inhibited in cells with wildtype Hsp104 activity, existing prion complexes marked with Sup35-GFP become undetectable by microscopy within hours; 97,103 however, upon Hsp104 inhibition, the same complexes persist. 97 Since Hsp104 does not alter the metabolic stability of Sup35, 97 the observed loss of fluorescence in wildtype cells provides an assay for fragmentation.…”

Section: Regeneration Of the Prion Template In Vivomentioning

confidence: 99%