Detection of swelling of single isolated mitochondrion with optical microscopy

Morikawa, Daisuke; Kanematsu, Keita; Shibata, Takahiro; Haseda, Keisuke; Umeda, Norihiro; Ohta, Yoshihiro

doi:10.1364/boe.5.000848

Cited by 17 publications

(9 citation statements)

References 15 publications

(15 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The obtained mitochondria were suspended in suspension buffer [10 mM Tris-HCl, 250 mM sucrose, 0.5 mM EGTA, (pH 7.4)]. Each preparation was assayed for ATP production [24] upon addition of 5 mM Namalate, giving a rate of 329 ± 34 nmol ATP/mg of protein/min (mean ± SE, n = 3), which was comparable to values reported previously [19] and which showed that mitochondria used for the experiments had a high potential for ATP production. Protein content was determined using a BCA protein assay with BSA as a standard.…”

Section: Preparation Of Isolated Mitochondriasupporting

confidence: 81%

“…DIC images and transmitted light images of individual mitochondria and beads were acquired with an inverted microscope (IX70, Olympus, Tokyo, Japan) equipped with a 20 × objective (UPlanSApo, Olympus), a 2 × relay lens (U-TVCAC, Olympus) and a cooled CCD camera (6.45 μm/pixel at 1x) (Sensicam QE, PCO AG; Kelheim, Germany). The wavelength used for the measurements was selected with a 10-nm bandpass filter centered at 546 nm [19], because the effects of redox changes of mitochondrial proteins on the absorption of mitochondrial suspensions are small within this range of the wavelength. To modulate the phase difference between beam a and beam b in Fig.…”

Section: Differential Interference Contrast (Dic) Imagingmentioning

confidence: 99%

See 1 more Smart Citation

Significant correlation between refractive index and activity of mitochondria: single mitochondrion study

Haseda¹,

Kanematsu²,

Noguchi³

et al. 2015

Biomed. Opt. Express

Self Cite

View full text Add to dashboard Cite

Measurements of refractive indices (RIs) of intracellular components can provide useful information on the structure and function of cells. The present study reports, for the first time, determination of the RI of an isolated mitochondrion in isotonic solution using retardation-modulated differential interference contrast microscopy. The value was 1.41 ± 0.01, indicating that mitochondria are densely packed with molecules having high RIs. Further, the RIs of each mitochondrion were significantly correlated with the mitochondrial membrane potential, an index of mitochondrial activity. These results will provide useful information on the structures and functions of cells based on the intracellular distribution of RIs.

show abstract

Section: Preparation Of Isolated Mitochondriasupporting

confidence: 81%

Section: Differential Interference Contrast (Dic) Imagingmentioning

confidence: 99%

Significant correlation between refractive index and activity of mitochondria: single mitochondrion study

Haseda¹,

Kanematsu²,

Noguchi³

et al. 2015

Biomed. Opt. Express

Self Cite

View full text Add to dashboard Cite

show abstract

“…The current values seen in these experiments are consistent with very few pores, but further experiments will be needed to confirm this interpretation. Further, very recent imaging data measuring single mitochondrial swelling under conditions similar to those presented in this work demonstrated that swelling occurs in a steplike manner, suggesting that it is induced by the activation of a relatively small number of pores ( Morikawa et al, 2014 ; Shibata et al, 2019 ). Interestingly, this study reported that despite the fact that all mitochondria undergo depolarization indicating PT activation, not all of them swell, suggesting an intriguing possibility that the high-conductance mode of PTP is not activated in every mitochondrion.…”

Section: Discussionsupporting

confidence: 64%

The very low number of calcium-induced permeability transition pores in the single mitochondrion

Neginskaya

Strubbe

Amodeo

et al. 2020

Journal of General Physiology

View full text Add to dashboard Cite

Mitochondrial permeability transition (PT) is a phenomenon of stress-induced increase in nonspecific permeability of the mitochondrial inner membrane that leads to disruption of oxidative phosphorylation and cell death. Quantitative measurement of the membrane permeability increase during PT is critically important for understanding the PT’s impact on mitochondrial function. The elementary unit of PT is a PT pore (PTP), a single channel presumably formed by either ATP synthase or adenine nucleotide translocator (ANT). It is not known how many channels are open in a single mitochondrion during PT, which makes it difficult to quantitatively estimate the overall degree of membrane permeability. Here, we used wide-field microscopy to record mitochondrial swelling and quantitatively measure rates of single-mitochondrion volume increase during PT-induced high-amplitude swelling. PT was quantified by calculating the rates of water flux responsible for measured volume changes. The total water flux through the mitochondrial membrane of a single mitochondrion during PT was in the range of (2.5 ± 0.4) × 10−17 kg/s for swelling in 2 mM Ca2+ and (1.1 ± 0.2) × 10−17 kg/s for swelling in 200 µM Ca2+. Under these experimental conditions, a single PTP channel with ionic conductance of 1.5 nS could allow passage of water at the rate of 0.65 × 10−17 kg/s. Thus, we estimate the integral ionic conductance of the whole mitochondrion during PT to be 5.9 ± 0.9 nS for 2 mM concentration of Ca2+ and 2.6 ± 0.4 nS for 200 µM of Ca2+. The number of PTPs per mitochondrion ranged from one to nine. Due to the uncertainties in PTP structure and model parameters, PTP count results may be slightly underestimated. However, taking into account that each mitochondrion has ∼15,000 copies of ATP synthases and ANTs, our data imply that PTP activation is a rare event that occurs only in a small subpopulation of these proteins.

show abstract

“…The cells were further incubated at 25°C for 50 min, and 50-l aliquots of the sample were held between a slide glass and a coverslip and placed on the stage of an inverted optical microscope (IX-70; Olympus; Tokyo, Japan) equipped with a 40ϫ objective lens (Uapo40ϫ/340, NA ϭ 0.9; Olympus) (19). Ten minutes after placement, images were photographed with a cooled CCD camera (Sensicam QE, PCO AG, Kelheim, Germany) every 2 s, and 300 images were collected over 10 min.…”

Section: Measurement Of D-ala-d-alamentioning

confidence: 99%

Biochemical and Biophysical Characterization of an Unexpected Bacteriolytic Activity of VanX, a Member of the Vancomycin-resistance vanA Gene Cluster

Sohya¹,

Kamioka²,

Fujita³

et al. 2014

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: VanX belongs to the vanA gene cluster, which confers vancomycin resistance by protecting the bacterial cell wall. Results: Bacteriolysis occurred when isolated VanX was expressed in E. coli, which originated from the dipeptidase activity of VanX. Conclusion: This is the first direct characterization of VanX-mediated bacteriolysis. Significance: This finding suggests a new class of therapeutic agents taking advantage of VanX-mediated bacteriolysis.

show abstract

Detection of swelling of single isolated mitochondrion with optical microscopy

Cited by 17 publications

References 15 publications

Significant correlation between refractive index and activity of mitochondria: single mitochondrion study

Significant correlation between refractive index and activity of mitochondria: single mitochondrion study

The very low number of calcium-induced permeability transition pores in the single mitochondrion

Biochemical and Biophysical Characterization of an Unexpected Bacteriolytic Activity of VanX, a Member of the Vancomycin-resistance vanA Gene Cluster

Contact Info

Product

Resources

About