Role of Red-Ox Cycle in Structural Oscillations and Solvation Dynamics in the Mitochondria of a Live Cell

Abstract

Structural oscillations and solvation dynamics in the mitochondria of a live cell are studied by time-resolved microscopy using a covalent fluorescence probe. We compared the dynamics in a human breast cancer cell (MCF-7) with that in a normal breast cell MCF-10A. The probe, CPM (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin), binds with the free thiol groups. In MCF-10A cell, CPM binds with the discrete mitochondria. In MCF-7, CPM labels the clustered mitochondria in the peri-nuclear region. Location of the CPM in the mitochondria is confirmed by colocalization with a mitochondria-tracker dye. The red-ox cycle in the mitochondria causes periodic fluctuation in the microenvironment in the discrete mitochondria. This is manifested in fluctuations in fluorescence intensity of CPM bound to mitochondria. The magnitude of oscillation is much less for CPM bound to the clustered mitochondria (in which the red-ox cycle is inefficient) in the cancer cell (MCF-7). In both of the cells (MCF-10A and MCF-7) CPM bound to thiol-containing proteins in mitochondria exhibits ultraslow response with average solvation time (⟨τs⟩) of 850 and 1400 ps in MCF-10A and MCF-7, respectively.

Structural Oscillations of Non–muscle Myosin II–C2: Time Resolved Confocal Microscopy

“…Representative graphs of such frequent oscillation were shown from three randomly selected time windows of 3 sec (Figure B, ii‐iv). The time period of fluorescence oscillation was calculated by determining the peak position by smoothening the intensity vs. time plot using a fast Fourier transform filter . We find that the period of oscillation is not constant and the half‐period varies from 0.35 to 0.55 sec, with an average of 0.45±0.1 sec.…”

Cytochrome c‐Capped Fluorescent Gold Nanoclusters: Imaging of Live Cells and Delivery of Cytochrome c

“…From Equation (1), the at 510–515 nm corresponds to n ≈13 Au atoms in the NC (assuming E f ≈5.53 eV, as in bulk Au) . The slight (≈5 nm) blueshift of of Au‐NCs in MCF7 cells relative to that in MCF10A cells might be due to slight variation in the microenvironment between the two cells …”

“…[5,6,[13][14][15] The slight( % 5nm) blueshift of l max em of Au-NCsi nM CF7 cells relative to that in MCF10A cells might be due to slight variation in the microenvironment between the two cells. [36] The somewhat drastic change ( % 35 nm redshift in l max em and change in size from Au 8 in bulk water to Au 13 inside the cell) in Au-NCsm ight be due to the interaction of cyt c-capped Au-NCs with different biomoleculesi nside the cell. One such candidate is GSH.…”

Spatial inhomogeneity in spectra and exciton dynamics in porphyrin micro-rods and micro-brushes: Confocal microscopy

“…The experimental set-up for confocal microscopy has been described in our previous publications. 40,41 To summarize, a combination of confocal microscope (Olympus IX-71) and TCSPC setup (PicoQuant, Micro-Time 200, numerical aperture (NA)≈1.2) and a pulsed picosecond diode laser (PDL 828-S "SEPIA II," Pico Quant, at 470 nm) were used. The diffraction limited spot size is 0.6λ/1.2∼ λ/2.…”

“…The emission spectra of TPPS supramolecular structures were recorded using an electron multiplying charge-coupled device (EMCCD, ANDOR Technology) attached to a spectrograph (ANDOR Technology, Shamrock series). 40,41 2.2c3 Fluorescence lifetime measurement: For recording instrument response function (IRF), we used a bare slide and collected the scattered laser light. The FWHM of the IRF for excitation at 470 nm is ∼70 ps.…”