Dynamics of Gene Silencing in a Live Cell: Stochastic Resonance

Binding of a specific siRNA to the target mRNA in a live cell (human breast cancer cell, MCF-7) is studied by confocal microscopy. The specific siRNA (labeled with a fluorophore, alexa 488) exhibits much higher intensity of fluorescence in the bound state than in the free (unbound) state. It is observed that repeated unbinding and rebinding of siRNA (to target mRNA) occur before gene silencing. 16 273 on-time periods (residence or dwell time of siRNA in bound form) are detected. They follow a strikingly simple pattern. All of the on-time periods are odd-integral multiples of 5.5 ± 0.05 ms. This is ascribed to stochastic resonance

Excited State Proton Transfer in the Lysosome of Live Lung Cells:Normal and Cancer Cells

Dynamics of excited state proton transfer(ESPT) in the lysosome region of live lung cells (normaland cancer) is studied by picosecond time-resolved confocal microscopy. For this, we used a fluorescent probe, pyranine (8- hydroxy-pyrene-1,3,6-trisulfonate, HPTS). From the colocalization of HPTS with a lysotracker dye (lysotracker yellow), we confirmed that HPTS resides in the lysosome for both of the cells. The diffusion coefficient (Dt) in the lysosome region was obtained from fluorescence correlation spectroscopy (FCS). From Dt, the viscosity of lysosome is estimated to be ∼40 and ∼30 cP in the cancer and normal cells, respectively. The rate constants of the elementary steps of ESPT in a normal lung cell (WI38) are compared with those in a lung cancer cell (A549). It is observed that the time constant of the initial proton transfer process in a normal cell (τPT = 40 ps) is similar to that in a cancer cell. The recombination of the geminate ion pair is slightly faster (τrec = 25 ps) in the normal cell than that (τrec = 30 ps) in a cancer cell. The time constant of the dissociation (τdiss) of the geminate ion pair for the cancer cell (τdiss = 80 ps) is 1.5 times faster compared to that (τdiss = 120 ps) in a normal cell