Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion
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Abstract
The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the
rheological properties of cell cytoplasm. GFP mutant S65T was purified from recombinant
bacteria for solution studies, and expressed in CHO cell cytoplasm. GFP-S65T was brightly
fluorescent in solution (lambda ex 492 nm, lambda em 509 nm) with a lifetime of 2.9
ns and a rotational correlation time (tc) of 20 ns. Recovery of GFP fluorescence after
photobleaching was complete with a half-time (t1/2) in aqueous saline of 30 +/- 2
ms (5-micron diameter spot), giving a diffusion coefficient of 8.7 x 10(-7) cm2/s.
The t1/2 was proportional to solution viscosity and was dependent on spot diameter.
In contrast to fluorescein. GFP photobleaching efficiency was not affected by solution
O2 content, triplet state quenchers, singlet oxygen scavengers, and general radical
quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process
was detected and ascribed to reversible photobleaching. The t1/2 for reversible photobleaching
was 1.5-5.5 ms (relative viscosity 5-250), was independent of spot diameter, and was
unaffected by O2 or quenchers. In cell cytoplasm, time-resolved microfluorimetry indicated
a GFP lifetime of 2.6 ns and a tc of 36 +/- 3 ns, giving a relative viscosity (cytoplasm
versus water) of 1.5. Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete
with a t1/2 of 83 +/- 6 ms, giving a relative viscosity of 3.2. GFP translational
diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2.
Taken together with measurements of GFP translation and rotation in aqueous dextran
solutions, the data in cytoplasm support the view that the primary barrier to GFP
diffusion is collisional interactions between GFP and macromolecular solutes.