Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange

被引:14
作者
Bizzarri, Ranieri [1 ,2 ]
Cardarelli, Francesco [3 ]
Serresi, Michela [3 ]
Beltram, Fabio [1 ,2 ,3 ]
机构
[1] Scuola Normale Super Pisa, NEST, I-56127 Pisa, Italy
[2] Ist Nanosci CNR, I-56127 Pisa, Italy
[3] Ist Italiano Tecnol, Ctr Nanotechnol Innovat NEST, I-56127 Pisa, Italy
关键词
Fluorescence/Luminescence; Kinetics; Optical sensors; Biological samples; Bioanalytical methods; ARGININE-RICH MOTIF; NUCLEAR-ENVELOPE; PROTEIN DYNAMICS; IMPORTIN-ALPHA; TRANSPORT; PERMEABILITY; TRANSLOCATION; DIFFUSION; MOBILITY;
D O I
10.1007/s00216-012-6025-4
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Fluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components. A peculiar aspect of this method is that it is based on the change of optical properties only, whereas dynamics and biochemistry of the molecules of interest are not perturbed. This makes FRAP particularly suitable for the study of protein translocation, e.g., between nucleus and cytoplasm. Here we present a comprehensive theoretical treatment of FRAP applied to protein nucleocytoplasmic translocation by passive diffusion and/or energy-driven processes across the nuclear envelope. Our mathematical model is validated by experimental FRAP studies with functionalized fluorescent protein chimeras. Using this approach we demonstrate that molecular crowding at the nuclear pore does not hamper passive diffusion and calculate the dimension of the nuclear pore size (5.33 nm). Additionally, our FRAP analysis reveals the biochemical parameters (maximum translocation rate and dissociation constant of the transport complex in cytoplasm) associated with the active import of a prototypical nuclear localization sequence (NLS of SV40) and related mutants. We demonstrate that transportin binding and active import into the nucleus are independent processes that can be separately modulated. The present results are discussed in light of their potential to help in engineering sequences for intracellular targeted delivery of sensors and/or therapeutic compounds. Finally, the limits of validity of our mathematical model are addressed.
引用
收藏
页码:2339 / 2351
页数:13
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