![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 15, Issue 10, 4749-4760, October 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, 1649-028 Lisbon, Portugal
Submitted June 17, 2004;
Accepted July 23, 2004
Monitoring Editor: Joseph Gall
Fluorescence recovery after photobleaching (FRAP) is a widely used tool for estimating mobility parameters of fluorescently tagged molecules in cells. Despite the widespread use of confocal laser scanning microscopes (CLSMs) to perform photobleaching experiments, quantitative data analysis has been limited by lack of appropriate practical models. Here, we present a new approximate FRAP model for use on any standard CLSM. The main novelty of the method is that it takes into account diffusion of highly mobile molecules during the bleach phase. In fact, we show that by the time the first postbleach image is acquired in a CLSM a significant fluorescence recovery of fast-moving molecules has already taken place. The model was tested by generating simulated FRAP recovery curves for a wide range of diffusion coefficients and immobile fractions. The method was further validated by an experimental determination of the diffusion coefficient of fluorescent dextrans and green fluorescent protein. The new FRAP method was used to compare the mobility rates of fluorescent dextrans of 20, 40, 70, and 500 kDa in aqueous solution and in the nucleus of living HeLa cells. Diffusion coefficients were lower in the nucleoplasm, particularly for higher molecular weight dextrans. This is most likely caused by a sterical hindrance effect imposed by nuclear components. Decreasing the temperature from 37 to 22°C reduces the dextran diffusion rates by 
30% in aqueous solution but has little effect on mobility in the nucleoplasm. This suggests that spatial constraints to diffusion of dextrans inside the nucleus are insensitive to temperature.
* Corresponding author. E-mail address: josebraga{at}fm.ul.pt.
This article has been cited by other articles:
|
|
 |
|
  E. C. Freundt, L. Yu, E. Park, M. J. Lenardo, and X.-N. Xu Molecular Determinants for Subcellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame 3b Protein J. Virol., July 1, 2009; 83(13): 6631 - 6640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Rino, J. M. P. Desterro, T. R. Pacheco, T. W. J. Gadella Jr., and M. Carmo-Fonseca Splicing Factors SF1 and U2AF Associate in Extraspliceosomal Complexes Mol. Cell. Biol., May 1, 2008; 28(9): 3045 - 3057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Grunwald, B. Spottke, V. Buschmann, and U. Kubitscheck Intranuclear Binding Kinetics and Mobility of Single Native U1 snRNP Particles in Living Cells Mol. Biol. Cell, December 1, 2006; 17(12): 5017 - 5027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. R. Darrah, M. Tlalka, A. Ashford, S. C. Watkinson, and M. D. Fricker The vacuole system is a significant intracellular pathway for longitudinal solute transport in basidiomycete fungi. Eukaryot. Cell, July 1, 2006; 5(7): 1111 - 1125. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Perez-Vilar, J. C. Olsen, M. Chua, and R. C. Boucher pH-dependent Intraluminal Organization of Mucin Granules in Live Human Mucous/Goblet Cells J. Biol. Chem., April 29, 2005; 280(17): 16868 - 16881. [Abstract] [Full Text] [PDF]
|
|
