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Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching.

ABSTRACT: Fluorescence recovery after photobleaching (FRAP) is an excellent tool to measure the chemical rate constants of fluorescently labeled proteins in living cells. Usually FRAP experiments are conducted with the protein concentrations being in a steady state, i.e., when the association and dissociation of the proteins are equilibrated. This is a strong limitation because situations in which rate constants change with time are of great scientific interest. In this study, we present an approach in which FRAP is used shortly after DNA damage introducing laser microirradiation, which results in the recruitment of the DNA clamp protein proliferating cell nuclear antigen (PCNA) to DNA lesions. We establish different kinetic models that are compatible with the observed PCNA recruitment data if FRAP is not used. By using FRAP at different time points during protein accumulation, we can not only exclude two out of three models, but we can also determine the rate constants with increased reliability. This study thus demonstrates the feasibility of using FRAP during protein recruitment and its application in the discrimination of possible kinetic models.

SUBMITTER: Lengert L

PROVIDER: S-EPMC4624168 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching.

Lengert Laurin L Lengert Nicor N Drossel Barbara B Cardoso M Cristina MC Muster Britta B Nowak Danny D Rapp Alexander A

Biophysical journal 20151001 8

Fluorescence recovery after photobleaching (FRAP) is an excellent tool to measure the chemical rate constants of fluorescently labeled proteins in living cells. Usually FRAP experiments are conducted with the protein concentrations being in a steady state, i.e., when the association and dissociation of the proteins are equilibrated. This is a strong limitation because situations in which rate constants change with time are of great scientific interest. In this study, we present an approach in wh ...[more]

PMID: 26488646

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Project description:Fluorescence Recovery After Photobleaching (FRAP) has been extensively used to understand molecular dynamics in cells. This technique when applied to soluble, globular molecules driven by diffusion is easily interpreted and well understood. However, the classical methods of analysis cannot be applied to anisotropic structures subjected to directed transport, such as cytoskeletal filaments or elongated organelles transported along microtubule tracks. A new mathematical approach is needed to analyze FRAP data in this context and determine what information can be obtain from such experiments. To address these questions, we analyze fluorescence intensity profile curves after photobleaching of fluorescently labelled intermediate filaments anterogradely transported along microtubules. We apply the analysis to intermediate filament data to determine information about the filament motion. Our analysis consists of deriving equations for fluorescence intensity profiles and developing a mathematical model for the motion of filaments and simulating the model. Two closed forms for profile curves were derived, one for filaments of constant length and one for filaments with constant velocity, and three types of simulation were carried out. In the first type of simulation, the filaments have random velocities which are constant for the duration of the simulation. In the second type, filaments have random velocities which instantaneously change at random times. In the third type, filaments have random velocities and exhibit pausing between velocity changes. Our analysis shows: the most important distribution governing the shape of the intensity profile curves obtained from filaments is the distribution of the filament velocity. Furthermore, filament length which is constant during the experiment, had little impact on intensity profile curves. Finally, gamma distributions for the filament velocity with pauses give the best fit to asymmetric fluorescence intensity profiles of intermediate filaments observed in FRAP experiments performed in polarized migrating astrocytes. Our analysis also shows that the majority of filaments are stationary. Overall, our data give new insight into the regulation of intermediate filament dynamics during cell migration.

Dataset Information

Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching.

Publications

Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching.

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