Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins

Carrero, Gustavo; McDonald, Darin; Crawford, Ellen; Vries, Gerda de; Hendzel, Michael J.

doi:10.1016/s1046-2023(02)00288-8

Cited by 178 publications

(192 citation statements)

References 46 publications

(84 reference statements)

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“…This result suggests that CIT-K affects midbody microtubules' stability independently from KIF14, especially in cells showing the highest sensitivity to CIT-K loss. To address more directly this phenotype, we evaluated the turnover rate of α-tubulin at the midbody of control cells and of CIT-Kdepleted cells by performing fluorescence recovery after photobleaching (FRAP) 30 in HeLa cells stably expressing RFP-tagged α-tubulin. This test revealed that knockdown of CIT-K reduces the half-time of recovery by 50% (Figure 2c).…”

Section: Resultsmentioning

confidence: 99%

“…Recovery kinetics were then measured and, after background subtraction, were normalized to pre-and last post-bleach frames. 30 HeLa cells were transfected with pEGFP-EB1 expression construct or with GFP-TUBB3 fusion constructs. Time-lapse imaging of EB1 comets was performed using 63 × 1.3 objective at 1 frame/500 ms for 2 min on an SP5 confocal microscope (Leica).…”

Section: Methodsmentioning

confidence: 99%

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Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation

et al. 2015

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Cytokinesis, the physical separation of daughter cells at the end of cell cycle, is commonly considered a highly stereotyped phenomenon. However, in some specialized cells this process may involve specific molecular events that are still largely unknown. In mammals, loss of Citron-kinase (CIT-K) leads to massive cytokinesis failure and apoptosis only in neuronal progenitors and in male germ cells, resulting in severe microcephaly and testicular hypoplasia, but the reasons for this specificity are unknown. In this report we show that CIT-K modulates the stability of midbody microtubules and that the expression of tubulin β-III (TUBB3) is crucial for this phenotype. We observed that TUBB3 is expressed in proliferating CNS progenitors, with a pattern correlating with the susceptibility to CIT-K loss. More importantly, depletion of TUBB3 in CIT-K-dependent cells makes them resistant to CIT-K loss, whereas TUBB3 overexpression increases their sensitivity to CIT-K knockdown. The loss of CIT-K leads to a strong decrease in the phosphorylation of S444 on TUBB3, a post-translational modification associated with microtubule stabilization. CIT-K may promote this event by interacting with TUBB3 and by recruiting at the midbody casein kinase-2α (CK2α) that has previously been reported to phosphorylate the S444 residue. Indeed, CK2α is lost from the midbody in CIT-K-depleted cells. Moreover, expression of the nonphosphorylatable TUBB3 mutant S444A induces cytokinesis failure, whereas expression of the phospho-mimetic mutant S444D rescues the cytokinesis failure induced by both CIT-K and CK2α loss. Altogether, our findings reveal that expression of relatively low levels of TUBB3 in mitotic cells can be detrimental for their cytokinesis and underscore the importance of CIT-K in counteracting this event.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation

et al. 2015

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“…Within the last 30 years, FRAP has become an important and versatile technique to study the dynamics in various systems, such as living cells, membranes and other biological environments [14]. In polymer physics, the photobleaching methods are employed to investigate diffusion in macromolecular systems, particularly in networks.…”

Section: Fluorescence Recovery After Photobleachingmentioning

confidence: 99%

Determination of the diffusion coefficients of small solutes in cheese: A review

Floury

Jeanson

Aly

et al. 2010

Dairy Sci. Technol.

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-In cheese technology, the mass transfer of small solutes, such as salt, moisture and metabolites during brining and ripening, is very important for the final quality of the cheese. This paper has the following objectives: (i) to review the data concerning the diffusion coefficients of solutes in different cheese types; (ii) to review the experimental methods available to model the mass transfer properties of small solutes in complex matrices such as cheese; and (iii) to consider some potential alternative approaches. Numerous studies have reported the transfer of salt in cheese during brining and ripening. Regardless of the type of cheese and its composition, the effective diffusion coefficients of salt have been reported to be between 1 and 5.3 × 10 −10 m 2 ·s −1 at 10-15°C. However, few papers have dealt with the mass transfer properties of other small solutes in cheese. Most of the reported effective diffusion coefficient values have been obtained by macroscopic and destructive concentration profile methods. More recently, some other promising techniques, such as nuclear magnetic resonance, magnetic resonance imaging or fluorescence recovery after photobleaching, are currently being developed to measure the mass transfer properties of solutes in heterogeneous media at microscopic scales. However, these methods are still difficult to apply to complex matrices such as cheese. Further research needs to focus on: (i) the development of nondestructive techniques to determine the mass transfer properties of small solutes at a microscopic level in complex matrices such as cheese; and (ii) the determination of the mass transfer properties of metabolites that are involved in enzymatic reactions during cheese ripening. cheese / mass transfer / diffusion / modelling / solute Article published by EDP SciencesRésumé -Détermination des coefficients de diffusion de petits solutés dans le fromage : une synthèse. En technologie fromagère, le transfert de petits solutés, tels que le sel, l'eau et les métabolites au cours du saumurage et de l'affinage, joue un rôle majeur sur la qualité finale du fromage. Cette revue bibliographique a pour objectifs principaux : (i) de faire le bilan des valeurs publiées des coefficients de diffusion de différents solutés dans les fromages ; (ii) de passer en revue les méthodes expérimentales disponibles pour déterminer les propriétés de transfert des petits solutés dans des milieux complexes comme le fromage ; (iii) de considérer les méthodes alternatives potentiellement applicables aux fromages. Dans la littérature, de nombreuses études ont été publiées au sujet du transfert de sel dans les fromages au cours du saumurage et de l'affinage. En fonction du type de fromage et de sa composition, les coefficients de diffusion effectifs du sel sont compris entre 1 et 5,3 × 10 −10 m 2 ·s −1 à des températures comprises entre 10 et 15°C. Très peu d'études concernant les propriétés de transfert d'autres petits solutés dans les fromages ont été publiées. La plupart des coefficients de diffusi...

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“…To determine whether the rate of ICP27 import into the nucleus is affected by hypomethylation, we performed Fluorescence Recovery After Photobleaching (FRAP) (5,10). HeLa cells were infected with vN-YFP-ICP27 (16) at an MOI of 5 in the absence or presence of AdOx added 1 h after infection.…”

mentioning

confidence: 99%

Arginine Methylation of the RGG Box Does Not Appear To Regulate ICP27 Import during Herpes Simplex Virus Infection

Souki

Hernandez

Sandri‐Goldin

2011

J Virol

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Arginine methylation can regulate protein import and export and can modulate protein interactions. Herpes simplex virus 1 (HSV-1) ICP27 is a shuttling protein involved in viral mRNA export. We previously reported that ICP27 is methylated on three arginines within its RGG box and that arginine methylation regulates ICP27 export and its interaction with SRPK1 and Aly/REF. Here, we report that ICP27 was efficiently imported into the nucleus when hypomethylated as determined by Fluorescence Recovery After Photobleaching (FRAP). Furthermore, coimmunoprecipitation of ICP27 with ␤-importin was not significantly affected by ICP27 hypomethylation. Thus, ICP27 import does not appear to be regulated by arginine methylation.

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Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins

Cited by 178 publications

References 46 publications

Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation

Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation

Determination of the diffusion coefficients of small solutes in cheese: A review

Arginine Methylation of the RGG Box Does Not Appear To Regulate ICP27 Import during Herpes Simplex Virus Infection

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