Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: FRAP analysis suggests that the deletion of the domains of transgelin-2 destabilizes its association to F-actin. (A) A part of individual SFs is bleached in 2D, and the resulting fluorescence recovery is probed by the line-scanning along the middle of the bleached region at a high temporal resolution. (B) Confocal images of transgelin-2 and its mutants taken before ( t = -2 s), just after ( t = 0 s), and after ( t = -7 s) the bleaching, with respective kymographs on the right. Scale, 1 μm. (C) Time course of for different mutants is shown by two ways: a linear scale plot (upper) and a log-log plot (lower). (D) k for different mutants. Asterisks represent statistically significant differences compared to FL (*, p< 0.01).

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: Transgelin-2 binds F-actin via the CH and AB domains. (A) Schematic diagram of the domain structure of transgelin-2 (encoded by the 7.4 □ kb TAGLN2 gene) and its mutants. mClover2 present at the N-terminus is omitted. (B) Confocal images of representative cells to analyze the intracellular colocalization between transgelin-2 or its mutant (mClover2) and F-actin (Lifeact). Scale, 20 μm. (C) The relationship of the fluorescence intensity between Lifeact (ordinate) and mClover2 (abscissa) measured at each pixel of the confocal images, with Pearson correlation coefficient R between the two fluorescent labels.

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques: Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: FRAP analysis suggests that F-actin in SFs works as an immobile scaffold for transgelin-2. (A) Confocal images of mClover2-beta-actin subjected to FRAP. Scale, 5 μm. (B) Time course of in the FRAP response of mClover2-beta-actin is shown by two ways – a linear scale plot (left, mean ± SD) and a log-log plot (right, mean) – showing that the recovery is obviously slower compared to that of transgelin-2 in .

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques:

Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: Individual domain-level determination of the reactive-diffusive properties. (A) Normalized fluorescence intensity F ( x, y = 0, t )/ F 0 is plotted as blue dots over the position in the longitudinal x for specific time t , with the regression curves (black) determined by the maximum likelihood method. Here, representative data of FL are shown. τ represents the time constant, i.e., the inverse of the recovery rate k determined in . (B) D eff (gray) and D pure (black) for each mutant. Asterisks represent statistically significant differences compared to FL within the D eff groups or between the specified pairs of D eff and D pure (*, p < 0.01; **, p < 0.05). (C) Immunoblots of transgelin-2 FL and its mutants with mClover2. (D) Equilibrium dissociation constant K for each mutant. Data are expressed as the mean ± coefficient of variation.

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques: Fluorescence, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: FRAP analysis suggests that the deletion of the domains of transgelin-2 destabilizes its association to F-actin. (A) A part of individual SFs is bleached in 2D, and the resulting fluorescence recovery is probed by the line-scanning along the middle of the bleached region at a high temporal resolution. (B) Confocal images of transgelin-2 and its mutants taken before ( t = -2 s), just after ( t = 0 s), and after ( t = -7 s) the bleaching, with respective kymographs on the right. Scale, 1 μm. (C) Time course of for different mutants is shown by two ways: a linear scale plot (upper) and a log-log plot (lower). (D) k for different mutants. Asterisks represent statistically significant differences compared to FL (*, p< 0.01).

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: Transgelin-2 binds F-actin via the CH and AB domains. (A) Schematic diagram of the domain structure of transgelin-2 (encoded by the 7.4 □ kb TAGLN2 gene) and its mutants. mClover2 present at the N-terminus is omitted. (B) Confocal images of representative cells to analyze the intracellular colocalization between transgelin-2 or its mutant (mClover2) and F-actin (Lifeact). Scale, 20 μm. (C) The relationship of the fluorescence intensity between Lifeact (ordinate) and mClover2 (abscissa) measured at each pixel of the confocal images, with Pearson correlation coefficient R between the two fluorescent labels.

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques: Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: FRAP analysis suggests that F-actin in SFs works as an immobile scaffold for transgelin-2. (A) Confocal images of mClover2-beta-actin subjected to FRAP. Scale, 5 μm. (B) Time course of in the FRAP response of mClover2-beta-actin is shown by two ways – a linear scale plot (left, mean ± SD) and a log-log plot (right, mean) – showing that the recovery is obviously slower compared to that of transgelin-2 in .

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques:

Journal: bioRxiv

Article Title: Determining the inherent reaction-diffusion properties of actin-binding proteins in cells by incorporating genetic engineering to FRAP-based framework

doi: 10.1101/2020.09.21.305615

Figure Lengend Snippet: Individual domain-level determination of the reactive-diffusive properties. (A) Normalized fluorescence intensity F ( x, y = 0, t )/ F 0 is plotted as blue dots over the position in the longitudinal x for specific time t , with the regression curves (black) determined by the maximum likelihood method. Here, representative data of FL are shown. τ represents the time constant, i.e., the inverse of the recovery rate k determined in . (B) D eff (gray) and D pure (black) for each mutant. Asterisks represent statistically significant differences compared to FL within the D eff groups or between the specified pairs of D eff and D pure (*, p < 0.01; **, p < 0.05). (C) Immunoblots of transgelin-2 FL and its mutants with mClover2. (D) Equilibrium dissociation constant K for each mutant. Data are expressed as the mean ± coefficient of variation.

Article Snippet: Expression plasmids encoding mClover2-tagged transgelin-2 (TAGLN2) and mRuby2-tagged Lifeact were constructed by inserting the PCR-amplified cDNAs (human TAGLN2, pFN21ASDA0120, Kazusa DNA Research Institute; Lifeact, Addgene plasmid # 54688; a gift from Michael Davidson) into the mClover2-C1 vector (Addgene plasmid #54577, a gift from Michael Davidson) and the mRuby2-N1 vector (Addgene plasmid #54614, a gift from Michael Davidson), respectively.

Techniques: Fluorescence, Mutagenesis, Western Blot