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SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation.

Takemoto K, Matsuda T, Sakai N, Fu D, Noda M, Uchiyama S, Kotera I, Arai Y, Horiuchi M, Fukui K, Ayabe T, Inagaki F, Suzuki H, Nagai T - Sci Rep (2013)

Bottom Line: Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest.Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division.Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

View Article: PubMed Central - PubMed

Affiliation: 1] Research Institute for Electronic Sciences, Hokkaido University, Kita-20 Nishi-10 Kita-ku, Sapporo, Hokkaido 001-0020, Japan [2] [3].

ABSTRACT
Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

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Molecular inactivation of cofilin with SuperNova.(a) COS7 cells co-expressing actin-GFP and cofilin-SuperNova were made. The fluorescence image of actin-GFP and cofilin-SuperNova before and after CALI is shown. Bleaching of SuperNova's fluorescence was clearly observed after CALI. The dashed square in the actin-GFP image is the pseudopod region selected in time lapse imaging and analysis. Scale bar, 10 μm. (b) Still frames obtained during time lapse imaging of actin-GFP motion in lamellipodia before and after CALI by cofilin-SuperNova are shown. The numbers indicate time (min) after light irradiation (9.6 W/cm2, 1 min with through a 580AF20 from a mercury lamp). Scale bar, 10 μm. (c) Histograms of the temporal correlation in the cells expressing no fluorescent protein-tagged cofilin (NO, n = 14), cofilin-CFP (CFP, n = 20 cells), cofilin-KillerRed (n = 14 cells) and cofilin-SuperNova (n = 19 cells). Temporal correlation after CALI normalized by the pre-CALI value was represented as fold change in temporal correlation, which indicates the duration time of initial fluorescence pattern of actin-GFP. Therefore, a higher value reflects the slower movement. p < 0.001 for NO vs SuperNova, p > 0.10 for NO vs CFP, p = 0.083 for NO vs KillerRed, t-test.
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f4: Molecular inactivation of cofilin with SuperNova.(a) COS7 cells co-expressing actin-GFP and cofilin-SuperNova were made. The fluorescence image of actin-GFP and cofilin-SuperNova before and after CALI is shown. Bleaching of SuperNova's fluorescence was clearly observed after CALI. The dashed square in the actin-GFP image is the pseudopod region selected in time lapse imaging and analysis. Scale bar, 10 μm. (b) Still frames obtained during time lapse imaging of actin-GFP motion in lamellipodia before and after CALI by cofilin-SuperNova are shown. The numbers indicate time (min) after light irradiation (9.6 W/cm2, 1 min with through a 580AF20 from a mercury lamp). Scale bar, 10 μm. (c) Histograms of the temporal correlation in the cells expressing no fluorescent protein-tagged cofilin (NO, n = 14), cofilin-CFP (CFP, n = 20 cells), cofilin-KillerRed (n = 14 cells) and cofilin-SuperNova (n = 19 cells). Temporal correlation after CALI normalized by the pre-CALI value was represented as fold change in temporal correlation, which indicates the duration time of initial fluorescence pattern of actin-GFP. Therefore, a higher value reflects the slower movement. p < 0.001 for NO vs SuperNova, p > 0.10 for NO vs CFP, p = 0.083 for NO vs KillerRed, t-test.

Mentions: Finally to prove the utility of SuperNova in CALI based interventions in living cells, we developed a simple assay based on actin turnover in lamellipodia in living mammalian cells. This process is known to be regulated by cofilin, which regulates actin dynamics at the leading edge of motile cells by stimulating actin filament disassembly and accelerating actin filament turnover16. We constructed a cofilin-SuperNova fusion protein and transiently expressed it with actin-EGFP in living COS7 cells (Figure 4a). During 10 minutes before CALI, actin filaments were freely moving (arrows in Figure 4b, upper panels). However following intense orange light irradiation, the motility of actin filaments in protrusive lamellipodia was suppressed qualitatively (arrowheads in Figure 4b, lower panels; supplemental movie), and quantitatively by temporal correlation coefficient analysis. In this assay higher temporal correlation equates to less active lamellipodia formation. The observed increase in temporal correlation after the CALI of cofilin is therefore consistent with its previously described function17. Importantly, the same effect was not observed in control experiments using cells lacking fluorescent protein tagged cofilin or cells expressing cofilin-CFP. These results suggest that the capacity to use SuperNova in CALI applications is comparable to that of KillerRed (Figure 4c) in terms of ROS generation, but significantly improved as a fusion partner by virtue of the engineered monomerisation


SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation.

Takemoto K, Matsuda T, Sakai N, Fu D, Noda M, Uchiyama S, Kotera I, Arai Y, Horiuchi M, Fukui K, Ayabe T, Inagaki F, Suzuki H, Nagai T - Sci Rep (2013)

Molecular inactivation of cofilin with SuperNova.(a) COS7 cells co-expressing actin-GFP and cofilin-SuperNova were made. The fluorescence image of actin-GFP and cofilin-SuperNova before and after CALI is shown. Bleaching of SuperNova's fluorescence was clearly observed after CALI. The dashed square in the actin-GFP image is the pseudopod region selected in time lapse imaging and analysis. Scale bar, 10 μm. (b) Still frames obtained during time lapse imaging of actin-GFP motion in lamellipodia before and after CALI by cofilin-SuperNova are shown. The numbers indicate time (min) after light irradiation (9.6 W/cm2, 1 min with through a 580AF20 from a mercury lamp). Scale bar, 10 μm. (c) Histograms of the temporal correlation in the cells expressing no fluorescent protein-tagged cofilin (NO, n = 14), cofilin-CFP (CFP, n = 20 cells), cofilin-KillerRed (n = 14 cells) and cofilin-SuperNova (n = 19 cells). Temporal correlation after CALI normalized by the pre-CALI value was represented as fold change in temporal correlation, which indicates the duration time of initial fluorescence pattern of actin-GFP. Therefore, a higher value reflects the slower movement. p < 0.001 for NO vs SuperNova, p > 0.10 for NO vs CFP, p = 0.083 for NO vs KillerRed, t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3775092&req=5

f4: Molecular inactivation of cofilin with SuperNova.(a) COS7 cells co-expressing actin-GFP and cofilin-SuperNova were made. The fluorescence image of actin-GFP and cofilin-SuperNova before and after CALI is shown. Bleaching of SuperNova's fluorescence was clearly observed after CALI. The dashed square in the actin-GFP image is the pseudopod region selected in time lapse imaging and analysis. Scale bar, 10 μm. (b) Still frames obtained during time lapse imaging of actin-GFP motion in lamellipodia before and after CALI by cofilin-SuperNova are shown. The numbers indicate time (min) after light irradiation (9.6 W/cm2, 1 min with through a 580AF20 from a mercury lamp). Scale bar, 10 μm. (c) Histograms of the temporal correlation in the cells expressing no fluorescent protein-tagged cofilin (NO, n = 14), cofilin-CFP (CFP, n = 20 cells), cofilin-KillerRed (n = 14 cells) and cofilin-SuperNova (n = 19 cells). Temporal correlation after CALI normalized by the pre-CALI value was represented as fold change in temporal correlation, which indicates the duration time of initial fluorescence pattern of actin-GFP. Therefore, a higher value reflects the slower movement. p < 0.001 for NO vs SuperNova, p > 0.10 for NO vs CFP, p = 0.083 for NO vs KillerRed, t-test.
Mentions: Finally to prove the utility of SuperNova in CALI based interventions in living cells, we developed a simple assay based on actin turnover in lamellipodia in living mammalian cells. This process is known to be regulated by cofilin, which regulates actin dynamics at the leading edge of motile cells by stimulating actin filament disassembly and accelerating actin filament turnover16. We constructed a cofilin-SuperNova fusion protein and transiently expressed it with actin-EGFP in living COS7 cells (Figure 4a). During 10 minutes before CALI, actin filaments were freely moving (arrows in Figure 4b, upper panels). However following intense orange light irradiation, the motility of actin filaments in protrusive lamellipodia was suppressed qualitatively (arrowheads in Figure 4b, lower panels; supplemental movie), and quantitatively by temporal correlation coefficient analysis. In this assay higher temporal correlation equates to less active lamellipodia formation. The observed increase in temporal correlation after the CALI of cofilin is therefore consistent with its previously described function17. Importantly, the same effect was not observed in control experiments using cells lacking fluorescent protein tagged cofilin or cells expressing cofilin-CFP. These results suggest that the capacity to use SuperNova in CALI applications is comparable to that of KillerRed (Figure 4c) in terms of ROS generation, but significantly improved as a fusion partner by virtue of the engineered monomerisation

Bottom Line: Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest.Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division.Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

View Article: PubMed Central - PubMed

Affiliation: 1] Research Institute for Electronic Sciences, Hokkaido University, Kita-20 Nishi-10 Kita-ku, Sapporo, Hokkaido 001-0020, Japan [2] [3].

ABSTRACT
Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

Show MeSH
Related in: MedlinePlus