A Rapid and Simple Method of Evaluating the Dimeric Tendency of Fluorescent Proteins in Living Cells Using a Truncated Protein of Importin α as Fusion Tag

Nakagawa, Chika; Nishimura, Shigenori; Senda-Murata, Kaori; Sugimoto, Kenji

doi:10.1271/bbb.110677

Cited by 9 publications

(9 citation statements)

References 17 publications

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“…Plasmids used for this study contain the CMV promoter with a short linker between an N-or C-terminal superfolder GFP (sfGFP), enhanced blue fluorescent protein 2 (EBFP2), or monomerized Venus (mVenus) tags (20,21). Monomeric sfGFP (msfGFP) contains a V206K monomerizing mutation (22)(23)(24). sfGFP (25) contains a valine residue encoded at position 206 in the fluorescent protein sequence, and GFP derivatives (e.g.…”

Section: Methodsmentioning

confidence: 99%

“…sfGFP (25) contains a valine residue encoded at position 206 in the fluorescent protein sequence, and GFP derivatives (e.g. EGFP, enhanced cyan fluorescent protein, and enhanced yellow fluorescent protein) with an alanine or valine at this position have a weak tendency to dimerize (22)(23)(24). Cx26 and Cx43 coding sequences used for fluorescent protein fusion constructs were from rats.…”

Section: Methodsmentioning

confidence: 99%

“…Oligomerization of fluorescent protein tags has long been a concern (32), and sensitive new methods indicate that sfGFP shows an increased tendency to aggregate/ oligomerize compared with sfGFP with a V206K monomerizing mutation (msfGFP) (22,33). When we tested for an effect of sfGFP on plaque arrangement stability, we found that Cx30 and Cx26 tagged with sfGFP had different mobility characteristics than connexins tagged with msfGFP (Fig.…”

Section: Mobility Of Gap Junction Channels Withinmentioning

confidence: 95%

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Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Stout¹,

Snapp

Spray³

2015

Journal of Biological Chemistry

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Background: Connexin proteins form gap junction channel clusters termed plaques. Results: Microscopy with fluorescent protein-tagged connexins showed that connexin26 and connexin30 plaque arrangement is dynamic, but gap junctions of connexin43 are stabilized by its C-terminal region. Conclusion:The arrangement of channels in gap junctions depends on connexin type. Significance: These findings help to clarify how gap junction plaque dynamics and composition could modulate intercellular communication.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Mobility Of Gap Junction Channels Withinmentioning

confidence: 95%

See 1 more Smart Citation

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Stout¹,

Snapp

Spray³

2015

Journal of Biological Chemistry

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“…10,11,17) The localization of DsRed-HP1α seemed to be the same as that of EGFP-HP1α (unpublished results), although the tetrameric DsRed fusion tag may hinder the precise localization of the protein of interest. 26) In Phase 2, HP1α began to diffuse into the nucleoplasm and its diffusion increased gradually. In Phase 3, HP1α diffused further towards the nuclear periphery.…”

Section: Discussionmentioning

confidence: 99%

Live-cell imaging of HP1α throughout the cell cycle of mouse C3H10T1/2 cells and rhythmical flickering of heterochromatin dots in interphase

Nakagawa

Kawakita

Fukada

et al. 2014

Bioscience, Biotechnology, and Biochemistry

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Heterochromatin protein 1 alpha (HP1α) localizes to heterochromatin in interphase and shows dynamic molecular behavior in living cells. We previously reported that during mitosis, the majority of HP1α diffused into the cytoplasm but some remained in centromere heterochromatin. Here, we further characterize the molecular behavior of HP1α throughout the cell cycle. Time-lapse imaging of DsRed-HP1α through two successive cell divisions indicated that interphase can be divided into four phases. HP1α forms heterochromatin dots in early G1, which are maintained without any apparent changes (Phase 1). However, the HP1α dots begin to diffuse into the nucleoplasm and start flickering with a rhythmical cycle (Phase 2). Then, the HP1α dots diffuse further towards the periphery of the nucleus (Phase 3), and uniformly diffuse throughout the entire nucleus (Phase 4). Rhythmical flickering of HP1α dots in the middle of interphase may be useful for following cell cycle progression in mouse living cells.

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“…Venus dimerization prevented import of the fluorescent protein fused to truncated importin α into the nucleus. The mutation A206L reduced Venus dimerization most efficiently compared to L221A and F223A (Nakagawa et al ).…”

Section: Introductionmentioning

confidence: 99%

New guidelines for fluorophore application in peroxisome targeting analyses in transient plant expression systems

Falter¹,

Thư²,

Pokhrel³

et al. 2019

JIPB

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Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein (YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co‐cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co‐transformation with the peroxisome marker, CFP‐PTS1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP‐PTS1, allowing piggy‐back import into peroxisomes. Indeed, if co‐expressed with monomeric Cerulean‐PTS1 (mCer‐PTS1), the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization‐based piggy‐back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co‐expressed with CFP‐PTS1 but not with mCer‐PTS1. By contrast, piggy‐back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.

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A Rapid and Simple Method of Evaluating the Dimeric Tendency of Fluorescent Proteins in Living Cells Using a Truncated Protein of Importin α as Fusion Tag

Cited by 9 publications

References 17 publications

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Live-cell imaging of HP1α throughout the cell cycle of mouse C3H10T1/2 cells and rhythmical flickering of heterochromatin dots in interphase

New guidelines for fluorophore application in peroxisome targeting analyses in transient plant expression systems

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