Multi-color fluorescence live-cell imaging in Dictyostelium discoideum
The cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imagi...
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Japan Society for Cell Biology
2024-12-01
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| Series: | Cell Structure and Function |
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| Online Access: | https://www.jstage.jst.go.jp/article/csf/49/2/49_24065/_html/-char/en |
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| author | Hidenori Hashimura Satoshi Kuwana Hibiki Nakagawa Kenichi Abe Tomoko Adachi Toyoko Sugita Shoko Fujishiro Gen Honda Satoshi Sawai |
| author_facet | Hidenori Hashimura Satoshi Kuwana Hibiki Nakagawa Kenichi Abe Tomoko Adachi Toyoko Sugita Shoko Fujishiro Gen Honda Satoshi Sawai |
| author_sort | Hidenori Hashimura |
| collection | DOAJ |
| description | The cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imaging of D. discoideum has greatly facilitated studies on fundamental topics, including cytokinesis, phagocytosis, and cell migration. Additionally, its unique life cycle places Dictyostelium at the forefront of understanding aggregative multicellularity, a recurring evolutionary trait found across the Opisthokonta and Amoebozoa clades. The use of multiple fluorescent proteins (FP) and labels with separable spectral properties is critical for tracking cells in aggregates and identifying co-occurring biomolecular events and factors that underlie the dynamics of the cytoskeleton, membrane lipids, second messengers, and gene expression. However, in D. discoideum, the number of frequently used FP species is limited to two or three. In this study, we explored the use of new-generation FP for practical 4- to 5-color fluorescence imaging of D. discoideum. We showed that the yellow fluorescent protein Achilles and the red fluorescent protein mScarlet-I both yield high signals and allow sensitive detection of rapid gene induction. The color palette was further expanded to include blue (mTagBFP2 and mTurquosie2), large Stoke-shift LSSmGFP, and near-infrared (miRFP670nano3) FPs, in addition to the HaloTag ligand SaraFluor 650T. Thus, we demonstrated the feasibility of deploying 4- and 5- color imaging of D. discoideum using conventional confocal microscopy. Key words: fluorescence imaging, organelle, cytoskeleton, small GTPase, Dictyostelium |
| format | Article |
| id | doaj-art-afa1c9033a244da194f71f1d29146a31 |
| institution | DOAJ |
| issn | 0386-7196 1347-3700 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Japan Society for Cell Biology |
| record_format | Article |
| series | Cell Structure and Function |
| spelling | doaj-art-afa1c9033a244da194f71f1d29146a312025-08-20T02:55:57ZengJapan Society for Cell BiologyCell Structure and Function0386-71961347-37002024-12-0149213515310.1247/csf.24065csfMulti-color fluorescence live-cell imaging in Dictyostelium discoideumHidenori Hashimura0Satoshi Kuwana1Hibiki Nakagawa2Kenichi Abe3Tomoko Adachi4Toyoko Sugita5Shoko Fujishiro6Gen Honda7Satoshi Sawai8Graduate School of Arts and Sciences, The University of TokyoGraduate School of Arts and Sciences, The University of TokyoGraduate School of Arts and Sciences, The University of TokyoDepartment of Biological Sciences, Graduate School of Sciences, The University of TokyoGraduate School of Arts and Sciences, The University of TokyoGraduate School of Arts and Sciences, The University of TokyoGraduate School of Arts and Sciences, The University of TokyoKomaba Institute for Science, Graduate School of Arts and Sciences, The University of TokyoGraduate School of Arts and Sciences, The University of TokyoThe cellular slime mold Dictyostelium discoideum, a member of the Amoebozoa, has been extensively studied in cell and developmental biology. D. discoideum is unique in that they are genetically tractable, with a wealth of data accumulated over half a century of research. Fluorescence live-cell imaging of D. discoideum has greatly facilitated studies on fundamental topics, including cytokinesis, phagocytosis, and cell migration. Additionally, its unique life cycle places Dictyostelium at the forefront of understanding aggregative multicellularity, a recurring evolutionary trait found across the Opisthokonta and Amoebozoa clades. The use of multiple fluorescent proteins (FP) and labels with separable spectral properties is critical for tracking cells in aggregates and identifying co-occurring biomolecular events and factors that underlie the dynamics of the cytoskeleton, membrane lipids, second messengers, and gene expression. However, in D. discoideum, the number of frequently used FP species is limited to two or three. In this study, we explored the use of new-generation FP for practical 4- to 5-color fluorescence imaging of D. discoideum. We showed that the yellow fluorescent protein Achilles and the red fluorescent protein mScarlet-I both yield high signals and allow sensitive detection of rapid gene induction. The color palette was further expanded to include blue (mTagBFP2 and mTurquosie2), large Stoke-shift LSSmGFP, and near-infrared (miRFP670nano3) FPs, in addition to the HaloTag ligand SaraFluor 650T. Thus, we demonstrated the feasibility of deploying 4- and 5- color imaging of D. discoideum using conventional confocal microscopy. Key words: fluorescence imaging, organelle, cytoskeleton, small GTPase, Dictyosteliumhttps://www.jstage.jst.go.jp/article/csf/49/2/49_24065/_html/-char/enfluorescence imagingorganellecytoskeletonsmall gtpasedictyostelium |
| spellingShingle | Hidenori Hashimura Satoshi Kuwana Hibiki Nakagawa Kenichi Abe Tomoko Adachi Toyoko Sugita Shoko Fujishiro Gen Honda Satoshi Sawai Multi-color fluorescence live-cell imaging in Dictyostelium discoideum Cell Structure and Function fluorescence imaging organelle cytoskeleton small gtpase dictyostelium |
| title | Multi-color fluorescence live-cell imaging in Dictyostelium discoideum |
| title_full | Multi-color fluorescence live-cell imaging in Dictyostelium discoideum |
| title_fullStr | Multi-color fluorescence live-cell imaging in Dictyostelium discoideum |
| title_full_unstemmed | Multi-color fluorescence live-cell imaging in Dictyostelium discoideum |
| title_short | Multi-color fluorescence live-cell imaging in Dictyostelium discoideum |
| title_sort | multi color fluorescence live cell imaging in dictyostelium discoideum |
| topic | fluorescence imaging organelle cytoskeleton small gtpase dictyostelium |
| url | https://www.jstage.jst.go.jp/article/csf/49/2/49_24065/_html/-char/en |
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