Visualizing multiple inter-organelle contact sites

Evaluating intracellular organelle (mitochondria-associated membrane)

Novel reporters of mitochondria-associated membranes (MAM), MAMtrackers, demonstrate MAM disruption as a common pathological feature in amyotrophic lateral sclerosis.
Sakai, S., Watanabe, S., Komine, O., Sobue, A., Yamanaka, K.
FASEB J. 35 (7): e21688 (2021). PubMed PMID 34143516.

Catalog no.	Name of clone	Descriptiion
RDB19895	pMAMtracker-Luc	A reporter plasmid based on split-Luciferase for evaluating intracellular organelle (mitochondria-associated membrane).
RDB19896	pMAMtracker-Green	A reporter plasmid based on split-GFP for evaluating intracellular organelle (mitochondria-associated membrane).

Fluorescent protein probe that can visualize the multiple inter-organelle contact sites in cells.

Divided GFP-fragment have a feature that they emit fluorescence again by reconstitution when each fragment is in close proximity. When labelled organelles such as mitochondria and ERs by the divided GFPs exist together, the contact sites in cells are visualized by the reconstituted fluorescent protein. The vectors of fluorescent protein probes developed by Dr. Yasushi Tamura of the Yamagata University, Dr. Toshiya Endo of the Kyoto Sangyo University and their colleague are available from the DNA Bank.
Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.
Kakimoto, Y., Tashiro, S., Kojima, R., Morozumi, Y., Endo, T., Tamura, Y.
Sci. Rep. 8 (1): 6175 (2018). PubMed PMID 29670150.
Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
Shirane, M., Wada, M., Morita, K., Hayashi, N., Kunimatsu, R., Matsumoto, Y., Matsuzaki, F., Nakatsumi, H., Ohta, K., Tamura, Y., Nakayama, K.I.
Nat. Commun. 11 (1): 4576 (2020). PubMed PMID 32917905.
Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells.
Tashiro, S., Kakimoto, Y., Shinmyo, M., Fujimoto, S., Tamura, Y.
Front. Cell Dev. Biol. 8: 571388 (2020). PubMed PMID 33330450.

Split-GFP probe plasmid usable in cultured cells

First ten beta-strands (GFP1-10)

Catalog #	Name of resource 1	Name of reource 2	Localization
RDB16041	pMM80	pcDNA3.1_Tom20N-3xFLAG-GFP1-10	mitochondria
RDB16042	pMM82	pcDNA3.1-Tom70N-3xFLAG-GFP1-10	mitochondria
RDB18663	pMM86	pcDNA3.1-ERj1N-V5-GFP1-10	endoplasmic reticulum

Last 11th beta-strand (GFP11)

Catalog #	Name of resource 1	Name of reource 2	Localization
RDB18662	pMM83	pcDNA3.1-Tom70N-3xFLAG-GFP11	mitochondria
RDB18665	pMM189	pIRESNeo3-TOMM70 1-70-3xFLAG-GFP11	mitochondria
RDB16047	pMM87	pcDNA3.1_ERj1N-V5-GFP11	endoplasmic reticulum
RDB16048	pMM89	pcDNA3.1_Sec63N-V5-GFP11	endoplasmic reticulum
RDB18655	pMM104	pcDNA3.1_Lamp1-HA_GFP11	lysosome

Split-GFP probe plasmid usable in budding yeast (for genome integration)

First ten beta-strands (GFP1-10)

Catalog #	Name of resource 1	Name of reource 2	Localization	DNA size (bp)
RDB18659	pYC141	pBS-GPDp-Ifa38-GFP1-10-CyC1ter-hphMX	endoplasmic reticulum	ca. 4,500
RDB18660	pYC143	pBS-GPDp-Tom71-GFP1-10-CyC1ter-hphMX	mitochondria	ca. 5,300
RDB18661	pYC144	pBS-GPDp-Pex3N-GFP1-10-CyC1ter-hphMX	peroxisome	ca. 3,600

Last 11th beta-strand (GFP11)

Catalog #	Name of resource 1	Name of reource 2	Localization	DNA size (bp)
RDB18656	pYC135	pBS-GPDp-Ifa38-V5-GFP11-CyC1ter-natNT2	endoplasmic reticulum	ca. 3,700
RDB18657	pYC136	pBS-GPDp-Erg6-V5-GFP11-CyC1ter-natNT2	lipid droplets	ca. 3,800
RDB18658	pYC137	pBS-GPDp-DPP1-V5-GFP11-CyC1ter-natNT2	vacuole	ca. 3,600

Split-GFP probe plasmid usable in budding yeast (for plasmid expressionn)

First ten beta-strands (GFP1-10)

Catalog #	Name of resource 1	Name of reource 2	Localization	Marker gene
RDB16037	pSFL22	pRS316-GPDp-Tom71-GFP1-10	mitochondria	URA3
RDB16043	pSFL16	pRS316-GPDp-Ifa38-GFP1-10	endoplasmic reticulum	URA3
RDB16049	pSFL19	pRS316-GPDp-Vph1-GFP1-10	vacuole	URA3
RDB16051	pSFL26	pRS316-GPDp-Pex3N-GFP1-10	peroxisome	URA3
RDB16053	pSFL28	pRS316-GPDp-Erg6-GFP1-10	lipid droplets	URA3
RDB18571	pSFL17	pRS315-GPDp-Ifa38-GFP1-10	endoplasmic reticulum	LEU2

Last 11th beta-strand (GFP11)

Catalog #	Name of resource 1	Name of reource 2	Localization	Marker gene
RDB16038	pSFL23	pRS314-GPDp-Tom71-GFP11	mitochondria	TRP1
RDB16039	pSFL75	pRS314-GPDp-Tom71-V5-GFP11	mitochondria	TRP1
RDB16040	pSFL15	pRS314-GPDp-YNR021W-GFP11	endoplasmic reticulum	TRP1
RDB16044	pSFL18	pRS314-GPDp-Ifa38-GFP11	endoplasmic reticulum	TRP1
RDB16045	pSFL86	pRS313-GPDp-Ifa38-3xFLAG-GFP11	endoplasmic reticulum	HIS3
RDB16046	pSFL59	pRS314-GPDp-Ifa38-mCherry-GFP11	endoplasmic reticulum	TRP1
RDB16050	pSFL108	pRS314-GPDp-Dpp1-V5-GFP11	vacuole	TRP1
RDB16052	pSFL96	pRS316-GPDp-Pex3N-3xFLAG-GFP11	peroxisome	URA3
RDB16054	pSFL67	pRS314-GPDp-Erg6-3xFLAG-GFP11	lipid droplets	TRP1
RDB18572	pSFL61	pRS316-GPDp-Ifa38-mCherry-GFP11	endoplasmic reticulum	URA3
RDB18573	pSFL66	pRS314-GPDp-Ifa38-3xFLAG-GFP11	endoplasmic reticulum	TRP1
RDB18574	pSFL68	pRS314-GPDp-Erg6-V5-GFP11	lipid droplets	TRP1
RDB18577	pSFL76	pRS314-GPDp-Ifa38-V5-GFP11	endoplasmic reticulum	TRP1

Backborn constructs

First ten beta-strands (GFP1-10)

Catalog #	Name of resource	Descriptiion
RDB18567	pSFL9 pRS316-GPDp-GFP1-10	Expression vector of the first ten beta-strands (GFP1-10).
RDB18568	pSFL10 pRS314-GPDp-GFP1-10	Expression vector of the first ten beta-strands (GFP1-10).

Last 11th beta-strand (GFP11)

Catalog #	Name of resource	Descriptiion
RDB18570	pSFL12 pRS314-GPDp-GFP11	Expression vector of the the last 11th beta-strand (GFP11).
RDB18575	pSFL73 pRS314-GPDp-3xFLAG-GFP11	Expression vector of 3xFLAG-GFP11.
RDB18576	pSFL74 pRS314-GPDp-V5-GFP11	Expression vector of V5-GFP11.

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GPI anchored Reconstitution-Activated Proteins Highlight Intercellular Connections (GRAPHIC)

Genetically Encoded Fluorescent Indicator GRAPHIC Delineates Intercellular Connections.
Kinoshita, N., Huang, A.J.Y., McHugh, T.J., Suzuki, S.C., Masai, I., Kim, I.H., Soderling, S.H., Miyawaki, A., Shimogori, T.
iScience 15: 28-38 (2019). PubMed PMID 31026667.

Diffusible GRAPHIC to visualize morphology of cells after specific cell-cell contact.
Kinoshita, N., Huang, A.J.Y., McHugh, T.J., Miyawaki, A., Shimogori, T.
Sci. Rep. 10 (1): 14437 (2020). PubMed PMID 32879377.

Catalog no.	Name of clone	Descriptiion
RDB15869	pCSII-CMV-MCS _n-GRAPHIC	Lentiviral expression vector of GRAPHIC probe (LZA).
RDB15870	pCSII-CMV-MCS_n-GRAPHIC T2A H2B-mCherry	Lentiviral expression vector of GRAPHIC probe (LZA).
RDB15871	pCSII-CMV-MCS_B (BFP type) n-GRAPHIC T2A H2B-mCherry	Lentiviral expression vector of GRAPHIC probe (LZA).
RDB15872	pCSII-CMV-MCS _c-GRAPHIC	Lentiviral expression vector of GRAPHIC probe (LZB).
RDB15873	pCSII-CMV-MCS _c-GRAPHIC T2A H2B-Azurite	Lentiviral expression vector of GRAPHIC probe (LZB).
RDB15874	pCSII-CMV-MCS _Y (YFP- type) c-GRAPHIC T2A H2B-Azurite	Lentiviral expression vector of GRAPHIC probe (LZB).
RDB19166	AAV:NT-probe-nuc-mCherry	Adeno-associated viral vector encoding the dGRAPHIC NT probe.
RDB19167	AAV:CT-probe-cyto-mCherry	Adeno-associated viral vector encoding the dGRAPHIC CT probe.

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2025.05.03 (T.M. GRP0061e)