Visualization of Rab5 activity in living cells using FRET microscopy

Emilia Galperin; Alexander Sorkin

doi:10.1016/S0076-6879(05)03011-9

Visualization of Rab5 activity in living cells using FRET microscopy

Emilia Galperin, Alexander Sorkin

Molecular and Cellular Biochemistry

Research output: Contribution to journal › Review article › peer-review

9 Scopus citations

Abstract

Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP-bound conformation in living cells. Rab5-binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5-tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP-Rab5 and YFP-Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP-bound Rab5. GTP-bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three-chromophore FRET (3-FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3-FRET analysis suggest that GTP-bound Rab5 is capable of oligomerization and present in multiprotein complexes.

Original language	English
Article number	11
Pages (from-to)	119-134
Number of pages	16
Journal	Methods in Enzymology
Volume	403
DOIs	https://doi.org/10.1016/S0076-6879(05)03011-9
State	Published - 2005

Bibliographical note

Funding Information:
This work was supported by grants from National Cancer Institute, National Institute of Drug Abuse (A.S.), and American Cancer Society (A.S. and E.G.), and a postdoctoral fellowship from the American Heart Association (to E.G.).

Funding

This work was supported by grants from National Cancer Institute, National Institute of Drug Abuse (A.S.), and American Cancer Society (A.S. and E.G.), and a postdoctoral fellowship from the American Heart Association (to E.G.).

Funders	Funder number
National Institute on Drug Abuse
American Cancer Society
National Childhood Cancer Registry – National Cancer Institute
American Heart Association

ASJC Scopus subject areas

Biochemistry
Molecular Biology

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10.1016/S0076-6879(05)03011-9

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title = "Visualization of Rab5 activity in living cells using FRET microscopy",

abstract = "Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP-bound conformation in living cells. Rab5-binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5-tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP-Rab5 and YFP-Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP-bound Rab5. GTP-bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three-chromophore FRET (3-FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3-FRET analysis suggest that GTP-bound Rab5 is capable of oligomerization and present in multiprotein complexes.",

author = "Emilia Galperin and Alexander Sorkin",

note = "Funding Information: This work was supported by grants from National Cancer Institute, National Institute of Drug Abuse (A.S.), and American Cancer Society (A.S. and E.G.), and a postdoctoral fellowship from the American Heart Association (to E.G.).",

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doi = "10.1016/S0076-6879(05)03011-9",

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T1 - Visualization of Rab5 activity in living cells using FRET microscopy

AU - Galperin, Emilia

AU - Sorkin, Alexander

N1 - Funding Information: This work was supported by grants from National Cancer Institute, National Institute of Drug Abuse (A.S.), and American Cancer Society (A.S. and E.G.), and a postdoctoral fellowship from the American Heart Association (to E.G.).

PY - 2005

Y1 - 2005

N2 - Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP-bound conformation in living cells. Rab5-binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5-tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP-Rab5 and YFP-Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP-bound Rab5. GTP-bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three-chromophore FRET (3-FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3-FRET analysis suggest that GTP-bound Rab5 is capable of oligomerization and present in multiprotein complexes.

AB - Rab5 is a member of the large family of small GTPases involved in membrane trafficking. Two genetically encoded sensors were developed to visualize Rab5 in its GTP-bound conformation in living cells. Rab5-binding fragments of Rabaptin5 or early endosomal antigen 1 (EEA.1) were fused to yellow fluorescent protein (YFP) and used in the fluorescent resonance energy transfer (FRET) assay together with Rab5-tagged cyan fluorescent protein (CFP). The presence of energy transfer between CFP-Rab5 and YFP-Rab5 binding fragments detected by sensitized FRET microscopy has validated the utility of these generated sensors to visualize the localization of GTP-bound Rab5. GTP-bound Rab5 was found in endosomes, often concentrated in distinct microdomains. Molecular architecture of the Rab5 microdomains was analyzed by three-chromophore FRET (3-FRET) microscopy, utilizing YFP, CFP, and monomeric red fluorescent proteins (mRFP.l). The results of the 3-FRET analysis suggest that GTP-bound Rab5 is capable of oligomerization and present in multiprotein complexes.

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DO - 10.1016/S0076-6879(05)03011-9

M3 - Review article

C2 - 16473582

AN - SCOPUS:32344439413

SN - 0076-6879

VL - 403

SP - 119

EP - 134

JO - Methods in Enzymology

JF - Methods in Enzymology

M1 - 11

ER -

Visualization of Rab5 activity in living cells using FRET microscopy

Abstract

Bibliographical note

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ASJC Scopus subject areas

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