A novel in situ assay for the identification and characterization of soluble nuclear mobility factors.
The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniques have revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged protein can be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus. However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absence of protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay that combines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a method we used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors. This assay can readily be adapted to identify and characterize other nuclear mobility factors.
Elbi
Cem
C
Laboratory of Receptor Biology and Gene Expression, Building 41, Room B602, National Cancer Institute, Bethesda, MD 20892-5055, USA.
Walker
Dawn A
DA
Lewis
Marcia
M
Romero
Guillermo
G
Sullivan
William P
WP
Toft
David O
DO
Hager
Gordon L
GL
DeFranco
Donald B
DB
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000230:Adenocarcinoma / Q000737:chemistry / Q000473:pathology; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry* / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000378:metabolism; D015674:Mammary Neoplasms, Animal / Q000737:chemistry / Q000473:pathology; D051379:Mice; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
D008164:Luminescent Proteins; D018832:Molecular Chaperones; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins