Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class I proteins in fibroblast cells

Ahmed A. Heikal

doi:10.1016/j.ymeth.2013.06.023

Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class I proteins in fibroblast cells

Ahmed A. Heikal

Chemistry & Biochemistry

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Major histocompatibility complex class I proteins, MHC(I), are expressed in almost all nucleated cells and synthesized in the endoplasmic reticulum (ER). The orientation and mobility of these complexes are crucial in their biological function in the immune system, i.e., the cytosolic pathogen peptides loading and their presentation to T-cell receptors at the plasma membrane, where cell destruction is triggered. Here, we investigate the structural flexibility and associations of GFP-encoded MHC(I) alleles (H2L^d), namely H2L^dGFP_in and H2L^dGFP_out, in cultured mouse fibroblast cells. Time-resolved fluorescence anisotropy of H2L^dGFP_in in the ER indicates a dominant overall tumbling motion of 56±7ns (ER), with a fast conformational flexibility, as compared with a restricted rotation of H2L^dGFP_out. At the single-molecule level, the diffusion coefficient of H2L^dGFP_in and H2L^dGFP_out in the ER is (1.8±0.5)×10^-9 and (2.1±0.6)×10^-9cm²/s, respectively, as revealed by fluorescence correlation spectroscopy. A complementary immunoblotting of H2L^dGFP constructs, isolated from mouse fibroblast cells, reveals band at 75kDa as compared with 29kDa of the free EGFP. These real-time dynamics provide new insights into the structural flexibility and intracellular associations of GFP-labeled MHC(I) alleles (H2L^d) in living cells.

Original language	English (US)
Pages (from-to)	283-291
Number of pages	9
Journal	Methods
Volume	66
Issue number	2
DOIs	https://doi.org/10.1016/j.ymeth.2013.06.023
State	Published - Mar 15 2014

Bibliographical note

Funding Information:
The stably-transfected fibroblast cells, expressing H2L d GFP in and H2L d GFP out , were a gift from Dr. Michael Edidin (Department of Biology, Johns Hopkins University) along with very helpful discussion. The author acknowledges the help of Florly Ariola and Minjoung Kyoung at the early stage of this project. Thanks to Keith Krise and Dr. Erin Sheets for their help with Western blot and to Dr. Joseph Johnson for helpful discussion. This work was supported, in part, by startup fund provided by the Department of Chemistry and Biochemistry and Department of Pharmacy Practice and Pharmaceutical Sciences provided by the University of Minnesota Duluth. Additional support was also provided by Grant-In-Aid of Research, Artistry and Scholarship (University of Minnesota) as well as NSF (MCB0718741) and NIH (AG030949) Grants.

Keywords

FCS
FLIM
MHC class I
Time-resolved anisotropy

Publisher link

10.1016/j.ymeth.2013.06.023

Find It

Find It at U of M Libraries

Cite this

@article{8e07322fd5144f1bb663bf5df4749b49,

title = "Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class I proteins in fibroblast cells",

abstract = "Major histocompatibility complex class I proteins, MHC(I), are expressed in almost all nucleated cells and synthesized in the endoplasmic reticulum (ER). The orientation and mobility of these complexes are crucial in their biological function in the immune system, i.e., the cytosolic pathogen peptides loading and their presentation to T-cell receptors at the plasma membrane, where cell destruction is triggered. Here, we investigate the structural flexibility and associations of GFP-encoded MHC(I) alleles (H2Ld), namely H2LdGFPin and H2LdGFPout, in cultured mouse fibroblast cells. Time-resolved fluorescence anisotropy of H2LdGFPin in the ER indicates a dominant overall tumbling motion of 56±7ns (ER), with a fast conformational flexibility, as compared with a restricted rotation of H2LdGFPout. At the single-molecule level, the diffusion coefficient of H2LdGFPin and H2LdGFPout in the ER is (1.8±0.5)×10-9 and (2.1±0.6)×10-9cm2/s, respectively, as revealed by fluorescence correlation spectroscopy. A complementary immunoblotting of H2LdGFP constructs, isolated from mouse fibroblast cells, reveals band at 75kDa as compared with 29kDa of the free EGFP. These real-time dynamics provide new insights into the structural flexibility and intracellular associations of GFP-labeled MHC(I) alleles (H2Ld) in living cells.",

keywords = "FCS, FLIM, MHC class I, Time-resolved anisotropy",

author = "Heikal, {Ahmed A.}",

note = "Funding Information: The stably-transfected fibroblast cells, expressing H2L d GFP in and H2L d GFP out , were a gift from Dr. Michael Edidin (Department of Biology, Johns Hopkins University) along with very helpful discussion. The author acknowledges the help of Florly Ariola and Minjoung Kyoung at the early stage of this project. Thanks to Keith Krise and Dr. Erin Sheets for their help with Western blot and to Dr. Joseph Johnson for helpful discussion. This work was supported, in part, by startup fund provided by the Department of Chemistry and Biochemistry and Department of Pharmacy Practice and Pharmaceutical Sciences provided by the University of Minnesota Duluth. Additional support was also provided by Grant-In-Aid of Research, Artistry and Scholarship (University of Minnesota) as well as NSF (MCB0718741) and NIH (AG030949) Grants.",

year = "2014",

month = mar,

day = "15",

doi = "10.1016/j.ymeth.2013.06.023",

language = "English (US)",

volume = "66",

pages = "283--291",

journal = "Methods",

issn = "1046-2023",

number = "2",

}

TY - JOUR

T1 - Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class I proteins in fibroblast cells

AU - Heikal, Ahmed A.

N1 - Funding Information: The stably-transfected fibroblast cells, expressing H2L d GFP in and H2L d GFP out , were a gift from Dr. Michael Edidin (Department of Biology, Johns Hopkins University) along with very helpful discussion. The author acknowledges the help of Florly Ariola and Minjoung Kyoung at the early stage of this project. Thanks to Keith Krise and Dr. Erin Sheets for their help with Western blot and to Dr. Joseph Johnson for helpful discussion. This work was supported, in part, by startup fund provided by the Department of Chemistry and Biochemistry and Department of Pharmacy Practice and Pharmaceutical Sciences provided by the University of Minnesota Duluth. Additional support was also provided by Grant-In-Aid of Research, Artistry and Scholarship (University of Minnesota) as well as NSF (MCB0718741) and NIH (AG030949) Grants.

PY - 2014/3/15

Y1 - 2014/3/15

N2 - Major histocompatibility complex class I proteins, MHC(I), are expressed in almost all nucleated cells and synthesized in the endoplasmic reticulum (ER). The orientation and mobility of these complexes are crucial in their biological function in the immune system, i.e., the cytosolic pathogen peptides loading and their presentation to T-cell receptors at the plasma membrane, where cell destruction is triggered. Here, we investigate the structural flexibility and associations of GFP-encoded MHC(I) alleles (H2Ld), namely H2LdGFPin and H2LdGFPout, in cultured mouse fibroblast cells. Time-resolved fluorescence anisotropy of H2LdGFPin in the ER indicates a dominant overall tumbling motion of 56±7ns (ER), with a fast conformational flexibility, as compared with a restricted rotation of H2LdGFPout. At the single-molecule level, the diffusion coefficient of H2LdGFPin and H2LdGFPout in the ER is (1.8±0.5)×10-9 and (2.1±0.6)×10-9cm2/s, respectively, as revealed by fluorescence correlation spectroscopy. A complementary immunoblotting of H2LdGFP constructs, isolated from mouse fibroblast cells, reveals band at 75kDa as compared with 29kDa of the free EGFP. These real-time dynamics provide new insights into the structural flexibility and intracellular associations of GFP-labeled MHC(I) alleles (H2Ld) in living cells.

AB - Major histocompatibility complex class I proteins, MHC(I), are expressed in almost all nucleated cells and synthesized in the endoplasmic reticulum (ER). The orientation and mobility of these complexes are crucial in their biological function in the immune system, i.e., the cytosolic pathogen peptides loading and their presentation to T-cell receptors at the plasma membrane, where cell destruction is triggered. Here, we investigate the structural flexibility and associations of GFP-encoded MHC(I) alleles (H2Ld), namely H2LdGFPin and H2LdGFPout, in cultured mouse fibroblast cells. Time-resolved fluorescence anisotropy of H2LdGFPin in the ER indicates a dominant overall tumbling motion of 56±7ns (ER), with a fast conformational flexibility, as compared with a restricted rotation of H2LdGFPout. At the single-molecule level, the diffusion coefficient of H2LdGFPin and H2LdGFPout in the ER is (1.8±0.5)×10-9 and (2.1±0.6)×10-9cm2/s, respectively, as revealed by fluorescence correlation spectroscopy. A complementary immunoblotting of H2LdGFP constructs, isolated from mouse fibroblast cells, reveals band at 75kDa as compared with 29kDa of the free EGFP. These real-time dynamics provide new insights into the structural flexibility and intracellular associations of GFP-labeled MHC(I) alleles (H2Ld) in living cells.

KW - FCS

KW - FLIM

KW - MHC class I

KW - Time-resolved anisotropy

UR - http://www.scopus.com/inward/record.url?scp=84896443923&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84896443923&partnerID=8YFLogxK

U2 - 10.1016/j.ymeth.2013.06.023

DO - 10.1016/j.ymeth.2013.06.023

M3 - Article

C2 - 23811298

AN - SCOPUS:84896443923

SN - 1046-2023

VL - 66

SP - 283

EP - 291

JO - Methods

JF - Methods

IS - 2

ER -

Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class I proteins in fibroblast cells

Abstract

Bibliographical note

Keywords

Publisher link

Find It

Other files and links

Fingerprint

Cite this