Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach

Jonathan R. Brewer, Henrik Seir Thoke, Robeto Stock , Luis A. Bagatolli

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.

OriginalsprogEngelsk
TidsskriftBiochimica et Biophysica Acta - Biomembranes
Vol/bind1859
Udgave nummer5
Sider (fra-til)888-895
ISSN0005-2736
DOI
StatusUdgivet - 2017

Fingeraftryk

Fluorescence
Polarization
Membranes
Imaging techniques
Microscopes
Fluorescent Dyes
Sphingomyelins
Fluorophores
Liquids
CCD cameras
Equipment and Supplies
Detectors
Scanning
Kinetics
Lasers

Citer dette

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title = "Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach",
abstract = "We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.",
keywords = "DiIC, Epitactic coupling, LAURDAN fluorescence, Lipid flip-flop, Membrane asymmetry, Membrane domains, Sphingomyelinase D",
author = "Brewer, {Jonathan R.} and Thoke, {Henrik Seir} and Robeto Stock and Bagatolli, {Luis A.}",
year = "2017",
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pages = "888--895",
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Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach. / Brewer, Jonathan R.; Thoke, Henrik Seir; Stock , Robeto; Bagatolli, Luis A.

I: Biochimica et Biophysica Acta - Biomembranes, Bind 1859, Nr. 5, 2017, s. 888-895.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach

AU - Brewer, Jonathan R.

AU - Thoke, Henrik Seir

AU - Stock , Robeto

AU - Bagatolli, Luis A.

PY - 2017

Y1 - 2017

N2 - We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.

AB - We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.

KW - DiIC

KW - Epitactic coupling

KW - LAURDAN fluorescence

KW - Lipid flip-flop

KW - Membrane asymmetry

KW - Membrane domains

KW - Sphingomyelinase D

U2 - 10.1016/j.bbamem.2017.01.024

DO - 10.1016/j.bbamem.2017.01.024

M3 - Journal article

VL - 1859

SP - 888

EP - 895

JO - B B A - Biomembranes

JF - B B A - Biomembranes

SN - 0005-2736

IS - 5

ER -