Emergent structures in mixed lipid membranes
Transcription
Emergent structures in mixed lipid membranes
Understanding Structure formation in Membranes Lateral Fluidity Out-of-plane dynamics Chemical heterogeneity Atul N. Parikh [email protected] Self-Assembly: Hydrophobic effect MULTILAMELLAR VESICLE UNILAMELLAR VESICLE AMPHIPHILE BILAYER LIQUID-CRYSTALLINE LAYERED STACKS 1 10 LOG (LENGTH SCALE), NM 100 1,000 (A ) D W ATER Supported Bilayer Lipid Membranes D D (B ) Figure 5 | Synthetic Model of the Cell Membrane. The cell membrane, being a self-assembled fluid, can be reconstituted from its components such as shown. By selecting appropriate lipids and proteins, a synthetic control of membrane structure and function can be achieved. Membrane Photolithography QUARTZ TEMPLATE CHROME LAYER AQUEOUS PHASE (PBS) HYDRATION LAYER SILICA SUBSTRATE 100 um UV (184-257 nm) PBS PBS PBS 100 um C. K. Yee, M. L. Amweg, A. N. Parikh, Adv. Mater. (2004) C. K. Yee, M. L. Amweg, A. N. Parikh, JACS (2004) Preserved fluidity of the unexposed bilayer Fluorescence Recovery after Photobleaching (FRAP) T = 0 min T = 3 min T = 5 min Unexposed region UV illuminated region 2750 2800 2850 2900 2950 Wavenumbers, cm-1 3000 3050 T = 9 min Laterally Confined Photochemistry (Wentworth et al, Science, 2002) “Most membrane proteins do not enjoy the continuous unrestricted lateral diffusion.... Instead, proteins diffuse in a more complicated way that indicates considerable lateral heterogeneity in membrane structure, at least on a nanometer scale” Jacobson, K., Sheets, E. D. & Simson, R. (1995) Science 268, 1441-1442 Multiphoton Photolithography 800 nm 100 fs Patterning with Femto-sec Laser Fluorescence images; 100X oil NA 1.45 97%POPC 3%NDB-PE 20μm Before Laser Exposure Parameters •λ = 800nm •Energy per Pulse (6-12nJ per pulse •Rep Rate = 540 KHz •Focus Diameter = 0.5μm After (modified) Multiphoton Modification of Lipids “Erasing” Barriers Repetition Rate Dependance • 2.7kHz • 1 laser pulse approx. every 10-4s • Able to pattern • 540kHz • 1 laser pulse approx. every 10-6s (~200x) • Able to pattern and with same power, “erase” • Same power thresholds Confined box Re-exposed perimeter Portion is removed time Box is now open Probing Membrane Heterogeniety and Dynamics using model bilayers PBS PBS PBS PBS PBS 100 um Before Backfilling T=0 min. T=1 min. T=5 min. T=10 min. T=20 min. A Biophysical tool for Understanding Lipid heterogeneity Designed reactive-diffusive fronts Lipid-lipid interdiffusion, compositional manipulation Phase dynamics and stability Engineering arrested diffusion Kinetically and chemically arrested Mixing for functional patterning HAEC Gm1 HL-60 CTB Bilayers CTB Engineering Lipid Rafts Sphingomyelin Cholesterol Gm1 Phospholipid PBS PBS PBS PBS PBS A. R. Sapuri, J. T. Groves, A. N. Parikh, in preparation (2004) TEMPLATED SELF-ASSEMBLY OF PHOSPHOLIPID MEMBRANES JACS 2005 a b d c e 1:1:1, DOPC:SM:Chol 3% NBD DHPE, 32%DOPC, 31% cholesterol, 32% sphingomyelin, 2% GM1 Ex 0.9sec a b c POPC GM1 +fitc CTB TR POPC GM1 + FITC CTB TR image Fitc image TR POPC GM1 + FITC CTB Green Channel image Corrected fret image Figure 3 a c b d e 100μm f c Buckling of PDMS Stamp Quartz Chrome PDMS Stamp Quartz Chrome Photomask UV/Ozone PDMS Quartz Chrome Buckling of the PDMS Stamp SiOx Peel the PDMS from the Mask Photochemically buckled PDMS substrates 80 60 1 µm µm 0 40 -1 20 -2 0 0 20 40 µm 60 80 500um Pattern 1.0 80 0.0 60 -0.5 1 0 40 µm µm Height 0.5 -1.0µm -1 20 0 20 40 60 µm 80 -2 100 0 0 20 40 µm 60 80 TR Labeled Lipid Incubation 2 minutes Anisotropic recovery of Photobleached spot Tm of DMPC is 23°C Epifluorescense image: 1% Texas red DHPE, 99% DMPC Comparison of FRAP on flat and Buckled Stamp Tm of DMPC is 23°C Epifluorescense image: 1% Texas red DHPE, 99% DMPC COWORKERS Dr. Chanel K. Yee, Materials Dr. Annapoorna R. Sapuri-Butti, Chemistry Dr. Sanhita Dixit, Physics Michael Howland, Materials Science Andreia Michelle Smith, Biophysics Calvin Yang, Biomedical Engineering Alan Szmodis, Biophysics Amy Anderson, Biophysics Ravi Butti, Materials Adrian Brozell, Applied Science Babak Sanii, Applied Science Rita El-Khouri, Chemistry Michelle Muha, Optical Science Allen T. Lee, Optical Science FUNDING BES, Department of Energy The NSF Center for Biophotonics, UC Davis NIH Lipid rafts in Cell signaling Dynamic assemblies rich in sphingomyelin, cholesterol, and saturated lipids Micro-environment for signaling molecules and their complex formations Signaling: bacterial recognition, adhesion, transport, localized oxidation, Drug-binding GPCR proteins Engineering Lipid Rafts Sphingomyelin Cholesterol Gm1 Phospholipid PBS PBS PBS PBS PBS A. R. Sapuri, J. T. Groves, A. N. Parikh, in preparation (2004) Detergent-Resistancy Raft-raft coalescence During apoptosis in HL-60 Cells A (Inducer: tBH, label: alexa-CTB) B C D Boiling, Yee, Lincoln, Gilchrist, Yeh, Parikh, Morse, Submitted, 2005 Orthogonal membrane phases by selective extraction of bilayer phases a b c d e Triton X100 experiment After triton Before triton Backfilled NBD POPC
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