Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM

Oreopoulos, John; Yip, Christopher M.

doi:10.1016/j.jsb.2009.02.011

Cited by 40 publications

(30 citation statements)

References 116 publications

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“…In control cells, the orientation of the absorption transition moment of the DiI is largely parallel to the membrane surface resulting in negative ͗P 2 ͘ values (Fig. 8B) (53,54). Having determined the average value in control cells, we next confirmed that dibucaine and valproate increased the fluidity of the cells.…”

Section: Transient Increase In Membrane Fluidity Does Not Alter Caveolaementioning

confidence: 58%

“…Fortunately, it has been reported that valproic acid, a branched chain weak organic acid, can rapidly increase membrane fluidity within minutes of addition to cells (51). To monitor the changes in membrane fluidity, we used polarized TIRF microscopy to access the degree of orientational order of the lipophilic membrane dye 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine (DiI) to determine the order parameter ͗P 2 ͘, as described previously (52,53). As depicted in Fig.…”

Section: Transient Increase In Membrane Fluidity Does Not Alter Caveolaementioning

confidence: 99%

“…The ͗P 2 ͘ values were calculated using an ImageJ macro that considers the evanescent electric field vector amplitude, excitation incident angle, and fluorescence detected dichroic rotation, see Ref. 53 for further details.…”

Section: Polarized Total Internal Reflection Microscopymentioning

confidence: 99%

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Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

Hirama

Das

Yang

et al. 2017

Journal of Biological Chemistry

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Edited by Dennis R. VoelkerCaveolae are bulb-shaped nanodomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have many physiological functions, including endocytic transport, mechanosensing, and regulation of membrane and lipid transport. Caveola formation relies on integral membrane proteins termed caveolins (Cavs) and the cavin family of peripheral proteins. Both protein families bind anionic phospholipids, but the precise roles of these lipids are unknown. Here, we studied the effects of phosphatidylserine (PtdSer), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) on caveolar formation and dynamics. Using live-cell, single-particle tracking of GFP-labeled Cav1 and ultrastructural analyses, we compared the effect of PtdSer disruption or phosphoinositide depletion with caveola disassembly caused by cavin1 loss. We found that PtdSer plays a crucial role in both caveola formation and stability. Sequestration or depletion of PtdSer decreased the number of detectable Cav1-GFP puncta and the number of caveolae visualized by electron microscopy. Under PtdSer-limiting conditions, the co-localization of Cav1 and cavin1 was diminished, and cavin1 degradation was increased. Using rapamycin-recruitable phosphatases, we also found that the acute depletion of PtdIns4P and PtdIns (4,5)P 2 has minimal impact on caveola assembly but results in decreased lateral confinement. Finally, we show in a model of phospholipid scrambling, a feature of apoptotic cells, that caveola stability is acutely affected by the scrambling. We conclude that the predominant plasmalemmal anionic lipid PtdSer is essential for proper Cav clustering, caveola formation, and caveola dynamics and that membrane scrambling can perturb caveolar stability.

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Section: Transient Increase In Membrane Fluidity Does Not Alter Caveolaementioning

confidence: 58%

Section: Transient Increase In Membrane Fluidity Does Not Alter Caveolaementioning

confidence: 99%

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Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

Hirama

Das

Yang

et al. 2017

Journal of Biological Chemistry

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“…To address this, we compared the mechanistic details of the interactions of two CAPs with contrasting core hydrophobicities (6K-F17 with a "low" core segment hydrophobicity (1.48) and 6K-F17-4L with a "high" core segment hydrophobicity (3.14)) (Table 1) in a bacterial membrane lipid bilayer model using a combination of "simultaneous" AFM and ATR-FTIR techniques. In a typical run, CAPs (8 M) were added to a lipid bilayer mixture composed of 3:1 POPE/DOPG, which resembles the phosphoethanolamine/phosphoglycerol ratio found in the inner membrane of Gram-positive and Gram-negative bacteria (26,27). The in situ AFM images revealed that direct fusion of the lipid vesicles onto mica resulted in a molecularly smooth intact surface with no or few defects (Fig.…”

Section: Caps In Peptide-membrane Interactionsmentioning

confidence: 99%

“…To examine the effects of the present CAPs on mammalian membranes, we performed in-tandem AFM and ATR-FTIR experiments on the four designed peptides with a mammalian membrane model (1:1:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol) that directly mimics the outer leaflet of eukaryotic cell membranes (27,33). Before peptide addition, fusion of this lipid mixture to mica resulted in DSPC/cholesterol-rich lipid-ordered domains surrounded by a DOPC-rich liquid-disordered phase, distinguishable by an ϳ2-nm height difference (Fig.…”

Section: Caps In Peptide-membrane Interactionsmentioning

confidence: 99%

Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions

Yin

Edwards

et al. 2012

Journal of Biological Chemistry

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339

230

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Background: Cationic antimicrobial peptides offer an alternative to conventional antibiotics, as they physically disrupt bacterial membranes, causing cell death. Results: Peptides designed with high hydrophobicity display strong self-association that is minimized by distribution of positive charges at both peptide termini. Conclusion: Balancing peptide hydrophobicity and charge distribution promotes efficient antimicrobial activity. Significance: Routes to optimization of peptide sequences are valuable for devising therapeutic strategies.

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Atomic Force Microscopy ( AFM )

Hyötylä

Lim

2012

Supramolecular Chemistry

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Atomic force microscopy (AFM) is a stylus‐based technique that uses sub‐nanonewton force sensitivity to image surfaces at sub‐nanometer resolution. As a surface‐sensitive tool, AFM is ideal for investigating the properties and local morphology of surface‐grafted supramolecules and (bio)polymers, which play important roles in mediating interfacial processes ranging from tribology to colloidal stability and biology. Moreover, the versatility of AFM to operate in diverse environments (i.e., air, liquid, or vacuum) adds to its appeal in comparison to other surface methods. Given its capability for high‐resolution imaging, force measurements, and nanomechanical manipulation, AFM is directly applicable to studies of stimuli‐responsive polymers, biomaterials, and polymeric nanostructures.

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Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM

Cited by 40 publications

References 116 publications

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane

Roles of Hydrophobicity and Charge Distribution of Cationic Antimicrobial Peptides in Peptide-Membrane Interactions

Atomic Force Microscopy ( AFM )

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