Distribution of MOP and GPI.

<p><b>A.</b> Section of an untreated MDA-MB-468 cell (scale bar 2 µm) and <b>B.</b> Magnified area (scale bar 500 nm) showing distribution of antibody detected MOP (in red) and paGFP-GPI (in green). Centers of peaks are shown. PALM images showing distribution of antibody detected MOP (no expression of paGFP-GPI) are given in Figure S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087225#pone.0087225.s001" target="_blank">File S1</a>. <b>C.</b> The cross-correlation curve indicates that MOP and GPI show partial co-localization in the steady state (gray squares, s.e.m, n = 23), suggesting that a fraction of MOP likely resides in GPI-enriched lipid rafts.</p

Elemental XPS spectra of beam protected (unexposed) and irradiated (exposed at different doses) PEG before and after protein incubation.

<p>Before (“unexp”) and after (“exp”) helium beam exposure, carbon C1s signals (A) show characteristic alkyl and ether peaks at 284.6 eV and 286.6 eV binding energies, respectively. The presence of oxygen O1s signals (B) at 532 eV and the absence of nitrogen N1s signals (C) at 400 eV also were observed. Subsequent incubation with avidin shows additional C1s peak at 288 eV (D), similar O1s signals at 532 eV (E) and existence of N1s peak at 400 eV (F) binding energies for beam exposed PEG.</p

A schematic of helium beam-induced protein patterning on PEG.

<p>Panels A and B show the electron images of 35 µm mesh and 300 nm masks, respectively. Top-down helium beam exposure through a mask (C) of grafted PEG on surface allowed proteins preferentially to attach on irradiated regions (D) to form patterns.</p

Differences in the distribution of paGFP-GPI and pa-mCherry1-actin, upon ethanol addition can be quantified using pcPALM.

<p><b>A.</b> Section of a MDA-MB-468 cell coexpressing paGFP-GPI (green) and pa-mCherry1-actin (red) in the absence of ethanol (lined in gray) and upon addition of 40 mM ethanol (lined in russet). Centers of peaks are shown. Scale bars 1 μm. <b>B.</b> The cross-correlation curve indicates that actin and GPI were uncorrelated in the steady state (gray squares, s.e.m, n = 24, c(r) ∼1). However, c(r) increased following ethanol addition (russet diamonds, s.e.m, n = 27) indicating partial spatial co-localization between actin and GPI.</p

Nanoscale Effects of Ethanol and Naltrexone on Protein Organization in the Plasma Membrane Studied by Photoactivated Localization Microscopy (PALM)

<div><p>Background</p><p>Ethanol affects the signaling of several important neurotransmitter and neuromodulator systems in the CNS. It has been recently proposed that ethanol alters the dynamic lateral organization of proteins and lipids in the plasma membrane, thereby affecting surface receptor-mediated cellular signaling. Our aims are to establish whether pharmacologically relevant levels of ethanol can affect the lateral organization of plasma membrane and cytoskeletal proteins at the nanoscopic level, and investigate the relevance of such perturbations for mu-opioid receptor (MOP) function.</p><p>Methodology/Principal Findings</p><p>We used Photoactivated Localization Microscopy with pair-correlation analysis (pcPALM), a quantitative fluorescence imaging technique with high spatial resolution (15–25 nm) and single-molecule sensitivity, to study ethanol effects on protein organization in the plasma membrane. We observed that short (20 min) exposure to 20 and 40 mM ethanol alters protein organization in the plasma membrane of cells that harbor endogenous MOPs, causing a rearrangement of the lipid raft marker glycosylphosphatidylinositol (GPI). These effects could be largely occluded by pretreating the cells with the MOP antagonist naltrexone (200 nM for 3 hours). In addition, ethanol induced pronounced actin polymerization, leading to its partial co-localization with GPI.</p><p>Conclusions/Significance</p><p>Pharmacologically relevant levels of ethanol alter the lateral organization of GPI-linked proteins and induce actin cytoskeleton reorganization. Pretreatment with the MOP antagonist naltrexone is protective against ethanol action and significantly reduces the extent to which ethanol remodels the lateral organization of lipid-rafts-associated proteins in the plasma membrane. Super-resolution pcPALM reveals details of ethanol action at the nanoscale level, giving new mechanistic insight on the cellular and molecular mechanisms of its action.</p></div

Gold nanoparticle probes and silver nanoparticle deposition on protein-PEG patterns (300 nm mask).

<p>Electron microscope images on the left column (with zoom-in pictures as insets) correspond to helium beam-patterned PEG incubated with polyclonal anti-mouse antibodies (A), streptavidin-polyHRP conjugates (B), or biotinylated lysozyme (C), while images on the right display beam-patterned PEG surfaces incubated with PBS buffer (no proteins); all samples were then passivated with BSA. Patterns were visualized by binding of 40 nm gold nanoparticles conjugated with D1.3 mouse antibodies (A), or silver staining through HRP conjugates (B). Captured biotinylated lysozyme (C) was detected by addition of streptavidin-polyHRP conjugates and silver staining.</p

ATR-FTIR spectra of beam protected (unexposed) and irradiated (exposed) PEG at different helium beam doses.

<p>Spectra of grafted PEG subjected to different beam doses and incubated in PBS for 1 hour show the characteristic ether and methoxy peaks at 1120 cm⁻¹ and 2870 cm⁻¹, respectively, which also are present in unexposed PEG.</p

Effect of ethanol and naltrexone on GPI distribution.

<p><b>A.</b> PALM image of the whole area (left, scale bar 5 µm) and a magnified area (right, scale bar 200 nm) showing GPI distribution in untreated MDA-MB-468 cells (control). <b>B.</b> PALM images of the whole area (left, scale bar 5 µm) and a magnified area (right, scale bar 200 nm) showing GPI distribution in MDA-MB-468 cells incubated with 40 mM ethanol for 20 min. Super-resolution images were generated by analyzing dataset using a standard PALM analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087225#pone.0087225-Betzig1" target="_blank">[25]</a>; peaks were grouped using maximum blinking time of 10 s for paGFP and group radius of 2.5 σ. Centers of peaks for panels A and B are given in Figure S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087225#pone.0087225.s001" target="_blank">File S1</a> and Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087225#pone.0087225.s001" target="_blank">File S1</a>. <b>C.</b> Average protein auto-correlation functions in untreated cells (gray, n = 31), after 20 min incubation in the presence of 20 mM ethanol (pink, n = 36), after 20 min incubation in the presence of 40 mM ethanol (russet, n = 36), upon 20 min incubation with 200 nM naltrexone (cyan, n = 33) and upon 3 h preincubation with 200 nM naltrexone followed subsequently by a 20 min incubation in the presence of 40 mM ethanol (black, n = 34). Insert shows magnified area at shorter radii with s.e.m. <b>D.</b> Increased local density of paGFP-GPI with s.e.m. Ethanol addition significantly decreased local density of GPI compared to that in untreated cells. <b>E.</b> Distribution of paGFP-GPI molecules per cluster (left) and cluster radius (right). Ethanol addition had significant increase in number of detected proteins per cluster and cluster radius compared to steady state, whereas no significant change is observed for other perturbation conditions.</p

Gold nanoparticle probes on protein-PEG patterns (35 µm mesh).

<p>Images on the left column show the schematic diagram (not to scale) of helium beam-patterned PEG incubated with avidin (A), polyclonal anti-rabbit antibodies (B), or BSA (C), followed by the addition of 100 nm gold nanoparticles conjugated with biotinylated rabbit antibodies. Electron microscope images on the right column show the protected and irradiated PEG regions after incubation with gold nanoparticle probes.</p

Formation of diformazan precipitates on protein-PEG patterns (35 µm mesh).

<p>Top row images show a schematic diagram (not to scale) of helium beam- patterned PEG incubated with avidin (A) or BSA (D) followed by addition of biotinylated alkaline phosphatase (AP) and substrate BCIP-NBT. Optical microscopic images below the diagram show the protected (light) and irradiated (dark) regions which display the specific patterned capture of biotinylated AP enzymes for avidin-incubated surface (B) and no pattern for BSA-incubated surface (E). Panels C and F show higher magnifications of images in B and E, respectively.</p