Dynamics in Cytoplasm, Nucleus, and Lipid Droplet of a Live CHO Cell: Time-Resolved Confocal Microscopy

Different regions of a single live Chinese hamster ovary (CHO) cell are probed by time-resolved confocal microscopy. We used coumarin 153 (C153) as a probe. The dye localizes in the cytoplasm, nucleus, and lipid droplets, as is clearly revealed by the image. The fluorescence correlation spectroscopy (FCS) data shows that the microviscosity of lipid droplets is ∼34 ± 3 cP. The microviscosities of nucleus and cytoplasm are found to be 13 ± 1 and 14.5 ± 1 cP, respectively. The average solvation time (⟨τ_s⟩) in the lipid droplets (3600 ± 50 ps) is slower than that in the nucleus (⟨τ_s⟩ = 750 ± 50 ps) and cytoplasm (⟨τ_s⟩ = 1100 ± 50 ps). From the position of emission maxima of C153, the polarity of the nucleus is estimated to be similar to that of a mixture containing 26% DMSO in triacetin (η ∼ 11.2 cP, ε ∼ 26.2). The cytoplasm resembles a mixture of 18% DMSO in triacetin (η ∼ 12.6 cP, ε ∼ 21.9). The polarity of lipid droplets is less than that of pure triacetin (η ∼ 21.7 cP, ε ∼ 7.11)

Ultrafast FRET in Ionic Liquid-P123 Mixed Micelles: Region and Counterion Dependence

Ultrafast fluorescence resonance energy transfer (FRET) in a mixed micelle containing a room-temperature ionic liquid (RTIL) is studied by picosecond and femtosecond emission spectroscopy. The mixed micelle consists of a triblock copolymer, (PEO)20-(PPO)70-(PEO)20 (Pluronic P123), and a RTIL, 1-pentyl-3-methyl-imidazolium tetra-flouroborate, ([pmim][BF4]) or 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]). Coumarin 480 (C480) is used as the donor, and the acceptor is rhodamine 6G (R6G). Multiple time scales of FRET were detectedan ultrashort component of 1−3 ps and two relatively long components (300−400 ps and 2500−3500 ps). The different time scales are attributed to different donor−acceptor distances. It is proposed that the ionic acceptor (R6G) is localized in the polar corona region of the mixed micelle, while the neutral donor (C480) is distributed over both corona and hydrophobic core regions. The ultrafast (1−3 ps) components are assigned to FRET at a close contact of donor and acceptor. This occurs for the donor in the polar corona region in close proximity of the acceptor. The longer components (300−400 ps and 2500−3500 ps) arise from long-distance FRET from the donor at the core and the acceptor at the corona region. The relative contribution of the ultrafast component of FRET (∼3 ps) increases from 5% at λex = 375 nm to 30% at λex = 435 nm in the 0.3 M [pmim][BF4] mixed micelle and from 25 to 100% in the 0.9 M [pmim][BF4] mixed micelle. It is suggested that, at λex = 435 nm, mainly the donor molecules present at the corona are excited, causing ultrafast FRET due to a short donor−acceptor distance. At shorter λex, the donor (C480) molecule at the core regions is excited, giving rise to a very long 3400 ps component (RDA ∼ 50 Å). Thus, λex variation leads to excellent spatial resolution. The counterion dependence (Br− vs BF4−) is attributed to the difference in the local polarity and size of the two mixed micelles

ApoE: In Vitro Studies of a Small Molecule Effector

Apolipoprotein E4 (apoE4), one of three isoforms of apoE, is the major risk factor for developing late onset Alzheimer’s disease. The only differences among these isoforms (apoE2, apoE3, and apoE4) are single amino acid changes. Yet these proteins are functionally very different. One approach to ameliorating the effect of apoE4 with respect to Alzheimer’s disease would be to find small molecular weight compounds that affect the behavior of apoE4. Few studies of this approach have been carried out in part because there was no complete structure of any full-length apoE isoform until 2011. Here, we focus on one small molecular weight compound, EZ-482, and explore the effects of its binding to apoE. Using hydrogen–deuterium exchange, we determined that EZ-482 binds to the C-terminal domains of both apoE3 and apoE4. The binding to apoE4, however, is accompanied by a unique N-terminal allosteric effect. Using fluorescence methods, we determined an apparent dissociation constant of approximately 8 μM. Although EZ-482 binds to the C-terminal domain, it blocks heparin binding to the N-terminal domain. The residues of apoE that bind heparin are the same as those involved in apoE binding to LDL and LRP-1 receptors. The methods and the data presented here may serve as a template for future studies using small molecular weight compounds to modulate the behavior of apoE