Plasma membrane asymmetry of lipid organization: fluorescence lifetime microscopy and correlation spectroscopy analysis.

A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.

Mitochondrial routing of glucose and sucrose polymers after pinocytotic uptake: avenues for drug delivery.

Mitochondria are key organelles organizing cellular metabolic flux. Therefore, a targeted drug delivery to mitochondria promises the advancement of medicine in fields that are associated with mitochondrial dysfunction. However, successful mitochondrial drug delivery is limited by complex transport steps across organelle membranes and fast drug efflux in cases of multidrug resistance. Strategies to deliver small-molecular-weight drugs to mitochondria are very limited, while the use of complex polymeric carriers is limited by a lack of clinical feasibility. We show here that clinically established macromolecules such as a sucrose copolymer (Ficoll 70/400 kDa) and polyglucose (dextran 70/500 kDa) are micropinocytosed swiftly by mesenchymal stem cells and subsequently routed to mitochondria. The intracellular level of Ficoll appears to decrease over time, suggesting that it does not persist within cells. After coupling to polysucrose, the low-molecular-weight photodynamic drug Rose Bengal reached mitochondria and thus exhibited an increased destructive potential after laser excitation. These findings support new opportunities to deliver already clinically approved drugs to mitochondria.

Interaction of an artificial antimicrobial peptide with lipid membranes.

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.

Study of interaction of hypericin and its pharmaceutical preparation by fluorescence techniques.

We report the detection of interactions between a photosensitizer, hypericin (HY), and its solvent system prepared with a formulation additive, polyvinylpyrrolidone (PVP), a commonly used pharmaceutical excipient. Fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) were used to study aggregation and binding of HY in the presence of PVP. Digitized fluorescence endoscopic imaging (DFEI) was used to study the effect of the pharmaceutical formulation in the in vivo tumor implanted chick chorioallantoic membrane (CAM) model. The results presented reveal the coordination of HY-PVP binding, HY disaggregation in the presence of PVP, and strengthened HY tumor uptake selectivity. PVP is thus suggested as a potential adjuvant to previously investigated N-methyl pyrrolidone (NMP) in the HY delivery system as well as a replacement for the conventionally used albumin in the HY bladder instillation fluids preparation for clinical use.

Synthetic sandwich culture of 3D hepatocyte monolayer.

The sandwich culture of hepatocytes, between double layers of extra-cellular matrix (ECM), is a well-established in vitro model for re-establishing hepatic polarity and maintaining differentiated functions. Applications of the ECM-based sandwich culture are limited by the mass transfer barriers induced by the top gelled ECM layer, complex molecular composition of ECM with batch-to-batch variation and uncontrollable coating of the ECM double layers. We have addressed these limitations of the ECM-based sandwich culture by developing an 'ECM-free' synthetic sandwich culture, which is constructed by sandwiching a 3D hepatocyte monolayer between a glycine-arginine-glycine-aspatic acid-serine (GRGDS)-modified polyethylene terephthalate (PET) track-etched membrane (top support) and a galactosylated PET film (bottom substratum). The bioactive top support and bottom substratum in the synthetic sandwich culture substituted for the functionalities of the ECM in the ECM-based sandwich culture with further improvement in mass transfer and optimal material properties. The 3D hepatocyte monolayer in the synthetic sandwich culture exhibited a similar process of hepatic polarity formation, better cell-cell interaction and improved differentiated functions over 14-day culture compared to the hepatocytes in collagen sandwich culture. The novel 3D hepatocyte monolayer sandwich culture using bioactive synthetic materials may readily replace the ECM-based sandwich culture for liver tissue engineering applications, such as drug metabolism/toxicity testing and hepatocyte-based bioreactors.

Molecular diffusion measurement in lipid bilayers over wide concentration ranges: a comparative study.

Molecular diffusion in biological membranes is a determining factor in cell signaling and cell function. In the past few decades, three main fluorescence spectroscopy techniques have emerged that are capable of measuring molecular diffusion in artificial and biological membranes at very different concentration ranges and spatial resolutions. The widely used methods of fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) can determine absolute diffusion coefficients at high (>100 microm(-2)) and very low surface concentrations (single-molecule level), respectively. Fluorescence correlation spectroscopy (FCS), on the other hand, is well-suited for the intermediate concentration range of about 0.1-100 microm(-2). However, FCS in general requires calibration with a standard dye of known diffusion coefficient, and yields only relative measurements with respect to the calibration. A variant of FCS, z-scan FCS, is calibration-free for membrane measurements, but requires several experiments at different well-controlled focusing positions. A recently established FCS method, electron-multiplying charge-coupled-device-based total internal reflection FCS (TIR-FCS), referred to here as imaging TIR-FCS (ITIR-FCS), is also independent of calibration standards, but to our knowledge no direct comparison between these different methods has been made. Herein, we seek to establish a comparison between FRAP, SPT, FCS, and ITIR-FCS by measuring the lateral diffusion coefficients in two model systems, namely, supported lipid bilayers and giant unilamellar vesicles.

Identification and characterization of a novel prespheroid 3-dimensional hepatocyte monolayer on galactosylated substratum.

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.

Novel use for polyvinylpyrrolidone as a macromolecular crowder for enhanced extracellular matrix deposition and cell proliferation.

Macromolecular crowding (MMC) is a biophysical effect that governs biochemical processes inside and outside of cells. Since standard cell culture media lack this effect, the physiological performance of differentiated and progenitor cells, including extracellular matrix (ECM) deposition, is impaired in vitro. To bring back physiological crowdedness to in vitro systems, we have previously introduced carbohydrate-based macromolecules to culture media and have achieved marked improvements with mixed MMC in terms of ECM deposition and differentiation of mesenchymal stem cells (MSCs). We show here that although this system is successful, it is limited, due to viscosity, to only 33% of the fractional volume occupancy (FVO) of full serum, which we calculated to have an FVO of approximately 54% v/v. We show here that full-serum FVO can be achieved using polyvinylpyrrolidone (PVP) 360 kDa. Under these conditions, ECM deposition in human fibroblasts and MSCs is on par, if not stronger than, with original MMC protocols using carbohydrates, but with a viscosity that is not significantly changed. In addition, we have found that the proliferation rate for bone marrow-derived MSCs and fibroblasts increases slightly in the presence of PVP360, similar to that observed with carbohydrate-based crowders. A palette of MMC compounds is now emerging that enables us to tune the crowdedness of culture media seamlessly from interstitial fluid (9% FVO), in which the majority of tissue cells might be based, to serum environments mimicking intravascular conditions. Despite identical FVO's, individual crowder size effects play a role and different cell types appear to have preferences in terms of FVO and the crowder that this is achieved with. However, in the quest of crowders that we have predicted to have a smoother regulatory approval path, PVP is a highly interesting compound, as it has been widely used in the medical and food industries and shows a novel promising use in cell culture and tissue engineering.

Phosphatidylserine dynamics in cellular membranes.

Much has been learned about the role of exofacial phosphatidylserine (PS) in apoptosis and blood clotting using annexin V. However, because annexins are impermeant and unable to bind PS at low calcium concentration, they are unsuitable for intracellular use. Thus little is known about the topology and dynamics of PS in the endomembranes of normal cells. We used two new probes-green fluorescent protein (GFP)-LactC2, a genetically encoded fluorescent PS biosensor, and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phospho-L-serine (TopFluor-PS), a synthetic fluorescent PS analogue-to examine PS distribution and dynamics inside live cells. The mobility of PS was assessed by a combination of advanced optical methods, including single-particle tracking and fluorescence correlation spectroscopy. Our results reveal the existence of a sizable fraction of PS with limited mobility, with cortical actin contributing to the confinement of PS in the plasma membrane. We were also able to measure the dynamics of PS in endomembrane organelles. By targeting GFP-LactC2 to the secretory pathway, we detected the presence of PS in the luminal leaflet of the endoplasmic reticulum. Our data provide new insights into properties of PS inside cells and suggest mechanisms to account for the subcellular distribution and function of this phospholipid.