Changes in Cholesterol Level Alter Integrin Sequestration in Raft-Mimicking Lipid Mixtures.

The influence of cholesterol (CHOL) level on integrin sequestration in raft-mimicking lipid mixtures forming coexisting liquid-ordered (lo) and liquid-disordered (ld) lipid domains is investigated using complementary, single-molecule-sensitive, confocal detection methods. Systematic analysis of membrane protein distribution in such a model membrane environment demonstrates that variation of CHOL level has a profound influence on lo-ld sequestration of integrins, thereby exhibiting overall ld preference in the absence of ligands and lo affinity upon vitronectin addition. Accompanying photon-counting histogram analysis of integrins in the different model membrane mixtures shows that the observed changes of integrin sequestration in response to variations of membrane CHOL level are not associated with altering integrin oligomerization states. Instead, our experiments suggest that the strong CHOL dependence of integrin sequestration can be attributed to CHOL-mediated changes of lipid packing and bilayer thickness in coexisting lo and ld domains, highlighting the significance of a biophysical mechanism of CHOL-mediated regulation of integrin sequestration. We envision that this model membrane study may help clarify the influence of CHOL in integrin functionality in plasma membranes, thus providing further insight into the role of lipid heterogeneities in membrane protein distribution and function in a cellular membrane environment.

NMR Spectra of Glycine Isotopomers in Anisotropic Media: Subtle Chiral Interactions.

NMR spectra of deuterated glycine-2-(13)C revealed interactions between chiral anisotropic gelatin and κ-carrageenan gels and the prochiral and chiral isotopomers. The (1)H, (2)H and (13)C NMR spectra of mixtures of racemic mono- and prochiral bis-deuterated glycine-2-(13)C were resolved and well simulated using distinct dipolar coupling constants DCαH and DCαD for the enantiomers and also for the -(13)CαD2- group (DC,DA, and DC,DB). The orientation of the proton or deuteron on the (13)Cα-atom of glycine was assigned by analogy with alanine and lactate assuming that the molecular orientation of glycine isotopomers is the same. The assignment of the prochiral sites was derived from chiral analogues.

Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.

Polymer-tethered lipid multi-bilayers: a biomembrane-mimicking cell substrate to probe cellular mechano-sensing.

Cells tiptoe through their environment forming highly localized and dynamic focal contacts. Experiments on polymeric gels of adjustable elasticity have shown that cells probe the viscoelasticity of their environment through an adaptive process of focal contact assembly/disassembly that critically affects cell adhesion, morphology, and motility. However, the specific mechanisms of this process have not yet been fully revealed. Here we report, for the first time, that fibroblast adhesion, morphology, and migration can also be controlled by altering the number of bilayers in a stack of multiple polymer-tethered lipid bilayers stabilized via maleimide-sulfhydral coupling chemistry. The observed changes in cell morphology, migration, and cytoskeletal organization in response to bilayer stacking correspond well with those previously observed on polymeric substrates of different polymer crosslinking density suggesting that variations in bilayer stacking are associated with changes in substrate viscoelasticity. This is in conceptual agreement with the existing knowledge about the structural, dynamic, and mechanical properties of polymer-lipid composite materials. Several distinct features, such as the lateral mobility of individual cell linkers and the immobilization of linker clusters, make the described substrates highly attractive tools for the study of dynamic, mechano-regulated cell linkages and cellular mechano-sensing.

Bilayer asymmetry influences integrin sequestering in raft-mimicking lipid mixtures.

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of αvß3 and α5ß1 integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase separations demonstrates that bilayer asymmetry has a profound influence on αvß3 and α5ß1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asymmetric bilayers, but to the ld phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.

Matrix-dependent modulation of anisotropic effects on NMR spectra from 7Li+ and 23Na+ encapsulated in cryptands.

(7)Li and (23)Na NMR spectra of the respective cations in gelatin and ι-carrageenan gels containing cryptand-[2.1.1] (for Li(+)) or cryptand-[2.2.2] (for Na(+)) displayed two transitions: the one at higher frequency corresponded to the cation surrounded by gel, the other to cation inside its appropriately sized cryptand. While binding to cryptands yielded much broader lines and shorter T (1) relaxation times, anisotropic splitting in first order (7)Li or (23)Na NMR spectra was not detected. Stretching the gels resulted in increasing the anisotropic electric field gradient tensor; thus, the NMR transitions of the cation in the gel were split (removal of degeneracy) to display its characteristic 3:4:3 triplet for spin = 3/2 nuclei. The transitions of the cryptand-bound cations (Li(+)-cryptand-[2.1.1] and Na(+)-cryptand-[2.2.2]) showed different extents of interaction with the electric field gradient tensor depending on the composition of the gel matrix. The NMR signal for (7)Li(+)-cryptand-[2.1.1] in stretched gelatin gel showed a five-fold increased splitting as compared to the (7)Li(+) signal in the reference gel. In stretched ι-carrageenan gels, no anisotropic splitting from the cryptand-bound Li(+) was recorded. Steady-state irradiation envelopes or z-spectra showed evidence of Li(+) exchange between isotropic (cryptand) and anisotropic (gel) sites only at higher temperatures (55 °C). For Na(+) bound to the cryptand-[2.2.2], anisotropic splitting (three-fold smaller compared with the (23)Na signal in the reference gel) was only recorded in stretched ι-carrageenan gels, whereas gelatin gels showed only anisotropic splitting for the (23)Na signal in the reference gel.

Iterative layer-by-layer assembly of polymer-tethered multi-bilayers using maleimide­thiol coupling chemistry.

The current study reports on the layer-by-layer assembly of a polymer-tethered lipid multi-bilayer stack using the iterative addition and roll out of giant unilamellar vesicles (GUVs) containing constituents with thiol and maleimide functional groups, respectively. Confocal microscopy and photobleaching experiments confirm stack integrity and stability over time, as well as the lateral fluidity of individual bilayers within the stacks. Complementary wide-field single molecule fluorescence microscopy and atomic force microscopy experiments show that increasing bilayer-substrate distances are associated with changes in lipid lateral mobility and bilayer morphology. Importantly, the described iterative approach can be employed to assemble multi-bilayer stacks with more than two bilayers, thus further reducing the influence of the underlying solid substrate on membrane behavior. Furthermore, the presence of lipopolymers within the multi-bilayer stacks results in fascinating membrane dynamics and organization properties, with interesting parallels to those found in plasma membranes. In that sense, the described multi-bilayer architecture represents an attractive model membrane platform for a variety of different biophysical studies.

Native ligands change integrin sequestering but not oligomerization in raft-mimicking lipid mixtures.

Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (α(v)ß(3) and α(5)ß(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for α(v)ß(3) and α(5)ß(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both α(v)ß(3) and α(5)ß(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, α(v)ß(3) sequesters strongly into raft-like domains and α(5)ß(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.

Hopanoids, like sterols, modulate dynamics, compaction, phase segregation and permeability of membranes.

In recent years, hopanoids, a group of pentacyclic compounds found in bacterial membranes, are in the spotlight since it was proposed that they induce order in lipid membranes in a similar way cholesterol do in eukaryotes, despite their structural differences. We studied here whether diplopterol (an abundant hopanoid) promoted similar effects on model membranes as sterols do. We analyzed the compaction, dynamics, phase segregation, permeability and compressibility of model membranes containing diplopterol, and compared with those containing sterols from animals, plants and fungi. We also tested the effect that the incubation with diplopterol had on hopanoid-lacking bacteria. Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes, while retaining membrane fluidity and compressibility. Furthermore, the exposition to this hopanoid decreases the permeability of the opportunistic pathogen Pseudomonas aeruginosa and increases the resistance to antibiotics. All effects promoted by diplopterol were similar to those generated by the sterols. Our observations add information on the functional significance of hopanoids as molecules that play an important role in membrane organization and dynamics in model membranes and in a bacterial system.

Design of quantum dot-conjugated lipids for long-term, high-speed tracking experiments on cell surfaces.

The current study reports the facile design of quantum dot (QD)-conjugated lipids and their application to high-speed tracking experiments on cell surfaces. CdSe/ZnS core/shell QDs with two types of hydrophilic coatings, 2-(2-aminoethoxy)ethanol (AEE) and a 60:40 molar mixture of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine and 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine- N-[methoxy(polyethylene glycol-2000], are conjugated to sulfhydryl lipids via maleimide reactive groups on the QD surface. Prior to lipid conjugation, the colloidal stability of both types of coated QDs in aqueous solution is confirmed using fluorescence correlation spectroscopy. A sensitive assay based on single lipid tracking experiments on a planar solid-supported phospholipid bilayer is presented that establishes conditions of monovalent conjugation of QDs to lipids. The QD-lipids are then employed as single-molecule tracking probes in plasma membranes of several cell types. Initial tracking experiments at a frame rate of 30 frames/s corroborate that QD-lipids diffuse like dye-labeled lipids in the plasma membrane of COS-7, HEK-293, 3T3, and NRK cells, thus confirming monovalent labeling. Finally, QD-lipids are applied for the first time to high-speed single-molecule imaging by tracking their lateral mobility in the plasma membrane of NRK fibroblasts with up to 1000 frames/s. Our high-speed tracking data, which are in excellent agreement with previous tracking experiments that used larger (40 nm) Au labels, not only push the time resolution in long-time, continuous fluorescence-based single-molecule tracking but also show that highly photostable, photoluminescent nanoprobes of 10 nm size can be employed (AEE-coated QDs). These probes are also attractive because, unlike Au nanoparticles, they facilitate complex multicolor experiments.

2H2O quadrupolar splitting used to measure water exchange in erythrocytes.

The 2H NMR resonance from HDO (D=2H) in human red blood cells (RBCs) suspended in gelatin that was held stretched in a special apparatus was distinct from the two signals that were symmetrically arranged on either side of it, which were assigned to extracellular HDO. The large extracellular splitting is due to the interaction of the electric quadrupole moment of the 2H nuclei with the electric field gradient tensor of the stretched, partially aligned gelatin. Lack of resolved splitting of the intracellular resonance indicated greatly diminished or absent ordering of the HDO inside RBCs. The separate resonances enabled the application of a saturation transfer method to estimate the rate constants of transmembrane exchange of water in RBCs. However both the theory and the practical applications needed modifications because even in the absence of RBCs the HDO resonances were maximally suppressed when the saturating radio-frequency radiation was applied exactly at the central frequency between the two resonances of the quadrupolar HDO doublet. More statistically robust estimates of the exchange rate constants were obtained by applying two-dimensional exchange spectroscopy (2D EXSY), with back-transformation analysis. A monotonic dependence of the estimates of the efflux rate constants on the mixing time, tmix, used in the 2D EXSY experiment were seen. Extrapolation to tmix=0, gave an estimate of the efflux rate constant at 15 degrees C of 31.5+/-2.2 s(-1) while at 25 degrees C it was approximately 50 s(-1). These values are close to, but less than, those estimated by an NMR relaxation-enhancement method that uses Mn2+ doping of the extracellular medium. The basis for this difference is thought to include the high viscosity of the extracellular gel. At the abstract level of quantum mechanics we have used the quadrupolar Hamiltonian to provide chemical shift separation between signals from spin populations across cell membranes; this is the first time, to our knowledge, that this has been achieved.

Prochiral and chiral resolution in 2H NMR spectra: solutes in stretched and compressed gelatin gels.

We demonstrate prochiral and chiral spectral resolution using residual (2)H NMR quadrupolar splittings over a wide range of anisotropic conditions in liquid samples. We use a reversible gel-stretching/compressing device in a conventional high-field NMR spectrometer. We show the stability of gelatin gels as well as their unique ability to switch between multiple stretched and compressed states, thus also changing the sign of residual dipolar couplings in (1)H and (13)C NMR. This flexibility will be important for resolving spectra of mixtures of other chiral compounds and for structure determination of selected peptides.

Fluorescence correlation spectroscopy of CdSe/ZnS quantum dot optical bioimaging probes with ultra-thin biocompatible coatings.

The current study reports on the colloidal stabilities and emission properties of CdSe/ZnS quantum dot (QD) optical probes capped with a variety of thin, hydrophilic surface coatings as studied using confocal fluorescence correlation spectroscopy. These coatings are based on mercaptoethanol, mercaptopropionic acid (with and without conjugated aminoethoxyethanol), lipopolymers (DSPE-PEG2000), cysteine (Cys), and a variety of Xaa-Cys dipeptides. The study shows that several types of QDs with thin hydrophilic coatings can be designed that combine good colloidal stability and excellent emission properties (brightness). Furthermore, there is a general correlation between colloidal stability and brightness. The experiments reported herein illustrate that QDs with multiple types of thin coatings can be created for optical imaging applications in a biological environment while also maintaining a size below 10 nm.

Tunable cell-surface mimetics as engineered cell substrates.

Most recent breakthroughs in understanding cell adhesion, cell migration, and cellular mechanosensitivity have been made possible by the development of engineered cell substrates of well-defined surface properties. Traditionally, these substrates mimic the extracellular matrix (ECM) environment by the use of ligand-functionalized polymeric gels of adjustable stiffness. However, such ECM mimetics are limited in their ability to replicate the rich dynamics found at cell-cell contacts. This review focuses on the application of cell surface mimetics, which are better suited for the analysis of cell adhesion, cell migration, and cellular mechanosensitivity across cell-cell interfaces. Functionalized supported lipid bilayer systems were first introduced as biomembrane-mimicking substrates to study processes of adhesion maturation during adhesion of functionalized vesicles (cell-free assay) and plated cells. However, while able to capture adhesion processes, the fluid lipid bilayer of such a relatively simple planar model membrane prevents adhering cells from transducing contractile forces to the underlying solid, making studies of cell migration and cellular mechanosensitivity largely impractical. Therefore, the main focus of this review is on polymer-tethered lipid bilayer architectures as biomembrane-mimicking cell substrate. Unlike supported lipid bilayers, these polymer-lipid composite materials enable the free assembly of linkers into linker clusters at cellular contacts without hindering cell spreading and migration and allow the controlled regulation of mechanical properties, enabling studies of cellular mechanosensitivity. The various polymer-tethered lipid bilayer architectures and their complementary properties as cell substrates are discussed.

Single-molecule fluorescence microscopy to determine phospholipid lateral diffusion.

The single-molecule detection (SMD) of individual fluorophores represents a powerful experimental technique that allows for the observation of individual membrane molecules in their different dynamic states without having to average over a large number of molecules. Spatial resolution in ensemble-averaging techniques such as fluorescence recovery after photobleaching, is limited by the diffraction limit of light ( approximately 250 nm). In contrast, SMD (as well as single-molecule tracking of gold-labeled biomolecules through Nanovid microscopy) provides a tracking accuracy of approx 10-30 nm (dependent on experimental conditions). This level of accuracy makes single-molecule tracking techniques much better suited to detect nanometer-size heterogeneous structures in membranes. SMD can be easily applied to model and cellular membranes using a variety of fluorescent labels including organic dyes, quantum dots, and dye/quantum dots-doped nanoparticles. The main focus of this chapter is to outline the SMD methodology to study lateral diffusion of lipids in model membranes. Subheading 1. provides an overview about the development of single-molecule tracking techniques, and explains the basic concept of single-molecule tracking. Subheading 2. lists all relevant chemicals necessary to successfully conduct lipid lateral diffusion studies on model membranes. Subheading 3. describes a typical experimental setup for SMD using wide-field illumination; thus, this setup can be utilized to track single-lipid tracers in solid-supported phospholipid bilayers and phospholipid monolayers at the air-water interface. Furthermore, some general considerations are included about different fluorescent labels for lipid-tracking studies. In addition a description of sample preparation procedures for the design of solid-supported phospholipid bilayers and Langmuir monolayers of phospholipids are described. Finally, Subheading 4. lists additional relevant information helpful for conducting SMD experiments on lipid membranes.

Lipopolymers from new 2-substituted-2-oxazolines for artificial cell membrane constructs.

We present the synthesis of novel 2-oxazoline monomers with different 2-substituents and their consecutive conversion into lipopolymers by living cationic polymerization. The side functions of these monomers were varied to realize different steric needs and hydrogen bonding interactions of the polymer side chains. 2-(2'-N-pyrrolidonyl-ethyl)-2-oxazoline, 2-(3'-methoxymonoethyleneglycol)propyl-2-oxazoline, and 2-(3'-methoxytriethyleneglycol)propyl-2-oxazoline were synthesized. All of the monomers could be converted into the corresponding lipopolymers by living cationic polymerization using 2,3-di-O-octadecyl-1-trifluormethansulfonyl-sn-glycerol as the initiator. The characterization of the 2,3-di-O-octadecyl-glycerol-poly(2-oxazoline) lipopolymers by NMR spectroscopy, IR spectroscopy, and gel permeation chromatography revealed that the targeted molar masses and compositions can be controlled by the initial initiator/monomer ([M](0)/[I](0)) ratio for all the synthesized lipopolymers. The polydispersities were found to be narrow (polydispersity indices from 1.06-1.3). The amphiphilic lipopolymers were spread at the air-water interface (Langmuir-Blodgett film balance) and the effect of the polymer side groups and chain lengths upon the Pi-area (A) isotherms of the corresponding lipopolymer monolayers were compared and analyzed. The impact of the polymer side functionalities on a 2D gel formation was examined using an interfacial rheometer operated in an oscillating stress-strain mode. Interestingly enough, none of the newly synthesized lipopolymers showed a rheological transition. This somewhat surprising result not only verified that these 2D gels are not established by hydrogen bonding among hydrophilic polymer moieties, as earlier proposed, but also supported the concept of jammed surface micelles as the more likely origin for the gelation phenomenon. [Diagram: see text]

Which hospitalisations are ambulatory care-sensitive, to what degree, and how could the rates be reduced? Results of a group consensus study in Germany.

BACKGROUND: Much has been written lately regarding hospitalisations for ambulatory care-sensitive conditions (ACSH) and their strengths and weaknesses as a quality management indicator. The idea underlying ambulatory care-sensitive conditions (ACSC) is that effective treatment of acute conditions, good management of chronic illnesses and immunisation against infectious diseases can reduce the risk of a specified set of hospitalisations. METHODS: The present paper applies group consensus methods to synthesise available evidence with expert opinion, thus identifying relevant ACSC. It contributes to the literature by evaluating the degree of preventability of ACSH and surveying the medical and systemic changes needed to increase quality for each diagnosis group. Forty physicians proportionally selected from all medical disciplines relevant to the treatment of ACSC participated in the three round Delphi survey. The setting of the study is Germany. RESULTS: The proposed core list is a subset of 22 ACSC diagnosis groups, covering 90% of all consented ACSH and conditions with a higher than 85% estimated degree of preventability. Of all 18.6 million German hospital cases in the year 2012, the panelists considered 5.04 million hospitalisations (27%) to be sensitive to ambulatory care, of which 3.72 (20%) were estimated to be actually preventable. If only emergencies are considered, the ACSH share reduces to less than 8%. The geographic distribution of ACSH indicates significant regional variation with particularly high rates and potential for improvement in the North Rhine region, in Thuringia, Saxony-Anhalt, northern and eastern Bavaria and the Saarland. The average degree of preventability was 75% across all diagnosis groups. By far the most often mentioned strategy for reducing ACSH was 'improving continuous care'. CONCLUSION: There are several good reasons why process indicators prevail in the assessment of ambulatory care. ACSH rates can however provide a more complete picture by adding useful information related to the overall patient outcome. The results of our analysis should be used to encourage debate and as a basis for further confirmatory work.

(1)H NMR z-spectra of acetate methyl in stretched hydrogels: quantum-mechanical description and Markov chain Monte Carlo relaxation-parameter estimation.

The (1)H NMR signal of the methyl group of sodium acetate is shown to be a triplet in the anisotropic environment of stretched gelatin gel. The multiplet structure of the signal is due to the intra-methyl residual dipolar couplings. The relaxation properties of the spin system were probed by recording steady-state irradiation envelopes ('z-spectra'). A quantum-mechanical model based on irreducible spherical tensors formed by the three magnetically equivalent spins of the methyl group was used to simulate and fit experimental z-spectra. The multiple parameter values of the relaxation model were estimated by using a Bayesian-based Markov chain Monte Carlo algorithm.

NMR of (133)Cs(+) in stretched hydrogels: One-dimensional, z- and NOESY spectra, and probing the ion's environment in erythrocytes.

(133)Cs nuclear magnetic resonance (NMR) spectroscopy was conducted on (133)Cs(+) in gelatin hydrogels that were either relaxed or stretched. Stretching generated a septet from this spin-7/2 nucleus, and its nuclear magnetic relaxation was studied via z-spectra, and two-dimensional nuclear Overhauser (NOESY) spectroscopy. Various spectral features were well simulated by using Mathematica and the software package SpinDynamica. Spectra of CsCl in suspensions of human erythrocytes embedded in gelatin gel showed separation of the resonances from the cation inside and outside the cells. Upon stretching the sample, the extracellular (133)Cs(+) signal split into a septet, while the intracellular peak was unchanged, revealing different alignment/ordering properties of the environment inside and around the cells. Differential interference contrast light microscopy confirmed that the cells were stretched when the overall sample was elongated. Analysis of the various spectral features of (133)Cs(+) reported here opens up applications of this K(+) congener for studies of cation-handling by metabolically-active cells and tissues in aligned states.