Hydrodynamic shear dissipation and transmission in lipid bilayers.

Vital biological processes, such as trafficking, sensing, and motility, are facilitated by cellular lipid membranes, which interact mechanically with surrounding fluids. Such lipid membranes are only a few nanometers thick and composed of a liquid crystalline structure known as the lipid bilayer. Here, we introduce an active, noncontact, two-point microrheology technique combining multiple optical tweezers probes with planar freestanding lipid bilayers accessible on both sides. We use the method to quantify both fluid slip close to the bilayer surface and transmission of fluid flow across the structure, and we use numerical simulations to determine the monolayer viscosity and the intermonolayer friction. We find that these physical properties are highly dependent on the molecular structure of the lipids in the bilayer. We compare ordered-phase with liquid disordered-phase lipid bilayers, and we find the ordered-phase bilayers to be 10 to 100 times more viscous but with 100 times less intermonolayer friction. When a local shear is applied by the optical tweezers, the ultralow intermonolayer friction results in full slip of the two leaflets relative to each other and as a consequence, no shear transmission across the membrane. Our study sheds light on the physical principles governing the transfer of shear forces by and through lipid membranes, which underpin cell behavior and homeostasis.

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry.

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels.

We report a simple, cost-effective, and reproducible method to form free-standing lipid bilayer membranes in microdevices made with Norland Optical Adhesive 81 (NOA81). Surface treatment with either alkylsilane or fluoroalkylsilane enables the self-assembly of stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC) membranes. Capacitance measurements are used to characterize the lipid bilayer and to follow its formation in real-time. With current recordings, we detect the insertion of single α-hemolysin pores into the bilayer membrane, demonstrating the possibility of using this device for single-channel electrophysiology sensing applications. Optical transparency of the device and vertical position of the lipid bilayer with respect to the microscope focal plane allows easy integration with other single-molecule techniques, such as optical tweezers. Therefore, this method to form long-lived lipid bilayers finds a wide range of applications, from sensing measurements to biophysical studies of lipid bilayers and associated proteins.

Growth, Distribution, and Photosynthesis of Chlamydomonas Reinhardtii in 3D Hydrogels.

Engineered living materials (ELMs) are a novel class of functional materials that typically feature spatial confinement of living components within an inert polymer matrix to recreate biological functions. Understanding the growth and spatial configuration of cellular populations within a matrix is crucial to predicting and improving their responsive potential and functionality. Here, this work investigates the growth, spatial distribution, and photosynthetic productivity of eukaryotic microalga Chlamydomonas reinhardtii (C. reinhardtii) in three-dimensionally shaped hydrogels in dependence of geometry and size. The embedded C. reinhardtii cells photosynthesize and form confined cell clusters, which grow faster when located close to the ELM periphery due to favorable gas exchange and light conditions. Taking advantage of location-specific growth patterns, this work successfully designs and prints photosynthetic ELMs with increased CO2 capturing rate, featuring high surface to volume ratio. This strategy to control cell growth for higher productivity of ELMs resembles the already established adaptations found in multicellular plant leaves.

Artificial Cell Membranes Interfaced with Optical Tweezers: A Versatile Microfluidics Platform for Nanomanipulation and Mechanical Characterization.

Cell lipid membranes are the site of vital biological processes, such as motility, trafficking, and sensing, many of which involve mechanical forces. Elucidating the interplay between such bioprocesses and mechanical forces requires the use of tools that apply and measure piconewton-level forces, e.g., optical tweezers. Here, we introduce the combination of optical tweezers with free-standing lipid bilayers, which are fully accessible on both sides of the membrane. In the vicinity of the lipid bilayer, optical trapping would normally be impossible due to optical distortions caused by pockets of the solvent trapped within the membrane. We solve this by drastically reducing the size of these pockets via tuning of the solvent and flow cell material. In the resulting flow cells, lipid nanotubes are straightforwardly pushed or pulled and reach lengths above half a millimeter. Moreover, the controlled pushing of a lipid nanotube with an optically trapped bead provides an accurate and direct measurement of important mechanical properties. In particular, we measure the membrane tension of a free-standing membrane composed of a mixture of dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) to be 4.6 × 10-6 N/m. We demonstrate the potential of the platform for biophysical studies by inserting the cell-penetrating trans-activator of transcription (TAT) peptide in the lipid membrane. The interactions between the TAT peptide and the membrane are found to decrease the value of the membrane tension to 2.1 × 10-6 N/m. This method is also fully compatible with electrophysiological measurements and presents new possibilities for the study of membrane mechanics and the creation of artificial lipid tube networks of great importance in intra- and intercellular communication.

Total body water measurement by a modification of the bioimpedance spectroscopy method.

We propose a method for calculating directly total body water (TBW) volumes (V (t)) from whole body resistance extrapolated at infinite frequency (R (infinity)) using a XITRON 4200 impedance meter. Mean TBW resistivities for men and women were determined from measurements of R (infinity) and fat-free mass (FFM(d)) measured by DXA in 58 healthy subjects assuming an average hydration coefficient of 73.2%. Mean differences between V (t) measured by our new method and those deduced from DXA data were +0.11 +/- 1.61 L for women and +0.13 +/- 2.16 L for men. For validation, this method was tested with the same resistivities against a 2nd group of 16 volunteers and the mean difference between V (t) from impedance and DXA was -0.80 +/- 1.43 L. Since the resistance at 50 kHz (R (50)) was found to be equal, in average, to 1.230 R (infinity) for men and 1.223 R (infinity) for women, this method can also be applied at 50 kHz with a similar accuracy by estimating R (infinity) from R (50). When our new method was applied to the monitoring of water loss during 28 dialysis runs performed on 13 patients, it predicted a mean water loss equal to 94% of ultrafiltered volume.

Evaluation of a foot-to-foot impedance meter measuring extracellular fluid volume in addition to fat-free mass and fat tissue mass.

OBJECTIVES: The first objective was to compare the accuracy of a foot-to-foot impedance meter with a multifrequency bioimpedance for measurements of fat-free mass (FFM) and fat mass (FM) using dual energy X-ray absorptiometry (DXA) as reference. The second objective was to validate measurements of extracellular water resistance and volume by the foot-to-foot impedance meter, using multifrequency bioimpedance as reference. METHODS: This investigation was carried out in 60 volunteers 18 to 71 y of age. Impedance meters were a Tefal Bodymaster Vision (foot-to-foot) that featured a square wave signal and a Xitron Hydra 4200 (5 to 1000 kHz) by using the bioimpedance spectroscopic method. RESULTS: Bland-Altman tests showed that FFM differences between Tefal and DXA data were 1.98 +/- 3.09 kg in men and -0.08+/-2.98 kg in women. Total body water was measured by the Xitron, and FFM as measured with the Xitron was calculated as total body water divided by 0.732. Mean differences between Xitron-measured and DXA-measured FFM were 2.37+/-3.03 kg for men and 2.84+/-2.40 kg for women, indicating a systematic underestimation by the Xitron of intracellular volume. Extracellular water resistances measured by Tefal were in good agreement with those measured by Xitron with electrodes pasted under the subject's feet (mean difference 8.5+/-31 Omega). Extracellular water volumes were calculated from Tefal-measured extracellular water resistances by using a modified bioimpedance spectroscopic method and differed from those measured with Xitron by-0.03+/-0.66 L. CONCLUSION: Limits of agreement with DXA-measured FFM produced by the foot-to-foot impedance meter tested are too large for clinical measurements in individuals, but they are sufficient to assess FFM in groups of subjects and for home use. Our prototype was also capable of estimating extracellular water volume with a similar accuracy as multifrequency bioimpedance in normal subjects.