A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions for fast plus-end directed fluid flows that facilitate mixing in a low Reynolds number regime.

Chemical shift anisotropy selective inversion.

Magic angle spinning (MAS) is used in solid-state NMR to remove the broadening effects of the chemical shift anisotropy (CSA). In this work we investigate a technique that can reintroduce the CSA in order to selectively invert transverse magnetization. The technique involves an amplitude sweep of the radio frequency field through a multiple of the spinning frequency. The selectivity of this inversion mechanism is determined by the size of the CSA. We develop a theoretical framework to describe this process and demonstrate the CSA selective inversion with numerical simulations and experimental data. We combine this approach with cross-polarization (CP) for potential applications in multi-dimensional MAS NMR.

Using a cross-coil to reduce RF heating by an order of magnitude in triple-resonance multinuclear MAS at high fields.

Four different coil designs for use with MAS in triple-resonance multi-nuclear experiments at high fields are compared, using a combination of finite element analysis (FEA) software and NMR experiments, with respect to RF field strength per unit power and relative sample heating, as governed by mean E/B(1) within the sample region. A commercial FEA package, Microwave Studio 5.1 by Computer Simulation Technology (CST) is shown to obtain remarkably accurate agreement with the experiments in Q(L), L, B, E, and mode frequencies in all cases. A simplified treatment of RF heating in NMR MAS samples is derived and shown to agree with the NMR experimental results within about 10% for two representative stator designs. The coil types studied include: (1) a variable-pitch solenoid outside a ceramic coilform, (2) a conventional solenoid very closely spaced to the MAS rotor, (3) a scroll coil, and (4) a segmented saddle cross coil (XC) for (1)H with an additional solenoid over it for the two lower-frequency channels. The XC/solenoid is shown to offer substantial advantages in reduced decoupler heating, improved S/N, and improved compatibility with multinuclear tuning and high-power decoupling. This seems largely because the division of labor between two orthogonal coils allows them each, and their associated circuitry, to be separately optimized for their respective regimes.

Reduction of spinning sidebands in proton NMR of human prostate tissue with slow high-resolution magic angle spinning.

High-resolution magic angle spinning (HRMAS) NMR spectroscopy has proven useful for analyzing intact tissue and permitting correlations to be made between tissue metabolites and disease pathologies. Extending these studies to slow-spinning methodologies helps protect tissue pathological structures from HRMAS centrifuging damage and may permit the study of larger objects. Spinning sidebands (SSBs), which are produced by slow spinning, must be suppressed to prevent the complication of metabolic spectral regions. In this study human prostate tissues, as well as gel samples of a metabolite mixture solution, were measured with continuous-wave (CW) water presaturation on a 14.1T spectrometer, with HRMAS spinning rates of 250, 300, 350, 600, and 700 Hz, and 3.0 kHz. Editing the spectra by means of a simple minimum function (Min(A, B, ..., N) for N spectra acquired at different but close spinning rates) produced SSB-free spectra. Statistically significant linear correlations were observed for metabolite concentrations quantified from the Min(A, B, ..., N)-edited spectra generated at low spinning rates, with concentrations measured from the 3 kHz spectra, and also with quantitative pathology. These results indicate the empirical utility of this scheme for analyzing intact tissue, which also may be used as an adjunct tool in pathology for diagnosing disease.

Proton high-resolution magic angle spinning NMR analysis of fresh and previously frozen tissue of human prostate.

The previously observed improvement in spectral resolution of tissue proton NMR with high-resolution magic angle spinning (HRMAS) was speculated to be due largely to freeze-thawing artifacts resulting from tissue storage. In this study, 12 human prostate samples were analyzed on a 14.1T spectrometer at 3 degrees C, with HRMAS rates of 600 and 700 Hz. These samples were measured fresh and after they were frozen for 12-16 hr prior to thawing. The spectral linewidths measured from fresh and previously frozen samples were identical for all metabolites except citrate and acetate. The metabolite intensities of fresh and freeze-thawed samples depend on the quantification procedures used; however, in this experiment the differences of means were <30%. As expected, it was found that tissue storage impacts tissue quality for pathological analysis, and HRMAS conditions alone are not sufficiently destructive to impair pathological evaluation. Furthermore, although storage conditions affect absolute metabolite concentrations in NMR analysis, relative metabolite concentrations are less affected.

High-resolution magic angle spinning proton NMR analysis of human prostate tissue with slow spinning rates.

The development of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy for intact tissue analysis and the correlations between the measured tissue metabolites and disease pathologies have inspired investigations of slow-spinning methodologies to maximize the protection of tissue pathology structures from HR-MAS centrifuging damage. Spinning sidebands produced by slow-rate spinning must be suppressed to prevent their complicating the spectral region of metabolites. Twenty-two human prostatectomy samples were analyzed on a 14.1T spectrometer, with HR-MAS spinning rates of 600 Hz, 700 Hz, and 3.0 kHz, a repetition time of 5 sec, and employing various rotor-synchronized suppression methods, including DANTE, WATERGATE, TOSS, and PASS pulse sequences. Among them, DANTE, as the simplest scheme, has shown the most potential in suppression of tissue water signals and spinning sidebands, as well as in quantifying metabolic concentrations.

Recent Advances in High-Resolution Solid-State NMR Spectroscopy.

Diverse complex systems may be studied by the new methods in solid-state NMR spectroscopy described herein. These methods use magic-angle spinning (MAS) on samples in oriented bilayers (right picture) and in orientationally disordered samples (left picture). Systems as diverse as uniformly 13 C,15 N-labeled proteins, model membrane systems, and silk, as produced by the silkworm, can be structurally characterized.