Structural basis of cytoplasmic streaming in Characean internodal cells. A hydrodynamic analysis

Summary Hydrodynamic equations were derived which relate the velocity profile of endoplasmic streaming with the motive force generated by active sliding of endoplasmic organelles in Characean internodal cells, under two implicit assumptions that (1) the sliding velocity of putative organelles is comparable to the streaming velocity of endoplasm, and (2) subcortical endoplasm is far less viscous than bulk endoplasm.The equations were extended so as to calculate the velocity profile in flattened or perfused internodal cells. Calculated profiles were basically consistent with reported patterns of streaming under these conditions.Utilizing published data, we deduce some hydrodynamic parameters of streaming, and predict the dimensions of putative organelles expected to drive entire cytoplasm. A revision for published values of the motive force of streaming is proposed.Hydrodynamic analyses made earlier on the spherical organelles are repeated. The results show that the organelles may generate streaming, depending on the configurationin vivo of fine filaments protruding from the body of the organelles.     

Ultrastructure of the vitreous body of the rabbit

Examination in the scanning and the transmission electron microscope showed three morphologically and structurally different types of cells in the vitreous body of the healthy rabbit eye: 1. cells with numerous cytoplasm processes, whose high metabolic activity is represented by the presence of a large number of organelles and which are capable of synthesizing fibrillar material; 2. elongate cells with a flattened nucleus, with long, narrow cytoplasm processes arising from both their poles and with only a few organelles in their cytoplasm; 3. large spherical cells with structureless contents, whose nucleus and few organelles are situated below the cell membrane. The organized component of the intercellular matter of the rabbit vitreous body is composed of collagen fibrils with a very variable diameter (24-180 nm), The collagen fibrils form the basis of the three-dimensional skeleton of the intercellular matter of the vitreous body.     

Intracellular Localization of Lunularic Acid and Prelunularic Acid in Suspension Cultured Cells of Marchantia polymorpha

Intracellular localization of lunularic acid and prelunularic acid in suspension cultured cells of Marchantia polymorpha L. was studied. The sum of both compounds was determined as lunularic acid group (LNAs) because of the instability of prelunularic acid to convert into lunularic acid.

Mechanical disruption of the cells followed by differential centrifugation showed that LNAs was associated with the supernatant of 100,000g centrifugation. Protoplasts isolated from the cells were osmotically ruptured and the distribution of LNAs among the organelles was examined by discontinuous density gradient centrifugation of the protoplast contents. Successful isolation of intact chloroplasts, mitochondria and peroxisomes free from cytoplasm indicated that LNAs was not accumulated in these organelles. Flotation techniques resulted in an efficient isolation of pure vacuoles and revealed that LNAs was distributed almost equally in the vacuoles and cytoplasm.

     

A New Coarse-Grained Model for E. coli Cytoplasm: Accurate Calculation of the Diffusion Coefficient of Proteins and Observation of Anomalous Diffusion

A new coarse-grained model of the E. coli cytoplasm is developed by describing the proteins of the cytoplasm as flexible units consisting of one or more spheres that follow Brownian dynamics (BD), with hydrodynamic interactions (HI) accounted for by a mean-field approach. Extensive BD simulations were performed to calculate the diffusion coefficients of three different proteins in the cellular environment. The results are in close agreement with experimental or previously simulated values, where available. Control simulations without HI showed that use of HI is essential to obtain accurate diffusion coefficients. Anomalous diffusion inside the crowded cellular medium was investigated with Fractional Brownian motion analysis, and found to be present in this model. By running a series of control simulations in which various forces were removed systematically, it was found that repulsive interactions (volume exclusion) are the main cause for anomalous diffusion, with a secondary contribution from HI.     

Anomalous Dynamics of Melanosomes Driven by Myosin-V in Xenopus laevis Melanophores

The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles spend ∼70% and 10% of the total time in active transport during dispersion and aggregation, respectively. Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V.     

Statistics of active transport in Xenopus melanophores cells

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 μm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 μm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.     

Intracellular Localization of Peptide Hydrolases in Wheat (Triticum aestivum L.) Leaves

Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.     

Visualizing protein motion in Couette flow by all-atom molecular dynamics

In living cells, biomacromolecules are exposed to a highly crowded environment. The cytoplasm, the nucleus, and other organelles are highly viscous fluids that differ from dilute in vitro conditions. Viscosity, a measure of fluid internal friction, directly affects the forces that act on immersed macromolecules. Although active motion of this viscous fluid – cytoplasmic streaming – occurs in many plant and animal cells, the effect of fluid motion (flow) on biomolecules is rarely discussed. Recently NMR experiments that apply a shearing flow in situ have been used for protein studies. While these NMR experiments have succeeded in spectroscopically tracking protein aggregation in real time, they do not provide a visual picture of protein motion under shear. To fill this gap, here we have used molecular dynamics simulations to study the motion of three proteins of different size and shape in a simple shearing flow. The proteins exhibit a superposition of random diffusion and shear-flow-induced rotational motion. Random rotational diffusion dominates at lower shear stresses, whereas an active “rolling motion” along the axis of the applied flow occurs at higher shear stress. Even larger shear stresses perturb protein secondary structure elements resulting in local and global unfolding. Apart from shear-induced unfolding, our results imply that, in an ideal Couette flow field biomolecules undergo correlated motion, which should enhance the probability of inter-molecular interaction and aggregation. Connecting biomolecular simulation with experiments applying shear flow in situ appears to be a promising strategy to study protein alignment, deformation, and dynamics under shear.     

Flow mixing enhancement from balloon pulsations in an intravenous oxygenator

Computational investigations of flow mixing and oxygen transfer characteristics in an intravenous membrane oxygenator (IMO) are performed by direct numerical simulations of the conservation of mass, momentum, and species equations. Three-dimensional computational models are developed to investigate flow-mixing and oxygen-transfer characteristics for stationary and pulsating balloons, using the spectral element method. For a stationary balloon, the effect of the fiber placement within the fiber bundle and the number of fiber rings is investigated. In a pulsating balloon, the flow mixing characteristics are determined and the oxygen transfer rate is evaluated. For a stationary balloon, numerical simulations show two well-defined flow patterns that depend on the region of the IMO device. Successive increases of the Reynolds number raise the longitudinal velocity without creating secondary flow. This characteristic is not affected by staggered or non-staggered fiber placement within the fiber bundle. For a pulsating balloon, the flow mixing is enhanced by generating a three-dimensional time-dependent flow characterized by oscillatory radial, pulsatile longitudinal, and both oscillatory and random tangential velocities. This three-dimensional flow increases the flow mixing due to an active time-dependent secondary flow, particularly around the fibers. Analytical models show the fiber bundle placement effect on the pressure gradient and flow pattern. The oxygen transport from the fiber surface to the mean flow is due to a dominant radial diffusion mechanism, for the stationary balloon. The oxygen transfer rate reaches an asymptotic behavior at relatively low Reynolds numbers. For a pulsating balloon, the time-dependent oxygen-concentration field resembles the oscillatory and wavy nature of the time-dependent flow. Sherwood number evaluations demonstrate that balloon pulsations enhance the oxygen transfer rate, even for smaller flow rates.     

Ultraviolet-Microbeam Irradiation of Regenerating Stentor*

SYNOPSIS. Regeneration of missing feeding organelles in the ciliate Stentor coeruleus can be delayed by ultraviolet (UV) irradiation. In an attempt to identify the radiation-sensitive cellular component(s), cells in various stages of regeneration were immobilized, flattened and irradiated with a microbeam of 265 nm UV light. Targets included regions of either the macronucleus or the cytoplasm at varying distances from the ramifying zone of the cortex, which is the region where the new oral organelles are formed. During the early stages of regeneration the macronucleus is more sensitive to UV-induced regeneration delay than the cytoplasm, but it is not possible to distinguish between direct and indirect effects. Damage to cortical primordia in middle or late stages leading to structural abnormalities appears to result only from direct effects.     

Effects of diffusion on the electrophoretic behavior of associating systems: The Gilbert-Jenkins theory revisited

The Gilbert-Jenkins theory predicts the asymptotic shape of moving-boundary sedimentation and electrophoretic patterns and broad zone molecular sieve chromatographic elution profiles for the class of interacting systems, A + B ⇄ C, in which two dissimilar macromolecules react reversibly to form a complex. A particularly provocative case is the one in which the complex has a greater migration velocity than that of either reactant, each of which has a different velocity. Depending upon conditions, this case predicts, for example, that in the asymptotic limit an ascending electrophoretic pattern or a frontal gel chromatographic elution profile can show two hypersharp reaction boundaries separated by a plateau. This prediction is now confirmed by numerical solution of transport equations which retain the second-order diffusional term and extrapolation of the computed patterns to zero diffusion coefficient. For finite diffusion coefficient, however, the two hypersharp reaction boundaries are separated by a weak negative gradient. These calculations are extended to an examination of the transitions between the three types of patterns admitted by the case under consideration in order to gain physical understanding and to define criteria for recognizing the transitions. Studies of this kind not only establish confidence in the Gilbert-Jenkins theory, but, in addition, they provide new insights which make for more effective application of the theory to real systems.     

Gel electrophoresis of micron-sized particles: a problem and a solution

The gel electrophoresis of spherical particles with a radius above 0.2 micron has not been reported yet. In the present study, video phase-contrast light microscopy is used to observe the motion of individual latex spheres, 0.52 micron in radius, during electrophoresis in 0.1% agarose gels. At 2 V/cm, the spheres initially migrate in the direction of the electrical field. However, each sphere eventually undergoes a cessation of all motion. Brownian motion is restored when the electrical potential gradient is reduced to zero. Arrest can be prevented by periodically inverting the direction of the electrical field. These observations are explained by electrical field-induced steric trapping of the spheres by gel fibers. Inversion of the electrical field should assist the application of agarose gel electrophoresis to micron-sized cellular organelles and cells.     

The Impact of Spinal Cord Nerve Roots and Denticulate Ligaments on Cerebrospinal Fluid Dynamics in the Cervical Spine

Cerebrospinal fluid (CSF) dynamics in the spinal subarachnoid space (SSS) have been thought to play an important pathophysiological role in syringomyelia, Chiari I malformation (CM), and a role in intrathecal drug delivery. Yet, the impact that fine anatomical structures, including nerve roots and denticulate ligaments (NRDL), have on SSS CSF dynamics is not clear. In the present study we assessed the impact of NRDL on CSF dynamics in the cervical SSS. The 3D geometry of the cervical SSS was reconstructed based on manual segmentation of MRI images of a healthy volunteer and a patient with CM. Idealized NRDL were designed and added to each of the geometries based on in vivo measurments in the literature and confirmation by a neuroanatomist. CFD simulations were performed for the healthy and patient case with and without NRDL included. Our results showed that the NRDL had an important impact on CSF dynamics in terms of velocity field and flow patterns. However, pressure distribution was not altered greatly although the NRDL cases required a larger pressure gradient to maintain the same flow. Also, the NRDL did not alter CSF dynamics to a great degree in the SSS from the foramen magnum to the C1 level for the healthy subject and CM patient with mild tonsillar herniation (∼6 mm). Overall, the NRDL increased fluid mixing phenomena and resulted in a more complex flow field. Comparison of the streamlines of CSF flow revealed that the presence of NRDL lead to the formation of vortical structures and remarkably increased the local mixing of the CSF throughout the SSS.     

Velocity sedimentation of organelles at low centrifugal force in an isokinetic gradient.

Mast-cell granules and polystyrene microspheres (0.600 and 1.011 micrometer in diameter) were sedimented in a previously described [Pretlow (1971) Anal. Biochem. 41, 248--255] isokinetic gradient in a low-speed centrifuge. For the analytical velocity sedimentation of organelles, this gradient offers several advantages over gradients that are commonly used for the sedimentation of organelles: (a) the density gradient (0.0008 g.ml-1.cm-1) is small, and the effective densities of organelles will change relatively little during sedimentation; (b) the densities at all points in the gradient (1.017--1.027 g/ml) are less than those in gradients commonly used for the sedimentation of organelles, the effective densities of sedimenting organelles are consequently relatively large, and the effect of density as a determinant of velocity of sedimentation is less limiting than in conventional gradients; (c) the small slope of the gradient is associated with a relatively slow increase in the viscosity encountered by the sedimenting organelle; (d) the iso-osmotic gradient is not significantly affected by the gradient medium (Ficoll), and the osmolarity can be adjusted to the desired value by the selection of an appropriate salt solution as the solvent for the Ficoll; (e) the gradient will be isokinetic for particles of densities similar to most organelles. An ultracentrifuge is not required for work with this gradient.     

Characteristics of weak base-induced vacuoles formed around individual acidic organelles

We have previously found that the weak base 4-aminopyridine induces Brownian motion of acidic organelles around which vacuoles are formed, causing organelle traffic disorder in neurons. Our present study investigated the characteristics of vacuoles induced by weak bases (NH(4)Cl, aminopyridines, and chloroquine) using mouse cells. Individual vacuoles included acidic organelles identified by fluorescent protein expression. Mitochondria and actin filaments were extruded outside the vacuoles, composing the vacuole rim. Staining with amine-reactive fluorescence showed no protein/amino acid content in vacuoles. Thus, serous vacuolar contents are probably partitioned by viscous cytosol, other organelles, and cytoskeletons, but not membrane. The weak base (chloroquine) was immunochemically detected in intravacuolar organelles, but not in vacuoles. Early vacuolization was reversible, but long-term vacuolization caused cell death. The vacuolization and cell death were blocked by the vacuolar H(+)-ATPase inhibitor and Cl--free medium. Staining with LysoTracker or LysoSensor indicated that intravacuolar organelles were strongly acidic and vacuoles were slightly acidic. This suggests that vacuolization is caused by accumulation of weak base and H(+) in acidic organelles, driven by vacuolar H(+)-ATPase associated with Cl(-) entering, and probably by subsequent extrusion of H(+) and water from organelles to the surrounding cytoplasm.     

Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming,Cell Expansion and Plant Development

Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.     

Transient state isoelectric focusing: theory

Mathematical theory is developed to describe the transient state of isoelectric focusing (pH-gradient electrophoresis) in a linear pH gradient under highly idealized conditions. This theory makes it possible to predict the concentration profile (distribution) for the protein or other amphoteric species of interest as a function of time, when the sample is applied in a zone of infinitesimal thickness at one end of the column, or in a uniform distribution throughout the column. Further, the position of the centroid, and the second moment around the mean, σ², (square of the standard deviation of peak width) are described as a function of time, irrespective of the initial distribution of the protein in the column. Three arbitrary stages of the “focusing” experiment are considered: (1) Focusing, wherein the sample is applied to a preformed pH gradient; (2) Defocusing, which occurs when the electrical field is abolished after an arbitrary time (usually after the concentration profile has begun to approach its steady state) and diffusion is allowed to occur. (3) Refocusing, which occurs after the electrical field is reapplied. Although stages 1 and 3 are conceptually identical aside from the difference in initial conditions, they may differ in several important respects in practice, both with regard to technical problems of measurement, and with regard to the closeness of conditions to the stated assumptions.This theory should make it possible to predict the time necessary to achieve any desired degree of focusing, i.e., approach to the steady-state distribution. Further, this theory and the techniques of analytical scanning isoelectric focusing provide the basis for measurement of the apparent diffusion coefficient (D), the derivative of velocity with respect to position, and if the field strength is known, the slope of the mobility-pH curve at the isoelectric point, {$\text{dM}\text{d(}\text{pH}\text{)}$}.     

The structure of cytoplasm in directly frozen cultured cells. II. Cytoplasmic domains associated with organelle movements

The relationship between organelle movement and cytoplasmic structure in cultured fibroblasts or epithelial cells was studied using video-enhanced differential interference contrast microscopy and electron microscopy of directly frozen whole mounts. Two functional cytoplasmic domains are characterized by these techniques. A central domain rich in microtubules is associated with directed as well as Brownian movements of organelles, while a surrounding domain rich in f-actin supports directed but often intermittent organelle movements more distally along small but distinct individual microtubule tracks. Differences in the organization of the cytoplasm near microtubules may explain why organelle movements are typically continuous in central regions but usually intermittent along the small tracks through the periphery. The central type of cytoplasm has a looser cytoskeletal meshwork than the peripheral cytoplasm which might, therefore, interfere less frequently with organelles moving along microtubules there.     

Characteristics of the flow velocity-pressure gradient relation in the assessment of stenoses: an in vitro study

Objectives. The flow velocity-pressure gradient (v-dp) relation is clinically used to assess coronary stenoses. This in vitro study aimed to investigate the ability to determine the impact of each individual stenosis in the setting of two consecutive stenoses, the effect of variable stenosis reference diameters and the impact of one or two wires in a stenosis, on the v-dp relation. Methods. The model consisted of a reservoir and different sized tubes and stenoses. Pressure gradient and flow velocity were assessed with a pressure and a Doppler wire. By plotting flow velocity and pressure gradient on an X-Y plot, the v-dp relation was determined. Results. The v-dp relation of a proximal stenosis was not influenced by a distal stenosis. The diameter of the segment where flow velocity was measured influenced the v-dp relation. This could be corrected by substituting flow velocity with volume flow. The presence of one or two wires in a stenosis made the v-dp relation substantially steeper. Conclusions. The v-dp relation can be used to determine the significance of each individual stenosis in arteries with consecutive stenoses, provided that the distance between the stenoses is large enough. The diameter of the segment where flow velocity is measured and the presence of one or two wires substantially affect the v-dp relation. (Neth Heart J 2008;16:156-62.)     

Flow-pressure drop measurement and calculation in a tapered femoral artery of a dog

This study describes the in vivo measurement of pressure drop and flow during the cardiac cycle in the femoral artery of a dog, and the computer simulation of the experiment based on the use of the measured flow, vessel dimensions and blood viscosity. In view of the experimental uncertainty in obtaining the accurate velocity profile at the wall region, the velocity pulse at the center was measured and numerical calculations were performed for the center Une instantaneous velocity and within the two limits of spatial distribution of inlet flow conditions: uniform and parabolic. Temporal and spatial variations of flow parameters, i.e., velocity profile, shear rate, non-Newtonian viscosity, wall shear stress, and pressure drop were calculated. There existed both positive and negative shear rates during a pulse cycle, i.e., the arterial wall experiences zero shear three times during a cardiac cycle. For the parabolic inlet condition, the taper of the artery not only increased the magnitude of the positive and negative shear rates, but caused a steep gradient in shear rate, a phenomenon which in turn affects wall shear stress and pressure. In contrast, for the uniform inlet condition, the flow through the tapered artery was predominantly the developing type, which resulted in reduction in magnitude of wall shear rate along the axial direction.