Computer-assisted analysis of particulate axoplasmic flow in organized CNS tissue cultures

Using organized nervous system tissue cultures of embryonic mouse spinal cord, time-lapse motion pictures of intra-axonal particle movements were made using Nomarski optics. The paths of 272 particles were followed and analyzed using digital computer methods. Particles had a bidirectional saltatory motility with net anterograde and retrograde velocities which were shown to be the same (about 1 micron per second). When anterograde moving and retrograde moving particles were analyzed separately for anterograde and retrograde saltations, the velocities of saltations in the direction of net movement were found to be 1.5 times greater than saltations against the direction of net movement. These differences were statistically significant. In addition, it was shown that there were regions within the axon where variations in particle motion were similar from particle to particle as they passed, and that more disturbed motion was found to occur near intra-axonal objects which were judged to be mitochondria. No decrease of particle velocity was noted in regions away from mitochondria nor was an increase in velocity noted near them. Computer-drawn particle plots illustrate the various facets of particle motion.     

Computer-assisted analysis of particulate axoplasmic flow in organized CNS tissue cultures.

Using organized nervous system tissue cultures of embryonic mouse spinal cord, time-lapse motion pictures of intra-axonal particle movements were made using Nomarski optics. The paths of 272 particles were followed and analyzed using digital computer methods. Particles had a bidirectional saltatory motility with net anterograde and retrograde velocities which were shown to be the same (about 1 micron per second). When anterograde moving and retrograde moving particles were analyzed separately for anterograde and retrograde saltations, the velocities of saltation in the direction of net movement were found to be 1.5 times greater than saltations against the direction of net movement. These differences were statistically significant. In addition, it was shown that there were regions within the axon where variations in particle motion were similar from particle to particle as they passed, and that more disturbed motion was found to occur near intra-axonal objects which were judged to be mitochondria. No decrease of particle velocity was noted in regions away from mitochondria nor was in increase in velocity noted near them. Computer-drawn particle plots illustrate the various facets of particle motion.     

Analysis of steric partition behavior of molecules in membranes using statistical physics. Application to gel chromatography and electrophoresis.

The principles of statistical physics are used to formulate general expressions for the steric partition behavior of molecules in both random and ordered membrane structures that may be applied to any shape of the solute and/or the volume-excluding element of the membrane. These expressions fully define partitioning in terms of the volume excluded to point molecules and to finite-sized molecules. The mean effective exclusion volume for a molecule is calculated as a function of a global interaction energy, which varies with position, conformation, and orientation of the molecule. It allows consideration of electrostatic and other nonsteric factors. To test the model, specific partition functions are derived for several simple geometries describing the membrane and solute. Frequently, the derived expressions agree with past analyses; however, a new expression describing partitioning within an random network of fibers is derived. It agrees with past results only in the limit of low exclusion volumes. With greater volume exclusions, past results greatly overestimate the partition function. It is applied to gel electrophoresis and chromatography and survives testing with available experimental data. Unlike past analyses, it predicts nonlinear Ferguson plots for agarose gel electrophoresis. In addition, an analytical expression predicting the minimum radius of a sphere excluded from a random fiber matrix is derived, tested, and found to agree with experimental data.     

Optically induced flow cytometry for continuous microparticle counting and sorting

This paper reports a new microfluidic device capable of performing optically induced flow cytometry (OIFC). This enables it to continuously count and to sort microparticles based on optically induced dielectrophoretic (ODEP) forces. Gravity was used to drive the particles instead of using syringe pumps. The particles were then focused inside a sample channel by the ODEP forces and then passed through a detection region. A pair of optical fibers were embedded into fiber channels to count the number of particles and analyze the particle size in real time. Using 20.9 and 9.7 microm polystyrene microparticles, the average light intensity were about 63.67 and 8.80 units, with a coefficient-of-variation (CV) of 7.46 and 25.57%, respectively. This demonstrated that these two particle sizes could be successfully distinguished. After detecting the number and size of the microparticles, an optically induced dynamic switch (ODS) was used to sort microparticles to downstream fluidic outlets. The ODS used ODEP forces generated by different illumination intensities or optical line widths. The ODS was composed of two virtual electrodes which controlled particle movement in two dimensions. The ODS can successfully sort microparticles with different sizes continuously. The development of the OIFC device is a major advancement in the design of microparticle counting and sorting devices. Applications in future biomedical applications for cell counting and manipulation are envisioned.     

植物微管特效解聚剂APM对紫露草雄蕊毛细胞胞质环流的影响

采用体外渗透和显微注射的方法。将植物微管特效解聚剂甲基氨草磷(APM)引入紫露草雄蕊毛细胞后,发现原来沿着胞质束运动的胞质颗粒运动速度渐慢,进而胞质束消失,颗粒运动停止。显微注射后,还发现APM可通过胞间通道由被注射的细胞向两侧细胞扩散,从而也导致两侧细胞胞质束消失,颗粒运动停止。APM对胞质环流的抑制作用是可逆的。结果表明微管可能是胞质束的重要组份之一,植物胞质环流与微管的聚合与解聚状态有密切关系。     

Commerical reference shape standards use in the study of particle shape effect on laser diffraction particle size analysis

The purpose of this paper is to describe the use of LGC Promochem AEA 1001 to AEA 1003 monosized fiberanalog shape standards in the study of the effect of particle shape on laser diffraction (LD) particle size analysis (psa). The psa of the AEA standards was conducted using LD psa systems from Beckman Coulter, Horiba, and Malvern Instruments. Flow speed settings, sample refractive index values, and sample cell types were varied to examine the extent to which the shape effect on LD psa results is modified by these variables. The volume and number probability plots resulting from these measurements were each characterized by a spread in the particle size distribution that roughly extended from the breadth to the longest dimension of the particles. For most of the selected sample refractive index values, the volume probability plots were characterized by apparent bimodal distributions. The results, therefore, provide experimental verification of the conclusions from theoretical studies of LD psa system response to monosized elliptical particles in which this apparent bimodality was the predicted result in the case of flow-oriented particles. The data support the findings from previous studies conducted over the past 10 years that have called into question the verity of the tenets of, and therefore the value of the application of, the equivalent spherical volume diameter theory and the random particle orientation model to the interpretation of LD psa results from measurements made on nonspherical particles.     

Computer-aided analysis of DNA curves on transverse gradient gels.

Transverse pore gradient polyacrylamide gel electrophoresis of DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than either the mere visual observation of crossing Ferguson curves or of a shift in mobility at a single gel concentration.     

Commercial reference shape standards use in the study of particle shape effect on laser diffraction particle size analysis

The purpose of this paper is to describe the use of LGC Promochem AEA 1001 to AEA 1003 monosized fiber-analog shape standards in the study of the effect of particle shape on laser diffraction (LD) particle size analysis (psa). The psa of the AEA standards was conducted using LD psa systems from Beckman Coulter, Horiba, and Malvern Instruments. Flow speed settings, sample refractive index values, and sample cell types were varied to examine the extent to which the shape effect on LD psa results is modified by these variables. The volume and number probability plots resulting from these measurements were each characterized by a spread in the particle size distribution that roughly extended from the breadth to the longest dimension of the particles. For most of the selected sample refractive index values, the volume probability plots were characterized by apparent bimodal distributions. The results, therefore, provide experimental verification of the conclusions from theoretical studies of LD psa system response to monosized elliptical particles in which this apparent bimodality was the predicted result in the case of flow-oriented particles. The data support the findings from previous studies conducted over the past 10 years that have called into question the verity of the tenets of, and therefore the value of the application of, the equivalent spherical volume diameter theory and the random particle orientation model to the interpretation of LD psa results from measurements made on nonspherical particles.     

Choosing orientation: influence of cargo geometry and ActA polarization on actin comet tails

Networks of polymerizing actin filaments can propel intracellular pathogens and drive movement of artificial particles in reconstituted systems. While biochemical mechanisms activating actin network assembly have been well characterized, it remains unclear how particle geometry and large-scale force balance affect emergent properties of movement. We reconstituted actin-based motility using ellipsoidal beads resembling the geometry of Listeria monocytogenes. Beads coated uniformly with the L. monocytogenes ActA protein migrated equally well in either of two distinct orientations, with their long axes parallel or perpendicular to the direction of motion, while intermediate orientations were unstable. When beads were coated with a fluid lipid bilayer rendering ActA laterally mobile, beads predominantly migrated with their long axes parallel to the direction of motion, mimicking the orientation of motile L. monocytogenes. Generating an accurate biophysical model to account for our observations required the combination of elastic-propulsion and tethered-ratchet actin-polymerization theories. Our results indicate that the characteristic orientation of L. monocytogenes must be due to polarized ActA rather than intrinsic actin network forces. Furthermore, viscoelastic stresses, forces, and torques produced by individual actin filaments and lateral movement of molecular complexes must all be incorporated to correctly predict large-scale behavior in the actin-based movement of nonspherical particles.     

The intraflagellar transport machinery of Chlamydomonas reinhardtii

First discovered in the green alga, Chlamydomonas , intraflagellar transport (IFT) is the bidirectional movement of protein particles along the length of eukaryotic cilia and flagella. Composed of ∼16 different proteins, IFT particles are moved out to the distal tip of the organelle by kinesin-II and are brought back to the cell body by cytoplasmic dynein 1b. Mutant analysis of the IFT motor and particle proteins using diverse organisms has revealed a conserved and essential role for IFT in the assembly and maintenance of cilia and flagella. IFT is thought to mediate this assembly through the delivery of axonemal precursors out to the distal tip of the growing organelle. Consistent with this model, the IFT particle proteins are rich in protein–protein binding motifs, suggesting that the particles may act as scaffolds for the binding of multiple cargoes. With most of the IFT proteins now identified at the level of the gene, this review will briefly examine both the structure and function of the IFT machinery of Chlamydomonas reinhardtii .     

Structural Aspects of Saltatory Particle Movement

A variety of cells possess particles which show movements statistically different from Brownian movements. They are characterized by discontinuous jumps of 2–30 µ at velocities of 0.5–5 µ/sec or more. A detailed analysis of these saltatory, jumplike movements makes it most likely that they are caused by transmission of force to the particles by a fiber system in the cell outside of the particle itself. Work with isolated droplets of cytoplasm from algae demonstrates a set of fibers involved in both cytoplasmic streaming and saltatory motion, suggesting that both phenomena are related to the same force-generating set of fibers. Analysis of a variety of systems in which streaming and/or saltatory movement occurs reveals two types of fiber systems spatially correlated with the movement, microtubules and 50 A microfilaments. The fibers in Nitella (alga) are of the microfilament type. In other systems (melanocyte processes, mitotic apparatus, nerve axons) microtubules occur. A suggestion is made, based on work on cilia, that a microtubule-microfilament complex may be present in those cases in which only microtubules appear to be present, with the microfilament closely associated with or buried in the microtubule wall. If so, then microfilaments, structurally similar to smooth muscle filaments, may be a force-generating element in a very wide variety of saltatory and streaming phenomena.     

A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin

BACKGROUND: Cell transfection requires cationic DNA complexes and heparan sulfate proteoglycans (HSPGs) at the cell surface. Syndecans are transmembrane HSPGs that are ubiquitously expressed on adherent cells. Their polyanionic heparan sulfate moieties are bound at the distal end of their ectodomain, thus facilitating interaction with large cationic particles. METHODS: We propose a model for cell entry involving syndecans as receptors for the DNA complexes by comparing transfection with bacteria uptake and using drug inhibition experiments along with confocal microscopy. RESULTS: When combined with results from the literature, our data suggest the following sequence of events: after initial particle binding, gradual electrostatic zippering of the plasma membrane onto the particle is sustained by lateral diffusion of syndecan molecules that cluster into cholesterol-rich rafts. Clustering in turn triggers PKC activity and linker protein-mediated actin binding to the cytoplasmic tail of the syndecans. Resulting tension fibers and a growing network of cortical actin may then pull the particle into the cell. CONCLUSIONS: Diversion of integrin- and syndecan-mediated cell adhesion processes for particle engulfment appears to be widely exploited by animals (chylomicrons), by pathogens (bacteria, viruses) and, as suggested here, by non-viral vectors.     

Wetting, percolation and morphogenesis in a model tissue system

Artificial tissues constructed of cells or polystyrene beads suspended in a solution of type I collagen will, under appropriate conditions, protrude into regions of similar matrices lacking particles, but containing the extracellular glycoprotein fibronectin. This phenomenon has been termed "matrix-driven translocation". Conditions required for the effect include the presence of heparin-like molecules on the cell or bead surfaces, appropriate concentrations of particles and collagen, and physiological ionic strength and pH. Here we consider the idea that the driving force for the concerted movement of matrix and suspended particles is the thermodynamically spontaneous spreading or wetting behavior of two immiscible fluids bounded by common substrata. Wetting theory is shown to be capable of accounting for the behavior of this model system, but this analysis requires that the two matrix regions constitute separate phases at thermodynamic coexistence. We show that one plausible mechanism for the generation of separate phases is the formation of a percolation network of collagen fibers on a lattice of cells or beads. It is argued that the concepts of wetting and percolation apply to properties in common between the model system and living tissues, and may therefore be used to provide a physical account of aspects of tissue morphogenesis.     

Adaptation at Specific Loci. I. Natural Selection on Phosphoglucose Isomerase of Colias Butterflies: Biochemical and Population Aspects

Electrophoretic variants of phosphoglucose isomerase (PGI) in Colias butterflies have been studied from field and laboratory viewpoints. The transmission pattern is that of a dimeric enzyme controlled by one structural gene locus. Populations usually harbor four to six allelic mobility classes. These mobility classes are shared among species complexes, though their frequencies differ widely. Preliminary Ferguson plot analysis of the variants has been carried out. Purified preparations of Colias PGI alleles are more effective in standardizing Ferguson plots than heterologous proteins, such as ferritin. Variation of Ferguson plot parameters is not an infallible guide to electrophoretically "cryptic alleles," as one putative case proved to be due to nonallele-specific effects. S, M, and F mobility classes in two Colias semispecies show the same retardation coefficients in Ferguson plots. Adults early in the flight periods of their nonoverlapping generations show genotype frequencies in Hardy-Weinberg equilibrium, but heterozygote excess develops as the insects age. Simple directional selection and large-scale population mixing are unlikely to be causes of this, although several other selection modes remain possible. Identical-by-descent lines of the four frequent-to-common alleles in C. eurytheme have been set up in culture, and enzyme has been purified from these for study of functional properties. Major differenecs in heat stability and in various kinetic parameters are found among the ten possible genotypes. In some cases, heterosis for kinetic parameters is seen; in other cases, opposing trends in kinetic function and heat stability create potential for net heterosis in function. Possible interpretations of these results in an adaptive metabolic context are discussed, and directions for further work are stated.     

Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels

The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.     

Co-Localization of Particle Transport with Microtubules in Cytoplasmic Exudates of the Siphonous Green Alga Bryopsis

Organelles in the cortical cytoplasm of the siphonous green alga Bryopsis display various types of motile activities. One of them, saltatory movement along axially oriented linear tracks is typical for mitochondria and other small particles. A method is described which allows in vitro observation of such movements in thin layers of cytoplasm extruded from the alga and attached to a poly-l -lysine coated glass surface. By comparing video recordings of motile activities with the position of cytoskeletal elements visualized by immunofluorescence in the same area of a cytoplasmic exudate, it can be shown that tracks along which particles have moved in vitro are identical with microtubules (MTs). Depolymerization of MTs in the cytoplasmic exudates by MT-specific inhibitors stops particle movement, whereas depolymerization of actin filaments with cytochalasin D disrupts actin bundles but has little effect on particle motility. These data are consistent with the model of MT guided particle transport.     

Physical properties of nucleoprotein cores from adenovirus type 5.

Analytical ultracentrifugation, thermal denaturation, and electron microscopy have been used to study nucleoprotein core particles, obtained from disrupted type 5 adenovirus and partially purified on glycerol density gradients. Electron microscopy at low salt concentrations has shown that the cores are homogeneous particles with characteristic structures, which vary with conditions of observation from a fairly loose network of fibers to a highly condensed, compact particle. Sedimentation measurements in the analytical ultracentrifuge, both by boundary and by band techniques, show that the cores are relatively homogeneous in solution and have sedimentation coefficients near 185 S at low salt concentrations, about 243 S in 1 or 2 M NaCl, and 376 S in 1 mM MgCl2. Correlation of sedimentation data with electron microscopic observations suggests that the 185 S particle has a loose, fibrous structure, while the faster species are more highly condensed particles. The melting temperature of the cores in 5 mM Tris/HCl is 79 degrees C, which is 10 degrees C higher than the Tm for purified, viral DNA. This indicates that the protein enhances the stability of DNA in the nucleoprotein complex.     

Motion of particles adhering to the leading lamella of crawling cells

Time-lapsed films of particle motion on the leading lamella of chick heart fibroblasts and mouse peritoneal macrophages were analyzed. The particles were composed of powdered glass or powdered aminated polystyrene and were 0.5-1.0 micrometer in radius. Particle motions were described by steps in position from one frame to the time-lapse movies to the next. The statistics of the step-size distribution of the particles were consistent with a particle in Brownian motion subject to a constant force. From the Brownian movement, we have calculated the two-dimensional diffusion coefficient of different particles. These vary by more than an order of magnitude (10(-11)-10(-10) cm2/s) even for particles composed of the same material and located very close to each other on the surface of the cell. This variation was not correlated with particle size but is interpretable as a result of different numbers of adhesive bonds holding the particles to the cells. The constant component of particle movement can be interpreted as a result of a constant force acting on each particle (0.1-1.0 x 10(-8) dyn). Variations in the fractional coefficient for particles close to each other on the cell surface do not yield corresponding differences in velocity, suggesting that the frictional coefficient and the driving force vary together. This is consistent with the hypothesis that the particles are carried by flow of the membrane as a whole or by flow of some submembrane material. The utility of our methods for monitoring cell motile behavior in biologically interesting situations, such as a chemotactic gradient, is discussed.     

Force acting on spheres adhered to a vessel wall

To evaluate the force and torque acting on leukocytes attached to the vessel wall, we numerically study the flow field around the leukocytes by using rigid spherical particles adhered to the wall of a circular cylindrical tube as a model of adherent leukocytes. The adherent particles are assumed to be placed regularly in the flow direction with equal spacings, in one row or two rows. The flow field of the suspending fluid is analyzed by a finite element method applied to the Stokes equations, and the drag force and torque acting on each particle, as well as the apparent viscosity, are evaluated as a function of the particle to tube diameter ratio and the particle arrangements. For two-row arrangements of adhered particles where neighboring particles are placed alternately on opposite sides of the vessel, the drag and the torque exerted on each particle are higher than those for single-row arrangements, for constant particle to tube diameter ratio and axial spacing between neighboring particles. This is enhanced for Larger particles and smaller axial spacings. The apparent viscosity of the flow through vessels with adhered particles is found to be significantly higher than that without adhered particles or when the particles are freely floating through the vessels.