Entropy driven self-assembly of nonamphiphilic colloidal membranes

We demonstrate that homogeneous monodisperse rods in the presence of attractive interactions assemble into equilibrium 2D fluid-like membranes composed of a one-rod length thick monolayer of aligned rods. Unique features of our system allow us to simultaneously investigate properties of these membranes at both continuum and molecular lengthscales. Analysis of thermal fluctuations at continuum lengthscales yields the membranes’ lateral compressibility and bending rigidity and demonstrates that the properties of colloidal membranes are comparable to those of traditional lipid bilayers. Fluctuations at molecular lengthscales, in which single rods protrude from the membrane surface, are directly measured by comparing the positions of individual fluorescently labeled rods within a membrane to that of the membrane’s continuum conformation. As two membranes approach each other in suspension, protrusion fluctuations are suppressed leading to effective repulsive interactions. Motivated by these observations, we propose an entropic mechanism that explains the stability of colloidal membranes and offers a general design principle for the self-assembly of 2D nanostructured materials from rod-like molecules.     

A van der Waals-type equation of state for fluids with associating molecules

The basic assumptions of van der Waals theory are contained in two well-known concepts: excluded volume (repulsive forces) and a homogeneous, isotropic field potential (attractive forces). We have superimposed on these, one more well-known concept: the existence of dimers, trimers, etc., at chemical equilibrium. With reasonable simplifying assumptions, we obtain a closed-form equation of state, applicable to all fluid densities, and potentially useful for fluids containing strongly polar or hydrogen-bonded molecules. At all temperatures and at high densities, the equation of state suggests a phase transition where, because of extensive association, a new (solid-like) phase is in equilibrium with a “normal” fluid phase. The boundary between these phases has no critical point.     

Electrical coupling between rods and cones in the tiger salamander retina.

Electrical coupling between rods and cones was studied in the salamander (Ambystoma tigrinum) retina by measuring the light responses and spectral sensitivities of rods and cones and by measuring the voltage responses from a rod to current pulses injected into a cone. A population of 10-20% of the photoreceptors exhibited a mixed-response waveform of the rod and the cone under dark-adapted conditions, and a response waveform closely resembled that of a cone in the presence of background illumination. Lucifer yellow injection revealed that these cells are morphologically identical to rods, and thus they are named rodcs. Dark-adapted rodcs exhibited a rod-like spectral sensitivity with a peak at approximately 520 nm that shifted to a cone-like spectral sensitivity with a peak at approximately 620 nm in response to background light (Purkinje shift). The voltage response of a rodc to a -1-nA current step injected into an adjacent cone is approximately 3.6 times larger than that of a rod to the same current step. These results indicate that there is a population of rods (rodcs) in the tiger salamander retina that is strongly coupled to the cones and that these cells allow significant mixture of rod and cone signals at the photoreceptor level.     

Transient electric dichroism of rod-like DNA molecules.

We report transient electric dichroism studies on monodisperse rod-like DNA molecules. By using restriction fragments and DNAs of known length, it is shown that the orientation time is accurately predicted by the theoretically calculated rotational diffusion coefficient. The field dependence of the steady-state dichroism values is not consistent with the induced electric dipole orientation mechanism, and the time dependence is not consistent with the presence of a permanent dipole moment. In order to explain the dependence of the dichroism on the electric field, the ionic strength of the medium, and the length of the macromolecule, we propose a new model in which anisotropic ion flow produces an asymmetric ion atmosphere around the polyelectrolyte, resulting in an orienting torque. From the limiting dichroism at high field, we estimate that the DNA bases are inclined at an angle of 73 degrees or less relative to the helix axis, in good agreement with the revised model of B-form DNA suggested by Levitt, in which the base pairs have a propeller-like twist. Our results establish transient electric dichroism measurements as a technique well suited for study of alterations in the length and base pair inclination of rod-like DNA molecules.     

A statistical approach to close packing of elastic rods and to DNA packaging in viral capsids

We propose a statistical approach for studying the close packing of elastic rods. This phenomenon belongs to the class of problems of confinement of low dimensional objects, such as DNA packaging in viral capsids. The method developed is based on Edwards' approach, which was successfully applied to polymer physics and to granular matter. We show that the confinement induces a configurational phase transition from a disordered (isotropic) phase to an ordered (nematic) phase. In each phase, we derive the pressure exerted by the rod (DNA) on the container (capsid) and the force necessary to inject (eject) the rod into (out of) the container. Finally, we discuss the relevance of the present results with respect to physical and biological problems. Regarding DNA packaging in viral capsids, these results establish the existence of ordered configurations, a hypothesis upon which previous calculations were built. They also show that such ordering can result from simple mechanical constraints.     

Self Assembly of Histone F2a1

Purified F₂a₁ histone molecules assemble into organized structures observable by electron microscopy. The basic structure observed at pH 8 and ionic strength of 0.15 has the shape of a bent rod with an average width of 22 Å. The average circumferential length of the rod is 220 Å and the average distance between the tips of the rod is 150 Å. When the ionic strength is increased the rods align lengthwise into intertwined fiber-like structures. In some cases bent rods assemble “face-to-face” to give circular structures. At high protein concentrations the long fibers form paracrystalline arrays. Examination of these arrays by optical diffraction yielded a meridional reflection with a spacing of 150 Å. The main additional reflections are compatible with a structure having a repeat unit of 300 Å.     

Deformation and Fracture Failure of a High-Speed Long Rod Intercepted by Linear Explosively Formed Penetrators Sequence

The fracture failure of a high-speed long rod has historically been a challenge. Since the flying plate and flying rod have a relatively low velocity, it is challenging to achieve a multi-stage fracture of the high-speed long rod within the range of existing technology. In this paper, the linear explosively formed penetrators (LEFPs) sequence with a stable flight velocity of 850 m/s were used to cut a high-speed long rod. We investigated the deformation and fracture of Φ10 mm tungsten alloy long rods having different length-diameter ratios (20, 26, 35) and different speeds (1200, 1400, 1600 m/s) by employing the LEFPs sequence with different spacings (0–40 mm) and different interception angles (30°, 60°). In the meantime, the fractured rods movement pattern was recorded with a high-speed camera to elucidate the change law of the length, speed, linear momentum, and angular momentum of fractured rods. It was found that the length loss rate of the fractured rods is as high as 27%. The fractured rods rotated around the center of mass, and the vertical speed change could reach up to 18% of the muzzle velocity of the long rod, and the greatest reduction of horizontal speed and momentum could reach 37%. The longer the interaction time between LEFPs sequence and the long rod, the more beneficial the failure of the long rod. The application of LEFPs sequence solved the difficult problem of disabling the high-speed long rod, and the quantitative analysis of the fracture failure of the long rod had an important sense for studying the terminal penetration effect of the fractured rods.     

Assembly of Tobacco Mosaic Virus In Vitro: Effect of State of Polymerization of the Protein Component

The in vitro assembly of tobacco mosaic virus from its constituent RNA and protein was followed by methods of electron microscopy. The effect of the state of polymerization of the protein upon the initiation of assembly of tobacco mosaic virus rods, and the subsequent rod elongation, was investigated. Protein in two identifiable states of polymerization was used: the 20S "disc", consisting of 34 monomers arrayed as a two-ring structure, and the 4S "A-protein", consisting of polymers in the trimer range of size. It is concluded, in confirmation of results of others, that rod assembly is initiated by the attachment of one end of the RNA chain of tobacco mosaic virus to one (or possibly a few) disc structure. Rod elongation, on the other hand, is found to take place by the sequential addition of structures of the size of A-protein, or smaller, to the previously initiated rods.     

Magnetic Anisotropy and the Orientation of Retinal Rods in a Homogeneous Magnetic Field

The reported orientation of retinal rods in a homogeneous magnetic field can be explained by the magnetic anisotropy of oriented molecules in the disc membranes of the rods.The energy of a single rod as a function of orientation in the magnetic field, the time required for alingment of the rod in a viscous medium, and the fluctuations of orientation are calculated. Arguments that rhodopsin is the constituent responsible for the effect are given. The possibility of orientation due to inhomogeneity of the magnetic field is ruled out. The application of magnetic anisotropy as an experimental tool in biology is indicated.     

Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit

Retinal photoreceptors use the heterotrimeric G protein transducin to couple rhodopsin to a biochemical cascade that underlies the electrical photoresponse. Several isoforms of each transducin subunit are present in the retina. Although rods and cones seem to contain distinct transducin subunits, it is not known whether phototransduction in a given cell type depends strictly on a single form of each subunit. To approach this question, we have deleted the gene for the rod transducin alpha-subunit in mice. In hemizygous knockout mice, there was a small reduction in retinal transducin alpha-subunit content but retinal morphology and the physiology of single rods were largely normal. In homozygous knockout mice, a mild retinal degeneration occurred with age. Rod-driven components were absent from the electroretinogram, whereas cone-driven components were retained. Every photoreceptor examined by single-cell recording failed to respond to flashes, with one exception. The solitary responsive cell was insensitive, as expected for a cone, but had a rod-like spectral sensitivity and flash response kinetics that were slow, even for rods. These results indicate that most if not all rods use a single transducin type in phototransduction.     

Fluorescence microscopy for simultaneous observation of 3D orientation and movement and its application to quantum rod-tagged myosin V

Single molecule fluorescence polarization techniques have been used for three-dimensional (3D) orientation measurements to observe the dynamic properties of single molecules. However, only few techniques can simultaneously measure 3D orientation and position. Furthermore, these techniques often require complex equipment and cumbersome analysis. We have developed a microscopy system and synthesized highly fluorescent, rod-like shaped quantum dots (Q rods), which have linear polarizations, to simultaneously measure the position and 3D orientation of a single fluorescent probe. The optics splits the fluorescence from the probe into four different spots depending on the polarization angle and projects them onto a CCD camera. These spots are used to determine the 2D position and 3D orientation. Q rod orientations could be determined with better than 10° accuracy at 33 ms time resolution. We applied our microscopy and Q rods to simultaneously measure myosin V movement along an actin filament and rotation around its own axis, finding that myosin V rotates 90° for each step. From this result, we suggest that in the two-headed bound state, myosin V necks are perpendicular to one another, while in the one-headed bound state the detached trailing myosin V head is biased forward in part by rotating its lever arm about its own axis. This microscopy system should be applicable to a wide range of dynamic biological processes that depend on single molecule orientation dynamics.     

Chiral symmetry breaking by spatial confinement in tactoidal droplets of lyotropic chromonic liquid crystals

In many colloidal systems, an orientationally ordered nematic (N) phase emerges from the isotropic (I) melt in the form of spindle-like birefringent tactoids. In cases studied so far, the tactoids always reveal a mirror-symmetric nonchiral structure, sometimes even when the building units are chiral. We report on chiral symmetry breaking in the nematic tactoids formed in molecularly nonchiral polymer-crowded aqueous solutions of low-molecular weight disodium cromoglycate. The parity is broken by twisted packing of self-assembled molecular aggregates within the tactoids as manifested by the observed optical activity. Fluorescent confocal microscopy reveals that the chiral N tactoids are located at the boundaries of cells. We explain the chirality induction as a replacement of energetically costly splay packing of the aggregates within the curved bipolar tactoidal shape with twisted packing. The effect represents a simple pathway of macroscopic chirality induction in an organic system with no molecular chirality, as the only requirements are orientational order and curved shape of confinement.     

Molecular characteristics of prion rods purified from scrapie-infected hamster brains

Purification of scrapie prions from hamster brains has demonstrated that the infectious particles contain one major protein, PrP 27-30. This protein, which is required for and inseparable from scrapie infectivity, polymerizes into heterogeneous rod-shaped particles measuring 10-20 nm in diameter and 100-200 nm in length. We attempted to identify the minimal infectious unit by disrupting aggregates of the rods. Prolonged sonication resulted in progressive fragmentation of the rods into spherical particles with a mean diameter of 19 nm and short rods with a mean length of 60 nm. No change in scrapie infectivity accompanied this profound alteration in rod morphology. In contrast, brief sonication disrupted the ultrastructure of the filamentous bacteriophage M13 and led to a marked loss in infectivity. No consistent correlation could be made between scrapie prion infectivity and disruption of the rods by a variety of treatments. Proteases, acid, base, chaotropic agents, detergents, and heat were examined for their ability to alter the morphology of the rods. The lack of correlation between ultrastructural morphology of the rods and titers of prions is consistent with the hypothesis that the rods are aggregates of prions and are not fundamental particles themselves.     

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca²⁺ current, suggesting interactions of transducin with the synaptic machinery.     

Reconstitution of rods from tobacco mosaic virus protein and RNA modified with bulky carcinogens.

Tobacco mosaic virus (TMV) RNA was treated with radioactive N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF) and (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BaP diol epoxide) to obtain 3-25 adducts per molecule. Modified full length 30S RNAs and unmodified RNA were reconstituted for various time periods with TMV protein. The particulate products were separated by ultracentrifugation, and the amounts of virus-like material were quantitated by UV spectrophotometry. The length distribution and general appearance of the virus-like rods were studied by electron microscopy. Neither type of carcinogen prevented typical rod formation, but the rate of formation and the maximal yield of reconstituted particles diminished with increasing modification by both agents. The rod length distribution also showed progressively lesser numbers of full-length virus rods. The particulate material contained approximately the same number of adducts as the modified RNA. Thus, it appears that these carcinogen modifications of guanine residues at the N-2 or C-8 atoms did not prevent orderly protein assembly on the RNA but instead slowed up this process and frequently stopped it, possibly at sites where adducts happen to be clustered.     

Electron microscopy of negatively stained and unstained fibrinogen.

Electron microscope images of negatively stained fibrinogen are predominantly asymmetric rods 450 A in length and about 60 A in width. The molecules appear to have considerable flexibility, and mass distribution along the major axis is not uniquely distinguished despite apparent beading in some particles. Scanning transmission electron microscopy of unstained fibrinogen again demonstrates that a majority of molecules are rodlike. The results differ from those obtained by negative staining in that a substantial fraction of images are trinodular with striking resemblance to those obtained by C. E. Hall and H. S. Slayter [J. Biophys. Biochem. Cytol. (1959) 5, 11--16] using the mica replica technique. The above results were obtained on glow-discharged carbon substrate films by a simple low-concentration, long-attachment-time modification of standard deposition methods that is diffusion controlled and depends on concentration and time but is independent of pH, buffer, and other staining conditions. Evidence is presented that standard attachment procedures result in artifactual images. Any models of fibrinogen in solution consequently must encompass properties that permit its visualization as an asymmetetric rod by electron microscopy as first suggested by Hall and Slayter 20 years ago.     

Length perception by dynamic touch: the effects of aging and experience

Two experiments investigated the effects of age and experience on length perception. A total of 46 participants were asked to wield and estimate the length of unseen rods by adjusting a movable board to equal their estimate of the reachable distance of the rod. The results demonstrated that (a) participants used the haptic subsystem of dynamic touch to perceive dissimilarities in object length and (b) experience playing racquet sports was more influential than the effect of age in perceptual judgments regarding object length. The results are discussed in the context of the ecological approach to haptic perception.     

Flexibility of myosin rod, light meromyosin, and myosin subfragment-2 in solution.

Myosin rod was prepared by papain proteolysis of myosin. The components of rod, light meromyosin (LMM) and subfragment-2 (S-2), were prepared by proteolysis of myosin and rod, respectively, using trypsin treated with tosylphenylalanine chloromethyl ketone. S-2, thus prepared, was of greater molecular weight than obtained previously, so that the combined molecular weights of LMM and S-2 were equal to that of rod, and S-2 contained virtually all of the region of the rod susceptible to trypsin. Electro-optical measurements were made on the three fragments in 2 mM sodium pyrophosphate, pH 9.3 at 3 degrees, over a large range of protein concentrations. Analysis of the relaxation of birefringence, at low protein concentration where there was no aggregation, showed that LMM (relaxation time 13.1 micros) behaves as a rigid cylinder. Rod (relaxation time 41.2 micros) and S-2 (relaxation time 6.0 micros) had relaxation rates that were too fast for rigid molecules of their dimensions, and therefore are not straight rods. This implies that myosin rod is flexible in the S-2 portion, presumably in the region susceptible to proteolysis. The implications of rod flexibility for the mechanism of muscle contraction are discussed.