Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.

Factors controlling the competition among rotational and vibrational energy transfer channels in glyoxal

The state-to-state transfer of rotational and vibrational energy has been studied for S1 glyoxal (CHOCHO) in collisions with D2, N2, CO and C2H4 using crossed molecular beams. A laser is used to pump glyoxal seeded in He to its S1 zero point level with zero angular momentum about its top axis (K′ = 0). The inelastic scattering to each of at least 26 S1 glyoxal rotational and rovibrational levels is monitored by dispersed S1–S0 fluorescence. Various collision partners are chosen to investigate the relative influences of reduced mass and the collision pair interaction potential on the competition among the energy transfer channels. When the data are combined with that obtained previously from other collision partners whose masses range from 2 to 84 amu, it is seen that the channel competition is controlled primarily by the kinematics of the collisional interaction. Variations in the intermolecular potential play strictly a secondary role.

High Resolution Detection of Mechanical Forces Exerted by Locomoting Fibroblasts on the Substrate

We have developed a new approach to detect mechanical forces exerted by locomoting fibroblasts on the substrate. Cells were cultured on elastic, collagen-coated polyacrylamide sheets embedded with 0.2-μm fluorescent beads. Forces exerted by the cell cause deformation of the substrate and displacement of the beads. By recording the position of beads during cell locomotion and after cell removal, we discovered that most forces were radially distributed, switching direction in the anterior region. Deformations near the leading edge were strong, transient, and variable in magnitude, consistent with active local contractions, whereas those in the posterior region were weaker, more stable, and more uniform, consistent with passive resistance. Treatment of cells with cytochalasin D or myosin II inhibitors caused relaxation of the forces, suggesting that they are generated primarily via actin–myosin II interactions; treatment with nocodazole caused no immediate effect on forces. Immunofluorescence indicated that the frontal region of strong deformation contained many vinculin plaques but no apparent concentration of actin or myosin II filaments. Strong mechanical forces in the anterior region, generated by locally activated myosin II and transmitted through vinculin-rich structures, likely play a major role in cell locomotion and in mechanical signaling with the surrounding environment.

Keratocytes Generate Traction Forces in Two PhasesV⃞

Forces generated by goldfish keratocytes and Swiss 3T3 fibroblasts have been measured with nanonewton precision and submicrometer spatial resolution. Differential interference contrast microscopy was used to visualize deformations produced by traction forces in elastic substrata, and interference reflection microscopy revealed sites of cell-substratum adhesions. Force ranged from a few nanonewtons at submicrometer spots under the lamellipodium to several hundred nanonewtons under the cell body. As cells moved forward, centripetal forces were applied by lamellipodia at sites that remained stationary on the substratum. Force increased and abruptly became lateral at the boundary of the lamellipodium and the cell body. When the cell retracted at its posterior margin, cell-substratum contact area decreased more rapidly than force, so that stress (force divided by area) increased as the cell pulled away. An increase in lateral force was associated with widening of the cell body. These mechanical data suggest an integrated, two-phase mechanism of cell motility: (1) low forces in the lamellipodium are applied in the direction of cortical flow and cause the cell body to be pulled forward; and (2) a component of force at the flanks pulls the rear margins forward toward the advancing cell body, whereas a large lateral component contributes to detachment of adhesions without greatly perturbing forward movement.

A model for individual and collective cell movement in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is a widely used model system for studying a variety of basic processes in development, including cell–cell signaling, signal transduction, pattern formation, cell motility, and the movement of tissue-like aggregates of cells. Many aspects of cell motion are poorly understood, including how individual cell behavior produces the collective motion of cells observed within the mound and slug. Herein, we describe a biologically realistic model for motile D. discoideum cells that can generate active forces, that interact via surface molecules, and that can detect and respond to chemotactic signals. We model the cells as deformable viscoelastic ellipsoids and incorporate signal transduction and cell–cell signaling by using a previously developed model. The shape constraint restricts the admissible deformations but makes the simulation of a large number of interacting cells feasible. Because the model is based on known processes, the parameters can be estimated or measured experimentally. We show that this model can reproduce the observations on the chemotactic behavior of single cells, streaming during aggregation, and the collective motion of an aggregate of cells driven by a small group of pacemakers. The model predicts that the motion of two-dimensional slugs [Bonner, J. T. (1998) Proc. Natl. Acad. Sci. USA 95, 9355–9359] results from the same behaviors that are exhibited by individual cells; it is not necessary to invoke different mechanisms or behaviors. Our computational experiments also suggest previously uncharacterized phenomena that may be experimentally observable.

A quantitative model for membrane fusion based on low-energy intermediates

The energetics of a fusion pathway is considered, starting from the contact site where two apposed membranes each locally protrude (as “nipples”) toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, because this would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of this latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.

Geometric incompatibility in a fault system.

Interdependence between geometry of a fault system, its kinematics, and seismicity is investigated. Quantitative measure is introduced for inconsistency between a fixed configuration of faults and the slip rates on each fault. This measure, named geometric incompatibility (G), depicts summarily the instability near the fault junctions: their divergence or convergence ("unlocking" or "locking up") and accumulation of stress and deformations. Accordingly, the changes in G are connected with dynamics of seismicity. Apart from geometric incompatibility, we consider deviation K from well-known Saint Venant condition of kinematic compatibility. This deviation depicts summarily unaccounted stress and strain accumulation in the region and/or internal inconsistencies in a reconstruction of block- and fault system (its geometry and movements). The estimates of G and K provide a useful tool for bringing together the data on different types of movement in a fault system. An analog of Stokes formula is found that allows determination of the total values of G and K in a region from the data on its boundary. The phenomenon of geometric incompatibility implies that nucleation of strong earthquakes is to large extent controlled by processes near fault junctions. The junctions that have been locked up may act as transient asperities, and unlocked junctions may act as transient weakest links. Tentative estimates of K and G are made for each end of the Big Bend of the San Andreas fault system in Southern California. Recent strong earthquakes Landers (1992, M = 7.3) and Northridge (1994, M = 6.7) both reduced K but had opposite impact on G: Landers unlocked the area, whereas Northridge locked it up again.

Mixed synapses discovered and mapped throughout mammalian spinal cord.

Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system.

Hyperplane arrangements, interval orders, and trees.

A hyperplane arrangement is a finite set of hyperplanes in a real affine space. An especially important arrangement is the braid arrangement, which is the set of all hyperplanes xi - xj = 1, 1 deformations of the braid arrangement are surveyed. In particular, there are unexpected connections with the theory of interval orders and with the enumeration of trees. For instance, the number of labeled interval orders that can be obtained from n intervals I1,..., In of generic lengths is counted. There is also discussed an arrangement due to N. Linial whose number of regions is the number of alternating (or intransitive) trees, as defined by Gelfand, Graev, and Postnikov [Gelfand, I. M., Graev, M. I., and Postnikov, A. (1995), preprint]. Finally, a refinement is given, related to counting labeled trees by number of inversions, of a result of Shi [Shi, J.-Y. (1986), Lecture Notes in Mathematics, no. 1179, Springer-Verlag] that a certain deformation of the braid arrangement has (n + 1)n-1 regions.