Control of Cyclin D1, p27Kip1, and Cell Cycle Progression in Human Capillary Endothelial Cells by Cell Shape and Cytoskeletal Tension

The extracellular matrix (ECM) plays an essential role in the regulation of cell proliferation during angiogenesis. Cell adhesion to ECM is mediated by binding of cell surface integrin receptors, which both activate intracellular signaling cascades and mediate tension-dependent changes in cell shape and cytoskeletal structure. Although the growth control field has focused on early integrin and growth factor signaling events, recent studies suggest that cell shape may play an equally critical role in control of cell cycle progression. Studies were carried out to determine when cell shape exerts its regulatory effects during the cell cycle and to analyze the molecular basis for shape-dependent growth control. The shape of human capillary endothelial cells was controlled by culturing cells on microfabricated substrates containing ECM-coated adhesive islands with defined shape and size on the micrometer scale or on plastic dishes coated with defined ECM molecular coating densities. Cells that were prevented from spreading in medium containing soluble growth factors exhibited normal activation of the mitogen-activated kinase (erk1/erk2) growth signaling pathway. However, in contrast to spread cells, these cells failed to progress through G1 and enter S phase. This shape-dependent block in cell cycle progression correlated with a failure to increase cyclin D1 protein levels, down-regulate the cell cycle inhibitor p27Kip1, and phosphorylate the retinoblastoma protein in late G1. A similar block in cell cycle progression was induced before this same shape-sensitive restriction point by disrupting the actin network using cytochalasin or by inhibiting cytoskeletal tension generation using an inhibitor of actomyosin interactions. In contrast, neither modifications of cell shape, cytoskeletal structure, nor mechanical tension had any effect on S phase entry when added at later times. These findings demonstrate that although early growth factor and integrin signaling events are required for growth, they alone are not sufficient. Subsequent cell cycle progression and, hence, cell proliferation are controlled by tension-dependent changes in cell shape and cytoskeletal structure that act by subjugating the molecular machinery that regulates the G1/S transition.

The effect of cultivation on the size, shape, and persistence of disease patches in fields

Epidemics of soil-borne plant disease are characterized by patchiness because of restricted dispersal of inoculum. The density of inoculum within disease patches depends on a sequence comprising local amplification during the parasitic phase followed by dispersal of inoculum by cultivation during the intercrop period. The mechanisms that control size, shape, and persistence have received very little rigorous attention in epidemiological theory. Here we derive a model for dispersal of inoculum in soil by cultivation that takes account into the discrete stochastic nature of the system in time and space. Two parameters, probability of movement and mean dispersal distance, characterize lateral dispersal of inoculum by cultivation. The dispersal parameters are used in combination with the characteristic area and dimensions of host plants to identify criteria that control the shape and size of disease patches. We derive a critical value for the probability of movement for the formation of cross-shaped patches and show that this is independent of the amount of inoculum. We examine the interaction between local amplification of inoculum by parasitic activity and subsequent dilution by dispersal and identify criteria whereby asymptomatic patches may persist as inoculum falls below a threshold necessary for symptoms to appear in the subsequent crop. The model is motivated by the spread of rhizomania, an economically important soil-borne disease of sugar beet. However, the results have broad applicability to a very wide range of diseases that survive as discrete units of inoculum. The application of the model to patch dynamics of weed seeds and local introductions of genetically modified seeds is also discussed.

Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand–DNA complexes: The interaction between DNA and calichearubicin B

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (φ) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand–DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin γ1I. The unwinding angle for CRB calculated by this method is −5.3 ± 0.5°. Its Ka value is 0.20 × 106 M−1. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.

Transition-state structure as a unifying basis in protein-folding mechanisms: Contact order, chain topology, stability, and the extended nucleus mechanism

I attempt to reconcile apparently conflicting factors and mechanisms that have been proposed to determine the rate constant for two-state folding of small proteins, on the basis of general features of the structures of transition states. Φ-Value analysis implies a transition state for folding that resembles an expanded and distorted native structure, which is built around an extended nucleus. The nucleus is composed predominantly of elements of partly or well-formed native secondary structure that are stabilized by local and long-range tertiary interactions. These long-range interactions give rise to connecting loops, frequently containing the native loops that are poorly structured. I derive an equation that relates differences in the contact order of a protein to changes in the length of linking loops, which, in turn, is directly related to the unfavorable free energy of the loops in the transition state. Kinetic data on loop extension mutants of CI2 and α-spectrin SH3 domain fit the equation qualitatively. The rate of folding depends primarily on the interactions that directly stabilize the nucleus, especially those in native-like secondary structure and those resulting from the entropy loss from the connecting loops, which vary with contact order. This partitioning of energy accounts for the success of some algorithms that predict folding rates, because they use these principles either explicitly or implicitly. The extended nucleus model thus unifies the observations of rate depending on both stability and topology.

How does a β-hairpin fold/unfold? Competition between topology and heterogeneity in a solvable model

We study the competition between topological effects and sequence inhomogeneities in determining the thermodynamics and the un/folding kinetics of a β-hairpin. Our work utilizes a new exactly solvable model that allows for arbitrary configurations of native contacts. In general, the competition between heterogeneity and topology results in a crossover of the dominant transition state. Interestingly, near this crossover, the single reaction coordinate picture can be seriously misleading. Our results also suggest that inferring the folding pathway from unfolding simulations is not always justified.

Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: Implications for retroviral assembly mechanisms

We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR−) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR− MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR− nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR− MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of ≈45 Å and a length of ≈200 Å. Because of the pleomorphic shape and paracrystalline packing of the Gag–RNA complexes, we raise the possibility that assembly of MuLV is driven by protein–RNA, as well as protein–protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.

Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34cdc2 mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Continuous and lurching traveling pulses in neuronal networks with delay and spatially decaying connectivity

Propagation of discharges in cortical and thalamic systems, which is used as a probe for examining network circuitry, is studied by constructing a one-dimensional model of integrate-and-fire neurons that are coupled by excitatory synapses with delay. Each neuron fires only one spike. The velocity and stability of propagating continuous pulses are calculated analytically. Above a certain critical value of the constant delay, these pulses lose stability. Instead, lurching pulses propagate with discontinuous and periodic spatio-temporal characteristics. The parameter regime for which lurching occurs is strongly affected by the footprint (connectivity) shape; bistability may occur with a square footprint shape but not with an exponential footprint shape. For strong synaptic coupling, the velocity of both continuous and lurching pulses increases logarithmically with the synaptic coupling strength gsyn for an exponential footprint shape, and it is bounded for a step footprint shape. We conclude that the differences in velocity and shape between the front of thalamic spindle waves in vitro and cortical paroxysmal discharges stem from their different effective delay; in thalamic networks, large effective delay between inhibitory neurons arises from their effective interaction via the excitatory cells which display postinhibitory rebound.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Primary Mesenchymal Cells Isolated from SPARC-null Mice Exhibit Altered Morphology and Rates of Proliferation

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.

Nuclear Pore Complex Number and Distribution throughout the Saccharomyces cerevisiae Cell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.

Structural invariance of constitutively active and inactive mutants of Acanthamoeba myosin IC bound to F-actin in the rigor and ADP-bound states

The three single-headed monomeric myosin I isozymes of Acanthamoeba castellanii (AMIs)—AMIA, AMIB, and AMIC—are among the best-studied of all myosins. We have used AMIC to study structural correlates of myosin’s actin-activated ATPase. This activity is normally controlled by phosphorylation of Ser-329, but AMIC may be switched into constitutively active or inactive states by substituting this residue with Glu or Ala, respectively. To determine whether activation status is reflected in structural differences in the mode of attachment of myosin to actin, these mutant myosins were bound to actin filaments in the absence of nucleotide (rigor state) and visualized at 24-Å resolution by using cryoelectron microscopy and image reconstruction. No such difference was observed. Consequently, we suggest that regulation may be affected not by altering the static (time-averaged) structure of AMIC but by modulating its dynamic properties, i.e., molecular breathing. The tail domain of vertebrate intestinal brush-border myosin I has been observed to swing through 31° on binding of ADP. However, it was predicted on grounds of differing kinetics that any such effects with AMIC should be small [Jontes, J. D., Ostap, E. M., Pollard, T. D. & Milligan, R. A. (1998) J. Cell Biol. 141, 155–162]. We have confirmed this hypothesis by observing actin-associated AMIC in its ADP-bound state. Finally, we compared AMIC to brush-border myosin I and AMIB, which were previously studied under similar conditions. In each case, the shape and angle of attachment to F-actin of the catalytic domain is largely conserved, but the domain structure and disposition of the tail is distinctively different for each myosin.

Synaptic regulation of protein synthesis and the fragile X protein

Protein synthesis occurs in neuronal dendrites, often near synapses. Polyribosomal aggregates often appear in dendritic spines, particularly during development. Polyribosomal aggregates in spines increase during experience-dependent synaptogenesis, e.g., in rats in a complex environment. Some protein synthesis appears to be regulated directly by synaptic activity. We use “synaptoneurosomes,” a preparation highly enriched in pinched-off, resealed presynaptic processes attached to resealed postsynaptic processes that retain normal functions of neurotransmitter release, receptor activation, and various postsynaptic responses including signaling pathways and protein synthesis. We have found that, when synaptoneurosomes are stimulated with glutamate or group I metabotropic glutamate receptor agonists such as dihydroxyphenylglycine, mRNA is rapidly taken up into polyribosomal aggregates, and labeled methionine is incorporated into protein. One of the proteins synthesized is FMRP, the protein that is reduced or absent in fragile X mental retardation syndrome. FMRP has three RNA-binding domains and reportedly binds to a significant number of mRNAs. We have found that dihydroxyphenylglycine-activated protein synthesis in synaptoneurosomes is dramatically reduced in a knockout mouse model of fragile X syndrome, which cannot produce full-length FMRP, suggesting that FMRP is involved in or required for this process. Studies of autopsy samples from patients with fragile X syndrome have indicated that dendritic spines may fail to assume a normal mature size and shape and that there are more spines per unit dendrite length in the patient samples. Similar findings on spine size and shape have come from studies of the knockout mouse. Study of the development of the somatosensory cortical region containing the barrel-like cell arrangements that process whisker information suggests that normal dendritic regression is impaired in the knockout mouse. This finding suggests that FMRP may be required for the normal processes of maturation and elimination to occur in cerebral cortical development.

Pattern and inhibition-dependent invasion of pyramidal cell dendrites by fast spikes in the hippocampus in vivo.

The invasion of sodium spikes from the soma into dendrites was studied in hippocampal pyramidal cells by simultaneous extracellular and intracellular recordings in anesthetized rats and by simultaneous extracellular recordings of the somatic and dendritic potentials in freely behaving animals. During complex-spike patterns, recorded in the immobile or sleeping animal, dendritic invasion of successive spikes was substantially attenuated. Complex-spike bursts occurred in association with population discharge of CA3-CA1 pyramidal cells (sharp wave field events). Synaptic inhibition reduced the amplitude of sodium spikes in the dendrites and prevented the occurrence of calcium spikes. These findings indicate that (i) the voltage-dependent calcium influx into the dendrites is under the control of inhibitory neurons and (ii) the temporal coincidence of synaptic depolarization and activation of voltage-dependent calcium conductances by the backpropagating spikes during sharp wave bursts may be critical for synaptic plasticity in the intact hippocampus.