Molecular switch in signal transduction: Reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the γ-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD–Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of α2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of α2 and facilitates the unwinding of a helical turn of α2 (residues 66–69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

A hierarchical neuronal network for planning behavior

Planning a goal-directed sequence of behavior is a higher function of the human brain that relies on the integrity of prefrontal cortical areas. In the Tower of London test, a puzzle in which beads sliding on pegs must be moved to match a designated goal configuration, patients with lesioned prefrontal cortex show deficits in planning a goal-directed sequence of moves. We propose a neuronal network model of sequence planning that passes this test and, when lesioned, fails in a way that mimics prefrontal patients’ behavior. Our model comprises a descending planning system with hierarchically organized plan, operation, and gesture levels, and an ascending evaluative system that analyzes the problem and computes internal reward signals that index the correct/erroneous status of the plan. Multiple parallel pathways connecting the evaluative and planning systems amend the plan and adapt it to the current problem. The model illustrates how specialized hierarchically organized neuronal assemblies may collectively emulate central executive or supervisory functions of the human brain.

Matchings and phylogenetic trees

This paper presents a natural coordinate system for phylogenetic trees using a correspondence with the set of perfect matchings in the complete graph. This correspondence produces a distance between phylogenetic trees, and a way of enumerating all trees in a minimal step order. It is useful in randomized algorithms because it enables moves on the space of trees that make random optimization strategies “mix” quickly. It also promises a generalization to intermediary trees when data are not decisive as to their choice of tree, and a new way of constructing Bayesian priors on tree space.

From snapshot to movie: φ analysis of protein folding transition states taken one step further

Kinetic anomalies in protein folding can result from changes of the kinetic ground states (D, I, and N), changes of the protein folding transition state, or both. The 102-residue protein U1A has a symmetrically curved chevron plot which seems to result mainly from changes of the transition state. At low concentrations of denaturant the transition state occurs early in the folding reaction, whereas at high denaturant concentration it moves close to the native structure. In this study we use this movement to follow continuously the formation and growth of U1A's folding nucleus by φ analysis. Although U1A's transition state structure is generally delocalized and displays a typical nucleation–condensation pattern, we can still resolve a sequence of folding events. However, these events are sufficiently coupled to start almost simultaneously throughout the transition state structure.

The Drosophila melanogaster Homologue of the Xeroderma Pigmentosum D Gene Product Is Located in Euchromatic Regions and Has a Dynamic Response to UV Light-induced Lesions in Polytene Chromosomes

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne’s syndrome, and trichothiodystrophy. XPD has a 5′- to 3′-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

Transduction of Basolateral-to-Apical Signals across Epithelial Cells: Ligand-stimulated Transcytosis of the Polymeric Immunoglobulin Receptor Requires Two Signals

Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA–pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate–sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

We present a new map showing dimeric kinesin bound to microtubules in the presence of ADP that was obtained by electron cryomicroscopy and image reconstruction. The directly bound monomer (first head) shows a different conformation from one in the more tightly bound empty state. This change in the first head is amplified as a movement of the second (tethered) head, which tilts upward. The atomic coordinates of kinesin·ADP dock into our map so that the tethered head associates with the bound head as in the kinesin dimer structure seen by x-ray crystallography. The new docking orientation avoids problems associated with previous predictions; it puts residues implicated by proteolysis-protection and mutagenesis studies near the microtubule but does not lead to steric interference between the coiled-coil tail and the microtubule surface. The observed conformational changes in the tightly bound states would probably bring some important residues closer to tubulin. As expected from the homology with kinesin, the atomic coordinates of nonclaret disjunctional protein (ncd)·ADP dock in the same orientation into the attached head in a map of microtubules decorated with dimeric ncd·ADP. Our results support the idea that the observed direct interaction between the two heads is important at some stages of the mechanism by which kinesin moves processively along microtubules.

The Dynamics of Golgi Protein Traffic Visualized in Living Yeast Cells

We describe for the first time the visualization of Golgi membranes in living yeast cells, using green fluorescent protein (GFP) chimeras. Late and early Golgi markers are present in distinct sets of scattered, moving cisternae. The immediate effects of temperature-sensitive mutations on the distribution of these markers give clues to the transport processes occurring. We show that the late Golgi marker GFP-Sft2p and the glycosyltransferases, Anp1p and Mnn1p, disperse into vesicle-like structures within minutes of a temperature shift in sec18, sft1, and sed5 cells, but not in sec14 cells. This is consistent with retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to early cisternae containing the target SNARE Sed5p. Strikingly, Sed5p itself moves rapidly to the endoplasmic reticulum (ER) in sec12 cells, implying that it cycles through the ER. Electron microscopy shows that Golgi membranes vesiculate in sec18 cells within 10 min of a temperature shift. These results emphasize the dynamic nature of Golgi cisternae and satisfy the kinetic requirements of a cisternal maturation model in which all resident proteins must undergo retrograde vesicular transport, either within the Golgi complex or from there to the ER, as anterograde cargo advances.

Time-Lapse Video Microscopy of Gliding Motility in Toxoplasma gondii Reveals a Novel, Biphasic Mechanism of Cell LocomotionV⃞

Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of intracellular parasites that share a unique form of gliding motility. Gliding is substrate dependent and occurs without apparent changes in cell shape and in the absence of traditional locomotory organelles. Here, we demonstrate that gliding is characterized by three distinct forms of motility: circular gliding, upright twirling, and helical rotation. Circular gliding commences while the crescent-shaped parasite lies on its right side, from where it moves in a counterclockwise manner at a rate of ∼1.5 μm/s. Twirling occurs when the parasite rights itself vertically, remaining attached to the substrate by its posterior end and spinning clockwise. Helical gliding is similar to twirling except that it occurs while the parasite is positioned horizontally, resulting in forward movement that follows the path of a corkscrew. The parasite begins lying on its left side (where the convex side is defined as dorsal) and initiates a clockwise revolution along the long axis of the crescent-shaped body. Time-lapse video analyses indicated that helical gliding is a biphasic process. During the first 180o of the turn, the parasite moves forward one body length at a rate of ∼1–3 μm/s. In the second phase, the parasite flips onto its left side, in the process undergoing little net forward motion. All three forms of motility were disrupted by inhibitors of actin filaments (cytochalasin D) and myosin ATPase (butanedione monoxime), indicating that they rely on an actinomyosin motor in the parasite. Gliding motility likely provides the force for active penetration of the host cell and may participate in dissemination within the host and thus is of both fundamental and practical interest.

Length suppression in histone messenger RNA 3′-end maturation: Processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA

Efficient 3′-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem–loop and a histone downstream element (HDE) that base pairs with the U7 small ribonuclearprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

Toward functional genomics in bacteria: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.

Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10−7 cm2/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10−7 cm2/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10−7 cm2/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.

Visualization of elongation factor G on the Escherichia coli 70S ribosome: The mechanism of translocation

During protein synthesis, elongation factor G (EF-G) binds to the ribosome and promotes the step of translocation, a process in which tRNA moves from the A to the P site of the ribosome and the mRNA is advanced by one codon. By using three-dimensional cryo-electron microscopy, we have visualized EF-G in a ribosome–EF-G–GDP–fusidic acid complex. Fitting the crystal structure of EF-G–GDP into the cryo density map reveals a large conformational change mainly associated with domain IV, the domain that mimics the shape of the anticodon arm of the tRNA in the structurally homologous ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. The tip portion of this domain is found in a position that overlaps the anticodon arm of the A-site tRNA, whose position in the ribosome is known from a study of the pretranslocational complex, implying that EF-G displaces the A-site tRNA to the P site by physical interaction with the anticodon arm.

Activity of individual ERCC1 and XPF subunits in DNA nucleotide excision repair

ERCC1–XPF is a structure-specific nuclease with two subunits, ERCC1 and XPF. The enzyme cuts DNA at junctions where a single strand moves 5′ to 3′ away from a branch point with duplex DNA. This activity has a central role in nucleotide excision repair (NER), DNA cross-link repair and recombination. To dissect the activities of the nuclease it is necessary to investigate the subunits individually, as studies of the enzyme so far have only used the heterodimeric complex. We produced recombinant ERCC1 and XPF separately in Escherichia coli as soluble proteins. Activity was monitored by a sensitive dual incision assay for NER by complementation of cell extracts. XPF and ERCC1 are unstable in mammalian cells in the absence of their partners but we found, surprisingly, that ERCC1 alone could confer some repair to extracts from ERCC1-defective cells. A version of ERCC1 lacking the first 88 non-conserved amino acids was also functional. This indicated that a small amount of active XPF was present in ERCC1 extracts, and immunoassays showed this to be the case. Some repair in XPF-defective extracts could be achieved by adding ERCC1 and XPF proteins together, but not by adding only XPF. The results show for the first time that functional ERCC1–XPF can be formed from separately produced subunits. Protein sequence comparison revealed similarity between the ERCC1 family and the C-terminal region of the XPF family, including the regions of both proteins that are necessary for the ERCC1–XPF heterodimeric interaction. This suggests that the ERCC1 and XPF families are related via an ancient duplication.

How amoeboids self-organize into a fruiting body: Multicellular coordination in Dictyostelium discoideum

When individual amoebae of the cellular slime mold Dictyostelium discoideum are starving, they aggregate to form a multicellular migrating slug, which moves toward a region suitable for culmination. The culmination of the morphogenesis involves complex cell movements that transform a mound of cells into a globule of spores on a slender stalk. The movement has been likened to a “reverse fountain,” whereby prestalk cells in the upper part form a stalk that moves downwards and anchors to the substratum, while prespore cells in the lower part move upwards to form the spore head. So far, however, no satisfactory explanation has been produced for this process. Using a computer simulation that we developed, we now demonstrate that the processes that are essential during the earlier stages of the morphogenesis are in fact sufficient to produce the dynamics of the culmination stage. These processes are cAMP signaling, differential adhesion, cell differentiation, and production of extracellular matrix. Our model clarifies the processes that generate the observed cell movements. More specifically, we show that periodic upward movements, caused by chemotactic motion, are essential for successful culmination, because the pressure waves they induce squeeze the stalk downwards through the cell mass. The mechanisms revealed by our model have a number of self-organizing and self-correcting properties and can account for many previously unconnected and unexplained experimental observations.