N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.

The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

Enzyme catalysis: a new definition accounting for noncovalent substrate- and product-like states.

Biological catalysis frequently causes changes in noncovalent bonding. By building on Pauling's assertion that any long-lived, chemically distinct interaction is a chemical bond, this article redefines enzyme catalysis as the facilitated making and/or breaking of chemical bonds, not just of covalent bonds. It is also argued that nearly every ATPase or GTPase is misnamed as a hydrolase and actually belongs to a distinct class of enzymes, termed here 'energases'. By transducing covalent bond energy into mechanical work, energases mediate such fundamental processes as protein folding, self-assembly, G-protein interactions, DNA replication, chromatin remodeling and even active transport.

Shigella actin-based motility in the presence of truncated vinculin.

Mounting evidence supports the role of truncated vinculin in the intracellular actin-based motility of Shigella flexneri. Vinculin's role was recently questioned by Goldberg [1997: Cell Motil Cytoskeleton 37:44-53] who observed Shigella motility in mouse embryonal carcinoma 5.51 cells, a genetically modified cell line that reputedly lacked vinculin. That challenge implicitly relied on the assumption that 5.51 cells had no detectable vinculin polypeptide and lacked full-length vinculin mRNA. Despite the appearance of being an unambiguous test of vinculin's role in Shigella motility, 5.51 cells were shown to contain adequate amounts of truncated vinculin (as well as the corresponding mRNA transcript) to support bacterial locomotion. We also examined Shigella locomotion in gamma229 cells, a related embryonal carcinoma cell line containing approximately one-half the vinculin content found in 5.51 cells. We observed that there was a commensurate twofold decrease in the Shigella motility rate, as compared to 5.51 cells; this finding raises the possibility that vinculin can become a rate-limiting factor under some circumstances. Immunofluorescence microscopy using vin 11-5 monoclonal antibody directed against the vinculin head domain showed intense staining of Shigella rocket tails in both gamma229 and 5.51 cells. Our findings clearly demonstrate that motility in 5.51 cells cannot be regarded as a valid criterion for evaluating the role of truncated vinculin in Shigella motility.

Profilin promotes barbed-end actin filament assembly without lowering the critical concentration.

The mechanism of profilin-promoted actin polymerization has been systematically reinvestigated. Rates of barbed-end elongation onto Spectrin.4.1. Actin seeds were measured by right angle light scattering to avoid confounding effects of pyrenyl-actin, and KINSIM was used to analyze elongation progress curves. Without thymosin-beta4, both actin and Profilin. Actin (P.A) are competent in barbed-end polymerization, and kinetic simulations yielded the same bimolecular rate constant ( approximately 10 x 10(6) M(-1) s(-1)) for actin monomer or Profilin. Actin. When measured in the absence of profilin, actin assembly curves over a 0.7-4 microM thymosin-beta4 concentration range fit a simple monomer sequestering model (1 microM K(D) for Thymosin-beta4. Actin). The corresponding constant for thymosin-beta4.pyrenyl-Actin, however, was significantly higher ( approximately 9-10 microM), suggesting that the fluorophore markedly weakens binding to thymosin-beta4. With solutions of actin (2 microM) and thymosin-beta4 (2 or 4 microM), the barbed-end assembly rate rose with increasing profilin concentration (0.7-2 microM). Actin assembly in presence of thymosin-beta4 and profilin fit a simple thermodynamic energy cycle, thereby disproving an earlier claim (D. Pantaloni and M.-F. Carlier (1993) Cell 75, 1007-1014) that profilin promotes nonequilibrium filament assembly by accelerating hydrolysis of filament-bound ATP. Our findings indicate that profilin serves as a polymerization catalyst that captures actin monomers from Thymosin-beta4. Actin and ushers actin as a Profilin. Actin complex onto growing barbed filament ends.

Disulfide-cross-linked tau and MAP2 homodimers readily promote microtubule assembly.

The neuronal proteins Tau and MAP2 use homologous C-terminal MT-binding regions (MTBRs) to interact with microtubules, F-actin, and intermediate filaments. Although Tau-MTBR is the principal component of pronase-treated Alzheimer paired helical filaments, both Tau and MAP2 form filaments in vitro from disulfide-linked homodimers. That the critical thiol lies within a domain needed for MT binding raised the question: Does disulfide formation block Tau-Tau or MAP2-MAP2 dimer binding to microtubules, thereby acting to divert dimers toward filament formation? We now report that cross-linked Tau and MAP2 homodimers readily promote tubulin polymerization and that monomer and dimer affinity for MTs is surprisingly similar. Therefore, disulfide cross-bridging into homodimers is unlikely to be a drive force for filament formation in Alzheimer's disease.

Actin-based motility of the intracellular pathogen Listeria monocytogenes: assessing the inhibitory specificity of ABM-1 peptide analogues.

Actin-Based Motility motifs [ABM-1 sequence = (D/E)FPPPPX(D/E), where X = P or T, and ABM-2 sequence = XPPPPP, where X denotes G, A, L, P, and S] facilitate assembly of an activated motility complex. Potent inhibition of intracellular motility of pathogens by ABM-1 and ABM-2 peptide analogues has served as a criterion for investigating actin-based motility. To assess the specificity of ABM-1 peptide inhibitors, we microinjected proline-rich peptides into Listeria-infected PtK2 host cells. Use of a combinatorial ABM-1 peptide library (empirical formula = D1E2F2P4T1) demonstrated that high-potency inhibition requires a precise sequence, and not merely a particular amino acid composition. Calculated concentrations of specific sequences in this library indicate that the entire (D/E)FPPPPX(D/E) motif is needed to achieve high-affinity inhibition in living cells. The failure of the well known proline-rich SH3 binding antagonists VSL-12 or APP-12 to inhibit Listeria motility also indicates that SH3 interactions are unlikely to control actin-based motility directly.

Vaccinia locomotion in host cells: evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2.

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of Listeria and Shigella actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in Listeria ActA and in the p90 host-cell vinculin fragment generated by Shigella infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 microM (intracellular) ABM-2 peptide (GPPPPP)3 blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of Listeria, Shigella, and now vaccinia.

Advances in the enzymology of glutamine synthesis.

Meister's proposal of a gamma-glutamyl-P intermediate in the glutamine synthetase reaction set the scene for understanding how the stepwise activation of the carboxyl group greatly increased its susceptibility toward nucleophilic attack and amide bond synthesis. Topics covered in this review include: the discovery of the enzymatic synthesis of glutamine; the role of glutamine synthetase in defining the thermodynamics of ATPases; early isotopic tracer studies of the synthetase reaction; the proposed intermediacy of gamma-glutamyl-phosphate; the mechanism of methionine sulfoximine inhibition; stereochemical mapping of the enzyme's active site; detection of enzyme reaction cycle intermediates; borohydride trapping of gamma-glutamyl-P; positional isotope exchanges catalyzed by glutamine synthetase; regulation of bacterial enzyme; and a brief account of how knowledge of the atomic structure of bacterial glutamine synthetase has clarified ligand binding interactions. Concluding remarks also address how the so-called "Protein Ligase Problem" may be solved by extending the catalytic versatility of carboxyl-group activating enzymes.

Vinculin proteolysis unmasks an ActA homolog for actin-based Shigella motility.

To generate the forces needed for motility, the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular pathogens Shigella flexneri and Listeria monocytogenes use molecular mimicry to create their own actin-based motors. We raised an antibody (designated FS-1) against the FEFPPPPTDE sequence of Listeria ActA, and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular locomotion upon microinjection of Shigella-infected cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL. Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length vinculin may be masked by its tail region and that this sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the head region of vinculin (Vin 11-5) localized to the back of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular motility, underscoring the functional importance of this sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected p90, but not full-length vinculin, accelerates rates of pathogen motility by a factor of 3 +/- 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based Shigella motility, and that supplementing cells with p90 stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-21885) and to vasodilator-stimulated phosphoprotein (VASP) (Brindle, N.P.J., M. R. Hold, J.E. Davies, C.J. Price, and D.R. Critchley. 1996. Biochem. J. 318:753-757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence. Unmasking of this site serves as a molecular switch that initiates assembly of an actin-based motility complex containing VASP and profilin.

Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): implications for actin-based Listeria motility.

Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.

ABM-1 and ABM-2 homology sequences: consensus docking sites for actin-based motility defined by oligoproline regions in Listeria ActA surface protein and human VASP.

Actin-based motility involves a cascade of binding interactions designed to assemble actin regulatory proteins into functional locomotory units. Listeria ActA surface protein contains a series of nearly identical EFPPPPTDE-type oligoproline sequences for binding vasodilator-stimulated phosphoprotein (VASP). The latter is a tetrameric protein with numerous GPP-PPP docking sites for profilin, a 15 kDa regulatory protein that promotes actin filament assembly. Analysis of known actin regulatory proteins led to the identification of distinct Actin-Based Motility homology sequences ABM-1; (D/E)FPPPPX(D/E); and ABM-2, XPPPPP (where X denotes G, A, L, and S).

In vitro polymerization of embryonic MAP-2c and fragments of the MAP-2 microtubule binding region into structures resembling paired helical filaments.

The microtubule-associated protein Tau is widely regarded as the principal component of paired helical filaments comprising Alzheimer neurofibrillary tangles. Tau fragments containing the non-identical repeat region formed structures resembling paired helical filaments (Schweers, O., Mandelkow, M., Biernat, J., and Mandelkow, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8463-8467). MAP-2, the other structurally related neuronal microtubule-associated protein, has not been implicated in paired helical filament formation. We now describe the assembly of paired helical filament-like structures from MAP-2 polypeptides containing only 100 residues. A dimeric species, stabilized by an interchain disulfide, appears to be involved in the assembly reaction. We also investigated the polymerization of embryonic MAP-2c, which, except for its microtubule binding region, is structurally distinct from Tau. Full-length MAP-2c formed paired helical filament-like polymers. Polymerized MAP-2c and the microtubule binding region fragment readily bound thioflavin-S, a dye that stains paired helical filaments in the histochemical diagnosis of Alzheimer's disease. Our unprecedented finding that a small MAP-2 microtubule binding region fragment and MAP-2c can form structures resembling straight filaments or Pronase-treated paired helical filaments raises fundamental questions concerning the role of MAP-2 in the pathobiology of Alzheimer disease.

Self-assembly of the brain MAP-2 microtubule-binding region into polymeric structures resembling Alzheimer filaments.

The neuronal microtubule-associated protein known as MAP-2 has not been considered to be a subunit of paired helical filaments (PHFs) in neurofibrillary tangles seen in Alzheimer's Disease. We now describe the assembly of paired helical filament-like structures from MAP-2's 203-residue microtubule-binding region (MTBR). SDS gel electrophoresis and equilibrium ultracentrifugation suggest that a dimeric form, cross-linked by an interchain disulfide, is involved in polymerization. MAP-2 MTBR polymers bind thioflavin-S, a dye used to histochemically localize Alzheimer neurofibrillary tangles. Our finding that PHF-like structures assemble from a MAP-2 fragment raises new questions about MAP-2's role in the etiology of Alzheimer's Disease.

Recognition of two classes of oligoproline sequences in profilin-mediated acceleration of actin-based Shigella motility.

The gram negative rod Shigella flexneri uses it surface protein IcsA to induce host cell actin assembly and to achieve intracellular motility. Yet, the IcsA protein lacks the oligoproline sequences found in ActA, the surface protein required for locomotion of the gram positive rod Listeria monocytogenes. Microinjection of a peptide matching the second ActA oligoproline repeat (FEFPPPPTDE) stops Listeria locomotion (Southwick, F.S., and D.L. Purich. 1994a. Proc. Natl. Acad. Sci. USA. 91:5168-5172), and submicromolar concentrations (intracellular concentration 80-800 nM) similarly arrest Shigella rocket-tail assembly and intracellular motility. Coinjection of a binary solution containing profilin and the ActA analogue increased the observed rates of intracellular motility by a factor of three (mean velocity 0.90 +/- 0.07 mu m/s, SD n=16 before injection vs 0.3 +/- 0.1 mu m/s, n=33 postinjection, intracellular concentration = 80 nM profilin plus 80 nM ActA analogue). Recent evidence suggests the ActA analogue may act by displacing the profilin-binding protein VASP (Pistor, S.C., T. Chakaborty, V. Walter, and J. Wehland. 1995. Curr. Biol. 5:517-525). At considerably higher intracellular concentrations (10 muM), the VASP oligoproline sequence (GPPPPP)3 thought to represent the profilin-binding site (Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B.M. Jockusch, and U. Walter. 1995. EMBO (Eur. Mol. Biol. Organ.) J. 14:1583-1589) also inhibited Shigella movement. A binary mixture of the VASP analogue and profilin (each 10 muM intracellular concentration) led to a doubling of Shigella intracellular migration velocity (0.09 +/- 0.06 mu m/s, n = 25 preinjection vs 0.18 +/- 0.10 mu m/s, n = 61 postinjection). Thus, the two structurally divergent bacteria, Listeria and Shigella, have adopted convergent mechanisms involving profilin recognition of VASP oligoproline sequences and VASP recognition of oligoproline sequences in ActA or an ActA-like host protein to induce host cell actin assembly and to provide the force for intracellular locomotion and cell-cell spread.

Non-cooperative binding of the MAP-2 microtubule-binding region to microtubules.

Microtubule-associated protein (MAP)-2 is a multi-domain cytoskeletal protein that copurifies with brain microtubules (MTs) through repeated cycles of warm polymerization and cold disassembly. Recent equilibrium binding studies of high molecular weight MAP-2ab to taxol-stabilized MTs suggest that the interactions are highly cooperative, as indicated by sigmoidal binding curves, non-linear Scatchard plots, and an apparent all-or-none response in MAP binding in titration experiments (Wallis, K. T., Azhar, S., Rho, M. B., Lewis, S. A., Cowan, N. J., and Murphy, D. B. (1993) J. Biol. Chem. 268, 15158-15167). To learn more about the mechanism of MAP-2 binding to MTs, we investigated the binding properties of bacterially expressed MT-binding region (MTBR) of bovine brain MAP-2. Scatchard plots of the binding data showed no evidence of cooperativity, as reflected by the linear plots of v/[MTBR]free versus v. The stoichiometry was 1-1.1 mol of MTBR/mol of tubulin dimer, and the dissociation constant for the MTBR was 1.1 microM. Bovine brain tau protein competitively inhibited MAP-2 binding, as evidenced by an increased Kd value for MTBR binding to MTs. Although the second repeat peptide m2 (VTSK-CGSLKNIRHRPGGG) is thought to play a dominant role in MAP-2 binding to MTs, a MTBR mutant (with m2 replaced by the third octadecapeptide repeat m3) displays an Kd of 2.8 +/- 0.1 microM and stoichiometry of 0.9 +/- 0.05 mol of MTBR/mol of tubulin dimer. Another mutant with additional copies of the second repeat, designated by us as MTBR[m12m2m32], displayed noncooperative binding with a Kd of 0.53 +/- 0.05 microM and a stoichiometry of 2.2 +/- 0.2 mol of mutant MTBR/tubulin dimer. Equilibrium sedimentation experiments demonstrated that the wild-type MTBR is monomeric, whereas MTBR[m12m2m32] self-associates to a stable dimer over the concentration range used in our MT binding studies. This finding indicates that only one of the two MT-binding sites on the dimer is probably linked to a microtubule at any given time.

Inhibition of Listeria locomotion by mosquito oostatic factor, a natural oligoproline peptide uncoupler of profilin action.

Mosquito oostatic factor, a naturally occurring decapeptide (YDPAPPPPPP), strikingly resembles the primary structure of oligoproline-rich regions within the protein ActA, a bacterial surface protein required for Listeria motility in host cells. When microinjected into Listeria-infected PtK2 cells, the insect oostatic factor rapidly blocks Listeria-induced actin rocket tail assembly as well as intracellular locomotion of this pathogen. At intracellular concentrations of about 90 nM, transient inhibition of rocket tail formation and bacterial locomotion occurs, followed by full recovery of tail length and motility. However, at 0.9 microM oostatic factor, both processes are permanently arrested. Introduction of oostatic factor by microinjection also causes PtK2 peripheral membrane retraction in both Listeria-infected and uninfected cells. Epifluorescence microscopy with bodipy-phallacidin reveals that cells microinjected with the insect factor lose all actin stress fibers and accumulate F-actin in regions of membrane retraction. When the insect peptide is combined with profilin as an equimolar binary solution (1 microM [final concentration] each), intracellular addition fails to inhibit Listeria rocket-tail formation, fails to block intracellular bacterial movement, and no longer causes marked membrane retraction. The ability of profilin to neutralize the inhibitory action of oostatic factor is consistent with complex formation, and this finding suggests that profilin may interact directly with ActA peptide as well as a host cell peripheral membrane component to promote actin filament assembly by locally generating ATP-actin. Dispersal of profilin from such sites by oligoproline-rich peptide inhibitors suggests that profilin is directly involved in intracellular pathogen locomotion and reorganization of actin cytoskeleton of the host cell peripheral membrane.

Dynamic remodeling of the actin cytoskeleton: lessons learned from Listeria locomotion.

The bacterial pathogen Listeria monocytogenes displays the remarkable ability to reorganize the actin cytoskeleton within host cells as a means for promoting cell-to-cell transfer of the pathogen, in a manner that evades humoral immunity. In a series of events commencing with the biosynthesis of the bacterial surface protein ActA, host cell actin and many actin-associated proteins self-assemble to form rocket-tail structures that continually grow at sites proximal to the bacterium and depolymerize distally. Widespread interest in the underlying molecular mechanism of Listeria locomotion stems from the likelihood that the dynamic remodeling of the host cell actin cytoskeleton at the cell's leading edge involves mechanistically analogous interactions. Recent advances in our understanding of these fundamental cytoskeletal rearrangements have been achieved through a clearer recognition of the central role of oligo-proline sequence repeats present in ActA, and these findings provide a basis for inferring the role of analogous host cell proteins in the force-producing and position-securing steps in pseudopod and lamellipod formation at the peripheral membrane.