Tail Assembly Research Articles

The 64-kilodalton capsid protein homolog of Beet yellows virus is required for assembly of virion tails.

The filamentous virion of the closterovirus Beet yellows virus (BYV) consists of a long body formed by the major capsid protein (CP) and a short tail composed of the minor capsid protein (CPm) and the virus-encoded Hsp70 homolog. By using nano-liquid chromatography-tandem mass spectrometry and biochemical analyses, we show here that the BYV 64-kDa protein (p64) is the fourth integral component of BYV virions. The N-terminal domain of p64 is exposed at the virion surface and is accessible to antibodies and mild trypsin digestion. In contrast, the C-terminal domain is embedded in the virion and is inaccessible to antibodies or trypsin. The C-terminal domain of p64 is shown to be homologous to CP and CPm. Mutation of the signature motifs of capsid proteins of filamentous RNA viruses in p64 results in the formation of tailless virions, which are unable to move from cell to cell. These results reveal the dual function of p64 in tail assembly and BYV motility and support the concept of the virion tail as a specialized device for BYV cell-to-cell movement.

The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA

At several E. coli promoters, initiation of transcription is repressed by a tight nucleoprotein complex formed by the assembly of the H-NS protein. In order to characterize the relationship between the structure of H-NS oligomers in solution and on relevant DNA fragments, we have compared wild-type H-NS and several transdominant H-NS mutants using gel shift assays, DNase I footprinting, analytical ultracentrifugation, and reactivity toward a cross-linking reagent. In solution, oligomerization occurs through two protein interfaces, one necessary to construct a dimeric core (and involving residues 1-64) and the other required for subsequent assembly of these dimers. We show that, as well as region 64-95, residues present in the NH(2)-terminal coiled coil domain also participate in this second interface. Our results support the view that the same interacting interfaces are also involved on the DNA. We propose that the dimeric core recognizes specific motifs, with the second interface being critical for their correct head to tail assembly. The COOH-terminal domain of the protein contains the DNA binding motif essential for the discrimination of this specific functional assembly over competitive nonspecific H-NS polymers.

Complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri.

Bacteriophage SfV is a temperate serotype-converting phage of Shigella flexneri. SfV encodes the factors involved in type V O-antigen modification, and the serotype conversion and integration-excision modules of the phage have been isolated and characterized. We now report on the complete sequence of the SfV genome (37,074 bp). A total of 53 open reading frames were predicted from the nucleotide sequence, and analysis of the corresponding proteins was used to construct a functional map. The general organization of the genes in the SfV genome is similar to that of bacteriophage lambda, and numerous features of the sequence are described. The superinfection immunity system of SfV includes a lambda-like repression system and a P4-like transcription termination mechanism. Sequence analysis also suggests that SfV encodes multiple DNA methylases, and experiments confirmed that orf-41 encodes a Dam methylase. Studies conducted to determine if the phage-encoded methylase confers host DNA methylation showed that the two S. flexneri strains analyzed encode their own Dam methylase. Restriction mapping and sequence analysis revealed that the phage genome has cos sites at the termini. The tail assembly and structural genes of SfV show homology to those of phage Mu and Mu-like prophages in the genome of Escherichia coli O157:H7 and Haemophilus influenzae. Significant homology (30% of the genome in total) between sections of the early, regulatory, and structural regions of the SfV genome and the e14 and KpLE1 prophages in the E. coli K-12 genome were noted, suggesting that these three phages have common evolutionary origins.

Quantification of Shigella IcsA required for bacterial actin polymerization.

Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500-2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella-infected cells.

Cell-to-cell movement and assembly of a plant closterovirus: roles for the capsid proteins and Hsp70 homolog.

Diverse animal and plant viruses are able to translocate their virions between neighboring cells via intercellular connections. In this work, we analyze the virion assembly and cell-to-cell movement of a plant closterovirus and reveal a strong correlation between these two processes. The filamentous virions of a closterovirus possess a long body formed by the major capsid protein (CP) and a short tail formed by the minor capsid protein (CPm). Genetic and biochemical analyses show that the functions of these virion components are distinct. A virion body is required primarily for genome protection, whereas a tail represents a specialized device for cell-to-cell movement. Furthermore, tail assembly is mediated by the viral Hsp70 homolog (Hsp70h) that becomes an integral part of the virion. Inactivation of the ATPase domain of Hsp70h results in assembly of tailless virions that are incapable of translocation. A dual role for the viral molecular chaperone Hsp70h in virion assembly and transport, combined with the previous finding of this protein in intercellular channels, allowed us to propose a model of closteroviral movement from cell to cell.

Cryoelectron-Microscopy Image Reconstruction of Symmetry Mismatches in Bacteriophage φ29

A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages. Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry. Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component. This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of φ29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch. A reconstruction of φ29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the φ29 DNA packaging motor.

It takes (more than) two to tango

For millennia, bacteria have begged, borrowed and stolen ideas from other organisms to colonize almost every available ecological niche. In particular, pathogenic bacteria, such as Listeria monocytogenes, show an astounding ability to exploit their hosts. After hitching a ride on a dairy product, Listeria invade mammalian cells (no mean feat in itself) and then use host cell proteins to assemble an actin ‘comet tail’ at one pole of the bacterium. Tail assembly propels the bacterium through the cytoplasm and eventually into neighbouring cells, spreading the infection away from the prying eyes of the host’s immune system. As Listeria exploit mammalian mechanisms for actin assembly, they have, in turn, been exploited by researchers as powerful tools in the study of the cytoskeleton. As a result, we now have a comprehensive view of the proteins involved in comet tail assembly and the role of the different bacterial proteins in driving the infection process. Only one bacterial protein, ActA, is required for tail production, meaning that ActA must be solely accountable for the recruitment of host cell proteins that are involved in tail assembly. And yet herein lies a dilemma as ActA has been shown to interact only with the Ena/VASP family of actin-binding proteins, which are themselves useful, but not essential, for tail production.

Pulse-chase analysis of the in Vivo assembly of the bacteriophage T4 tail

The in vivo assembly pathway of the complex tail of bacteriophage T4 virus was determined using pulse-chase analysis as a non-invasive alternative to the in vitro experiments previously used to map assembly. Bacteriophage T4 mutants defective in head assembly were used to infect cultures of Escherichia coli in order to study tail assembly in isolation. Beginning with the onset of late protein synthesis, the cultures were labeled continuously with [ 3H]leucine to normalize against subsequent sample losses. After completed tails had begun to accumulate at a constant rate, the cultures were pulsed with [ 35S]methionine, and then chased. Completed tails were purified at one minute intervals for the next 30 minutes and their proteins separated electrophoretically and counted by liquid scintillation. Total 35S incorporation into each protein rose and then leveled off as the chase of unlabeled methionine flushed the label through the pools of soluble proteins and assembly intermediates and into completed tails. The inflection point in the sigmoidal 35S-incorporation curve of each protein marks the maximal uptake of 35S within that pool just before the effect of the chase becomes apparent and the curve begins to level off. The length of the delay in the apparent chase time reflects the position of that protein in the pathway. The closer the assembly point to the end of the pathway, the sooner the chase appears, revealing the relative order of assembly. As predicted, tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest inflection, while those earlier in the pathway take longer to chase. Of the 17 tail proteins analyzed, 14 are in agreement with the established in vitro pathway. The other three, gp15, gp10 and gp53, have helped us to develop a model that offers a plausible explanation for their altered chase times.

Bacteriophage T4 self-assembly: localization of gp3 and its role in determining tail length.

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

Mapping and cloning recombinant breakpoints demarcating the hybrid sterility 6-specific sperm tail assembly defect.

Variants of the mouse t complex known as t haplotypes (t) express factors that perturb sperm differentiation, resulting in the non-Mendelian transmission of t from +/t heterozygous males and the sterility of t/t homozygous males. Previous studies of mice carrying heterospecific combinations of the t complex have revealed a 1-cM candidate locus, Hst6, for the distal-most of these factors, Tcd/Tcs2. Males heterozygous for the M. spretus allele of Hst6 and a t haplotype (Hst6(s)/t) are sterile, expressing an abnormality in sperm flagellar curvature ("curlicue") indistinguishable from one exhibited by sperm from t/t homozygotes. Hst6(s)/Hst6(s) males are also sterile; however, sperm produced by these males are completely immotile owing to the absence of assembled flagella. Recent studies have shown that the complete presentation of "curlicue" derives from expression of at least two factors within the locus, Curlicue a (Ccua) proximally and Curlicue b (Ccub) distally, with a factor affecting sperm-oolemma penetration, Stop1p, mapping between them. In the present report, we have examined expression of the Hst6-specific flagellar assembly phenotype in sperm from mice homozygous for M. spretus-M. m. domesticus recombinant Chr 17 homologs whose breakpoints map within the Hst6 locus. SSLP analysis of these homologs has demonstrated that the flagellar assembly defect maps to less than 0.2 cM between D17Mit61 and D17Mit135, coincident with Ccua. SSR content analysis of 23 BACs mapping to four contigs within the Hst6 locus has resulted in isolation of proximal and distal recombinant breakpoints circumscribing the flagellar assembly phenotype/Ccua factor. In addition, we have provided increased high-resolution mapping of the Stop1p and Ccub factors. These new data enhance our ability to isolate and characterize candidates for Tcd/Tcs2.

Functional analysis of a rickettsial OmpA homology domain of Shigella flexneri icsA.

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into "actin tails" at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the beta domain), such that the amino-terminal 706 amino acid residues (the alpha domain) are exposed on the exterior of the bacillus. The alpha domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the alpha domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.

Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells.

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.

Neural Wiskott-Aldrich syndrome protein is implicated in the actin-based motility of Shigella flexneri.

Shigella, the causative agent of bacillary dysentery, is capable of directing its own movement in the cytoplasm of infected epithelial cells. The bacterial surface protein VirG recruits host components mediating actin polymerization, which is thought to serve as the propulsive force. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP), which is a critical target for filopodium formation downstream of Cdc42, is required for assembly of the actin tail generated by intracellular S.flexneri. N-WASP accumulates at the front of the actin tail and is capable of interacting with VirG in vitro and in vivo, a phenomenon that is not observed in intracellular Listeria monocytogenes. The verprolin-homology region in N-WASP was required for binding to the glycine-rich repeats domain of VirG, an essential domain for recruitment of F-actin on intracellular S.flexneri. Overexpression of a dominant-negative N-WASP mutant greatly inhibited formation of the actin tail by intracellular S.flexneri. Furthermore, depletion of N-WASP from Xenopus egg extracts shut off Shigella actin tail assembly, and this was restored upon addition of N-WASP protein, suggesting that N-WASP is a critical host factor for the assembly of the actin tail by intracellular Shigella.

Crystallization and preliminary X-ray analysis of human platelet profilin complexed with an oligo proline peptide.

Profilin is an actin-monomer binding protein that regulates the distribution and dynamics of the actin cytoskeleton. Profilin binds poly-L-proline and proline-rich peptides in vitro and co-localizes with proline-rich proteins in focal adhesions and at the site of actin tail assembly on the surface of intracellular parasites such as Listeria monocytogenes. The crystallization of the complex between human platelet profilin (HPP) and an L-proline decamer [(Pro)10] is reported here. Diffraction from these crystals is consistent with the space group P21212 with unit-cell constants a = 68.25, b = 97.64, c = 39.10 A. The crystals contain two HPP molecules per asymmetric unit and diffract to 2.2 A.

Characterization of the vaccinia virus F8L protein.

Vaccinia virus infection dramatically affects the host actin cytoskeleton by inducing disassembly of actin stress fibres and formation of actin tails which propel the virus intra- and intercellularly. The viral factors responsible for these actin rearrangements remain unknown. Sequence analysis reveals significant homology between the vaccinia F8L ORF and the proline repeats of iActA, the protein which initiates actin tail assembly and motility in the bacterial pathogen Listeria ivanovii. We characterized the F8L gene product to examine its possible role in vaccinia rearrangements of the host actin cytoskeleton. F8L is a approximately 8 kDa protein expressed early during infection and is found throughout the cytoplasm, with no discernible association with viral or cellular structures. Furthermore, the F8L deletion strain, WR deltaF8L, forms particles and actin tails indistinguishable from WR. Our observations demonstrate that F8L is not required for vaccinia virus morphogenesis or the actin rearrangements observed during infection.

Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails.

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.

Vaccinia virus: a model system for actin-membrane interactions.

Our understanding of the interactions between the actin cytoskeleton and cellular membranes at the molecular level is rudimentary. One system that offers an opportunity to examine these interactions in greater detail is provided by vaccinia virus, which induces the nucleation of actin tails from the outer membrane surrounding the virion. To further understand the mechanism of their formation and how they generate motility, we have examined the structure of these actin tails in detail. Actin filaments in vaccinia tails are organized so they splay out at up to 45 degrees from the centre of the tail and are up to 0.74 micron in length, which is considerably longer than those reported in the Listeria system. Actin filaments show unidirectional polarity with their barbed filament ends pointing towards the surface of the virus particle. Rhodamine-actin incorporation experiments show that the first stage of tail assembly involves a polarized recruitment of G-actin, and not pre-formed actin filaments, to the membrane surrounding the virion. Incorporation of actin into the tail only occurs by nucleation from the viral surface, suggesting filament ends in the tail are blocked against further actin addition. As virus particles fuse with the plasma membrane during the extention of projections, actin nucleation sites previously in the viral membrane become localized to the plasma membrane, where they are able to nucleate actin polymerization in a manner analogous to the leading edge of motile cells.

In vitro assembly of infectious virions of double-stranded DNA phage phi 29 from cloned gene products and synthetic nucleic acids.

Up to 6 x 10(7) PFU of infectious virions of the double-stranded DNA bacteriophage phi 29 per ml were assembled in vitro, with 11 proteins derived from cloned genes and nucleic acids synthesized separately. The genomic DNA-gp3 protein conjugate was efficiently packaged into a purified recombinant procapsid with the aid of a small viral RNA (pRNA) transcript, a DNA-packaging ATPase (gp16), and ATP. The DNA-filled capsids were subsequently converted into infectious virions after the addition of four more recombinant proteins for neck and tail assembly. Electron microscopy and genome restriction mapping confirmed the identity of the infectious phi 29 virions synthesized in this system. A nonstructural protein, gp13, was indispensable for the assembly of infectious virions. The overproduced tail protein gp9 was present in solution in mostly dimeric form and was purified to homogeneity. The purified gp9 was biologically active for in vitro phi 29 assembly. Higher-order concentration dependence of in vitro phi 29 assembly on gp9 suggests that a complete tail did not form before attaching to the DNA-filled capsid, a result contrary to earlier findings for phages T4 and lambda. The work described here constitutes an extremely sensitive assay system for the analysis of components in phi 29 assembly and dissection of functional domains of structural components, enzymes, and pRNA (C.-S. Lee and P. Guo, Virology 202:1039-1042, 1995). Efficient packaging of foreign DNA in vitro and synthesis of viral particles from recombinant proteins facilitate the development of phi 29 as an in vivo gene delivery system. The finding that purified tail protein was able to incorporate into infectious virions might allow the construction of chimeric phi 29 carrying a tail fused to ligands for specific receptor of human cells.

Modeling nonlinear features of V tail aircraft using MNN

The nonlinear stability and control modeling involves a very popular high performance general aviation aircraft that uses a tail assembly instead of the traditional inverted T tail. The nonlinear response features of this aircraft result from a well developed Dutch roll mode and are caused by a dynamic stall phenomenon that occurs on the V tail during the maneuver. A new approach using multilayered neural network (MNN) for modeling the nonlinear features of this aircraft is suggested here. Both the conventional backpropagation (BP) and the extended Kalman filter (EKF)-based learning algorithm are used for training the neural network. Simulation results that confirm the efficacy of the method are given. Further, performance comparison of the EKF-based and the conventional BP algorithm is made to highlight the effectiveness of the EKF-based learning algorithm. >

Listeria monocytogenes intracellular migration: inhibition by profilin, vitamin D-binding protein and DNase I.

Infection of host cells by Listeria monocytogenes results in the recruitment of cytoplasmic actin into a tail-like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthens, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results in Listeria cell-to-cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial-actin tail interface. We have studied the consequences of microinjecting three different actin monomer-binding proteins on the actin tail assembly and Listeria intracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11-22 microM) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer-sequestering proteins, Vitamin D-binding protein (DBP; 1-2 microM) and DNase I (6-7 microM) completely block bacterial-induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10-20 min) indicating that the mechanisms by which these proteins interfere with Listeria-induced actin assembly are likely to be complex. To exclude the possibility that Listeria recruits preformed actin filaments to generate the tails and that these monomer-binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 microM). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate that Listeria-induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and that Listeria's ability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer-binding proteins.