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Biomedical subjects

Julie A Theriot

Publications and source records attributed to Julie A Theriot.

At least 19 recordsLinked to original sources
PubMed2025

Galvanin (TMEM154) is an electric-field sensor for directed cell migration.

Directed cell migration of immune and epithelial cells is critical for their rapid response to tissue injury or infection. Endogenous electric fields generated by disruption of the transepithelial potential across the skin have been postulated to play an important role in guiding cells to wound sites, though how individual cells sense these tissue-scale physical cues remains largely unknown. We have identified Galvanin (TMEM154), a previously uncharacterized single-pass transmembrane protein, as being required for electric-field-guided migration of individual rapidly moving cells. Galvanin functions in both immune and epithelial cell types. Upon exposure of cells to an electric field, Galvanin rapidly relocalizes to the anodal side of a cell, and the net charge on its extracellular domain is necessary and sufficient to drive this spatial relocalization. Furthermore, expression of Galvanin is sufficient to confer electric field-guided migration on otherwise non-responsive epithelial cells. In human neutrophils, we show that Galvanin relocalization is immediately followed by changes in the spatial pattern of cellular protrusion and retraction. The strong directional response of these cells is lost upon truncation of Galvanin's intracellular domain, suggesting that Galvanin acts as a direct sensor of the electric field, transducing spatial information about a cell's electrical environment to the intracellular migratory apparatus. This sensor relocalization mechanism of cell steering defines a new paradigm for directed cell migration.

Journal Article↗
PubMed2006

Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion.

Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier.

Journal Article↗
PubMed2006

A correlation-based approach to calculate rotation and translation of moving cells.

We present a noniterative image cross-correlation approach to track translation and rotation of crawling cells in time-lapse video microscopy sequences. The method does not rely on extracting features or moments, and therefore does not impose specific requirements on the type of microscopy used for imaging. Here we use phase-contrast images. We calculate cell rotation and translation from one image to the next in two stages. First, rotation is calculated by cross correlating the images' polar-transformed magnitude spectra (Fourier magnitudes). Rotation of the cell about any center in the original images results in translation in this representation. Then, we rotate the first image such that the cell has the same orientation in both images, and cross correlate this image with the second image to calculate translation. By calculating the rotation and translation over each interval in the movie, and thereby tracking the cell's position and orientation in each image, we can then map from the stationary reference frame in which the cell was observed to the cell's moving coordinate system. We describe our modifications enabling application to nonidentical images from video sequences of moving cells, and compare this method's performance with that of a feature extraction method and an iterative optimization method.

Algorithms↗
PubMed2006

Mechanism of polarization of Listeria monocytogenes surface protein ActA.

The polar distribution of the ActA protein on the surface of the Gram-positive intracellular bacterial pathogen, Listeria monocytogenes, is required for bacterial actin-based motility and successful infection. ActA spans both the bacterial membrane and the peptidoglycan cell wall. We have directly examined the de novo ActA polarization process in vitro by using an ActA-RFP (red fluorescent protein) fusion. After induction of expression, ActA initially appeared at distinct sites along the sides of bacteria and was then redistributed over the entire cylindrical cell body through helical cell wall growth. The accumulation of ActA at the bacterial poles displayed slower kinetics, occurring over several bacterial generations. ActA accumulated more efficiently at younger, less inert poles, and proper polarization required an optimal balance between protein secretion and bacterial growth rates. Within infected host cells, younger generations of L. monocytogenes initiated motility more quickly than older ones, consistent with our in vitro observations of de novo ActA polarization. We propose a model in which the polarization of ActA, and possibly other Gram-positive cell wall-associated proteins, may be a direct consequence of the differential cell wall growth rates along the bacterium and dependent on the relative rates of protein secretion, protein degradation and bacterial growth.

Bacterial Proteins↗
PubMed2006

Fine-scale time-lapse analysis of the biphasic, dynamic behaviour of the two Vibrio cholerae chromosomes.

Using fluorescent repressor-operator systems in live cells, we investigated the dynamic behaviour of chromosomal origins in Vibrio cholerae, whose genome is divided between two chromosomes. We have developed a method of analysing fine-scale motion in the curved co-ordinate system of vibrioid bacteria. Using this method, we characterized two different modes of chromosome behaviour corresponding to periods between segregation events and periods of segregation. Between segregation events, the origin positions are not fixed but rather maintained within ellipsoidal caged domains, similar to eukaryotic interphase chromosome territories. These domains are approximately 0.4 microm wide and 0.6 microm long, reflecting greater restriction in the short axis of the cell. During segregation, movement is directionally biased, speed is comparable between origins, and cell growth can account for nearly 20% of the motion observed. Furthermore, the home domain of each origin is positioned by a different mechanism. Specifically, the oriC(I) domain is maintained at a constant actual distance from the pole regardless of cell length, while the oriC(II) domain is maintained at a constant relative position. Thus the actual position of oriC(II) varies with cell length. While the gross behaviours of the two origins are distinct, their fine-scale dynamics are remarkably similar, indicating that both experience similar microenvironments.

Cell Cycle↗
PubMed2006

Listeria monocytogenes traffics from maternal organs to the placenta and back.

Infection with Listeria monocytogenes is a significant health problem during pregnancy. This study evaluates the role of trafficking between maternal organs and placenta in a pregnant guinea pig model of listeriosis. After intravenous inoculation of guinea pigs, the initial ratio of bacteria in maternal organs to placenta was 10(3)-10(4):1. Rapid increase of bacteria in the placenta changed the ratio to 1:1 after 24 h. Utilizing two wild-type strains, differentially marked by their susceptibility to erythromycin, we found that only a single bacterium was necessary to cause placental infection, and that L. monocytogenes trafficked from maternal organs to the placenta in small numbers. Surprisingly, bacteria trafficked in large numbers from the placenta to maternal organs. Bacterial growth, clearance, and transport between organs were simulated with a mathematical model showing that the rate of bacterial clearance relative to the rate of bacterial replication in the placenta was sufficient to explain the difference in the course of listeriosis in pregnant versus nonpregnant animals. These results provide the basis for a new model where the placenta is relatively protected from infection. Once colonized, the placenta becomes a nidus of infection resulting in massive reseeding of maternal organs, where L. monocytogenes cannot be cleared until trafficking is interrupted by expulsion of the infected placental tissues.

Animals↗
PubMed2005

Two independent spiral structures control cell shape in Caulobacter.

The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.

Bacterial Proteins↗
PubMed2005

Loading history determines the velocity of actin-network growth.

Directional polymerization of actin filaments in branched networks is one of the most powerful force-generating systems in eukaryotic cells. Growth of densely cross-linked actin networks drives cell crawling, intracellular transport of vesicles and organelles, and movement of intracellular pathogens such as Listeria monocytogenes. Using a modified atomic force microscope (AFM), we obtained force-velocity (Fv) measurements of growing actin networks in vitro until network elongation ceased at the stall force. We found that the growth velocity of a branched actin network against increasing forces is load-independent over a wide range of forces before a convex decline to stall. Surprisingly, when force was decreased on a growing network, the velocity increased to a value greater than the previous velocity, such that two or more stable growth velocities can exist at a single load. These results demonstrate that a single Fv relationship does not capture the complete behaviour of this system, unlike other molecular motors in cells, because the growth velocity depends on loading history rather than solely on the instantaneous load.

Actin Cytoskeleton↗
PubMed2005

Adhesion controls bacterial actin polymerization-based movement.

As part of its infectious life cycle, the bacterial pathogen Listeria monocytogenes propels itself through the host-cell cytoplasm by triggering the polymerization of host-cell actin near the bacterial surface, harnessing the activity of several cytoskeletal proteins used during actin-based cell crawling. To distinguish among several classes of biophysical models of actin-based bacterial movement, we used a high-throughput tracking technique to record the movement of many individual bacteria during temperature shifts. The speed of each bacterium varied strongly with temperature, closely following the Arrhenius rate law. Among bacteria, the prefactor A of the Arrhenius dependence unexpectedly varied exponentially with apparent activation energy, E(a), over a wide range (8-21 kcal/mol), reminiscent of the "rate compensation effect" of classical catalytic reactions. Average E(a) were increased for mutant bacteria deficient in binding Ena/VASP proteins and bacteria moving in diluted extract. These two effects were additive. The observed temperature and rate compensation effects are consistent with a class of simple kinetic models in which the bacterium advances through the thermally driven, cooperative breakage of groups of adhesive bonds on its surface. The estimated number of coupled adhesive bonds N on the bacterial surface varies between 10 and 40 bonds. In contrast to other models, this model correctly predicts an experimentally observed negative correlation between bacterial speed and actin gel density. The idea that speed depends on adhesion, rather than polymerization, suggests several alternative mechanisms by which known cytoskeletal regulatory proteins could control cellular movement.

Actins↗
PubMed2005

Bacterial shape and ActA distribution affect initiation of Listeria monocytogenes actin-based motility.

We have examined the process by which the intracellular bacterial pathogen Listeria monocytogenes initiates actin-based motility and determined the contribution of the variable surface distribution of the ActA protein to initiation and steady-state movement. To directly correlate ActA distributions to actin dynamics and motility of live bacteria, ActA was fused to a monomeric red fluorescent protein (mRFP1). Actin comet tail formation and steady-state bacterial movement rates both depended on ActA distribution, which in turn was tightly coupled to the bacterial cell cycle. Motility initiation was found to be a highly complex, multistep process for bacteria, in contrast to the simple symmetry breaking previously observed for ActA-coated spherical beads. F-actin initially accumulated along the sides of the bacterium and then slowly migrated to the bacterial pole expressing the highest density of ActA as a tail formed. Early movement was highly unstable with extreme changes in speed and frequent stops. Over time, saltatory motility and sensitivity to the immediate environment decreased as bacterial movement became robust at a constant steady-state speed.

Actins↗
PubMed2005

Large-scale quantitative analysis of sources of variation in the actin polymerization-based movement of Listeria monocytogenes.

During the actin polymerization-based movement of Listeria monocytogenes, individual bacteria are rapidly propelled through the host cell cytoplasm by the growth of a filamentous actin tail. The rate of propulsion varies significantly among individuals and over time. To study this variation, we used a high-throughput tracking technique to record the movement of a large number (approximately 7900) of bacteria in Xenopus frog egg extract. Most bacteria (70%) appeared to maintain an individual characteristic speed over several minutes, suggesting that the major source of variation in average speed is intrinsic to the bacterium. Thirty percent of bacteria had significant changes in speed over time spans of a few minutes, including 17% that appeared to collide with obstacles and 13% that moved with a significant periodic component. For the latter, the peak frequency was proportional to speed, suggesting a mechanism with a fixed spatial scale of approximately 0.6 bacterial length. Near the rear of the bacterium, temporal fluctuations in actin density were positively correlated with fluctuations in speed, whereas near the front the correlation was negative. A comparison of the performance of linear models that predict motion given actin density suggests that the mechanism has a history of 5-10 s, and that fluctuations in actin density near the front of the bacteria contain more predictive information than the rear. Our results are consistent with physical models where bacterial speed is governed by the rate of dissociation of bonds between the bacterial surface and the actin tail, and individual variation is determined by long-lived intrinsic variability in bacterial surface properties.

Actins↗
PubMed2004

Repeated cycles of rapid actin assembly and disassembly on epithelial cell phagosomes.

We have found that early in infection of the intracellular pathogen Listeria monocytogenes in Madin-Darby canine kidney epithelial cells expressing actin conjugated to green fluorescent protein, F-actin rapidly assembles (approximately 25 s) and disassembles (approximately 30 s) around the bacteria, a phenomenon we call flashing. L. monocytogenes strains unable to perform actin-based motility or unable to escape the phagosome were capable of flashing, suggesting that the actin assembly occurs on the phagosome membrane. Cycles of actin assembly and disassembly could occur repeatedly on the same phagosome. Indirect immunofluorescence showed that most bacteria were fully internalized when flashing occurred, suggesting that actin flashing does not represent phagocytosis. Escherichia coli expressing invA, a gene product from Yersinia pseudotuberculosis that mediates cellular invasion, also induced flashing. Furthermore, polystyrene beads coated with E-cadherin or transferrin also induced flashing after internalization. This suggests that flashing occurs downstream of several distinct molecular entry mechanisms and may be a general consequence of internalization of large objects by epithelial cells.

Actin-Related Protein 2↗
PubMed2004

Listeria monocytogenes actin-based motility varies depending on subcellular location: a kinematic probe for cytoarchitecture.

Intracellular Listeria monocytogenes actin-based motility is characterized by significant individual variability, which can be influenced by cytoarchitecture. L. monocytogenes was used as a probe to transmit information about structural variation among subcellular domains defined by mitochondrial density. By analyzing the movement of a large population of L. monocytogenes in PtK2 cells, we found that mean speed and trajectory curvature were significantly larger for bacteria moving in mitochondria-containing domains (generally perinuclear) than for bacteria moving in mitochondria-free domains (generally peripheral). Analysis of bacteria that traversed both mitochondria-containing and mitochondria-free domains revealed that these motile differences were not intrinsic to bacteria themselves. Disruption of mitochondrial respiration did not affect bacterial mean speed, speed persistence, or trajectory curvature. In contrast, microtubule depolymerization lead to decreased mean speed per bacterium and increased mean speed persistence of L. monocytogenes moving in mitochondria-free domains compared with untreated cells. L. monocytogenes were also observed to physically collide with mitochondria and push them away from the bacterial path of motion, causing bacteria to slow down before rapidly resuming their speed. Our results show that subcellular domains along with microtubule depolymerization may influence the actin cytoskeleton to affect L. monocytogenes speed, speed persistence, and trajectory curvature.

Actins↗
PubMed2004

Biophysical parameters influence actin-based movement, trajectory, and initiation in a cell-free system.

Using a biochemically complex cytoplasmic extract to reconstitute actin-based motility of Listeria monocytogenes and polystyrene beads coated with the bacterial protein ActA, we have systematically varied a series of biophysical parameters and examined their effects on initiation of motility, particle speed, speed variability, and path trajectory. Bead size had a profound effect on all aspects of motility, with increasing size causing slower, straighter movement and inhibiting symmetry-breaking. Speed also was reduced by extract dilution, by addition of methylcellulose, and paradoxically by addition of excess skeletal muscle actin, but it was enhanced by addition of nonmuscle (platelet) actin. Large, persistent individual variations in speed were observed for all conditions and their relative magnitude increased with extract dilution, indicating that persistent alterations in particle surface properties may be responsible for intrinsic speed variations. Trajectory curvature was increased for smaller beads and also for particles moving in the presence of methylcellulose or excess skeletal muscle actin. Symmetry breaking and movement initiation occurred by two distinct modes: either stochastic amplification of local variation for small beads in concentrated extracts, or gradual accumulation of strain in the actin gel for large beads in dilute extracts. Neither mode was sufficient to enable spherical particles to break symmetry in the cytoplasm of living cells.

Actins↗
PubMed2004

Differentiation and developmental pathways of uropathogenic Escherichia coli in urinary tract pathogenesis.

Uropathogenic Escherichia coli (UPEC) are capable of forming complex intracellular bacterial communities (IBC) within the superficial umbrella cells of the bladders of C3H and BALB/c mice. By using time-lapse fluorescence videomicroscopy to observe infected mouse bladder explants, we discovered that IBCs formed by uropathogenic E. coli progressed through four distinct developmental stages that differed with respect to growth rate, bacterial length, colony organization, motility, and its eventual dispersal. In the first phase, bacteria in the IBC were nonmotile, rod shaped, and grew rapidly in loosely organized colonies free in the cytoplasm of the bladder superficial umbrella cells. In the second phase, the loose collection of bacteria in the IBC matured into a slower growing, highly organized biofilm-like community consisting of coccoid bacteria that ultimately filled most of the cytoplasm. In the third phase, bacteria in the biofilm-like state in the IBC switched to a motile rod-shaped phenotype allowing detachment from the community and eventual fluxing out of the host cell. During the fourth phase, the bacteria filamented. Filamentation appeared to be in response to a Toll-like receptor 4-mediated innate defense mechanism. Bacteria that fluxed out of the superficial umbrella cells were able to reenter the IBC developmental cascade but with slower kinetics and ultimately a quiescent reservoir was established. Intracellular growth and filamentation provided an advantage to the bacteria in evading infiltrating polymorphonuclear leukocytes. This work has developed a technique to observe live infected organs and revealed a complex differentiation pathway that facilitates bacterial persistence in the urinary tract.

Animals↗
PubMed2004

High affinity, paralog-specific recognition of the Mena EVH1 domain by a miniature protein.

Many protein domains involved in cell signaling contain or interact with proline-rich sequences, and the design of molecules that perturb signaling pathways represents a foremost goal of chemical biology. Previously we described a protein design strategy in which the well-folded alpha-helix in avian pancreatic polypeptide (aPP) presents short alpha-helical recognition epitopes. The miniature proteins designed in this way recognize even shallow protein clefts with high affinity and specificity. Here we show that the well-folded type-II polyproline helix in aPP can present the short PPII-helical recognition epitope within the ActA protein of Listeria monocytogenes. Like miniature proteins that use an alpha-helix for protein recognition, the miniature protein designed in this way displays high affinity for a natural ActA target, the EVH1 domain Mena1-112, and achieves the elusive goal of paralog specificity, discriminating well between EVH1 domains Mena1-112, VASP1-115, and Evl1-112. Most importantly, the miniature protein competed with ActA in Xenopus laevis egg cytoplasmic extracts, decreasing actin-dependent motility of L. monocytogenes and causing extreme speed variations and discontinuous tail formation. Our results suggest that miniature proteins based on aPP may represent an excellent framework for the design of ligands that differentiate the roles of EVH1 domains in vitro and in vivo.

Amino Acid Sequence↗
PubMed2004

An introduction to cell motility for the physical scientist.

Directed, purposeful movement is one of the qualities that we most closely associate with living organisms, and essentially all known forms of life on this planet exhibit some type of self-generated movement or motility. Even organisms that remain sessile most of the time, like flowering plants and trees, are quite busy at the cellular level, with large organelles, including chloroplasts, constantly racing around within cellular boundaries. Directed biological movement requires that the cell be able to convert its abundant stores of chemical energy into mechanical energy. Understanding how this mechanochemical energy transduction takes place and understanding how small biological forces generated at the molecular level are marshaled and organized for large-scale cellular or organismal movements are the focus of the field of cell motility. This tutorial, aimed at readers with a background in physical sciences, surveys the state of current knowledge and recent advances in modeling cell motility.

Animals↗