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// Leire Neri 1 , Marta Lasa 1 , Alberto Elosegui-Artola 2 , Delia D'Avola 3, 4 , Beatriz Carte 1 , Cristina Gazquez 1 , Sara Alve 5 , Pere Roca-Cusachs 2, 6 , Mercedes Inarrairaegui 3, 4 , Jose Herrero 3, 4 , Jesus Prieto 1, 3 , Bruno Sangro 3, 4 and Rafael Aldabe 1, 4 1 Gene Therapy and Regulation of Gene Expression Program, Centro de Investigacion Medica Aplicada, Universidad de Navarra, Pamplona, Spain 2 Institute for Bioengineering of Catalonia, Barcelona, Spain 3 Liver Unit, Clinica Universidad de Navarra, Centro de Investigacion Biomedica en Red en el Area Tematica de Enfermedades Hepaticas y Digestivas (Ciberehd), Pamplona, Spain 4 Instituto de Investigacion Sanitaria de Navarra (IdiSNA), Pamplona, Spain 5 Department of Biology, CBMA-Centre of Molecular and Environmental Biology, University of Minho, Campus de Gualtar, Braga, Portugal 6 University of Barcelona, Barcelona, Spain Correspondence to: Rafael Aldabe, email: raldabe@unav.es Keywords: tropomyosin, CDK2, focal adhesions, cell-cell junctions, cell cycle arrest Received: December 21, 2016 Accepted: April 04, 2017 Published: April 21, 2017 ABSTRACT The identification of new targets for systemic therapy of hepatocellular carcinoma (HCC) is an urgent medical need. Recently, we showed that hNatB catalyzes the N-α-terminal acetylation of 15% of the human proteome and that this action is necessary for proper actin cytoskeleton structure and function. In tumors, cytoskeletal changes influence motility, invasion, survival, cell growth and tumor progression, making the cytoskeleton a very attractive antitumor target. Here, we show that hNatB subunits are upregulated in in over 59% HCC tumors compared to non-tumor tissue and that this upregulation is associated with microscopic vascular invasion. We found that hNatB silencing blocks proliferation and tumor formation in HCC cell lines in association with hampered DNA synthesis and impaired progression through the S and the G2/M phases. Growth inhibition is mediated by the degradation of two hNatB substrates, tropomyosin and CDK2, which occurs when these proteins lack N-α-terminal acetylation. In addition, hNatB inhibition disrupts the actin cytoskeleton, focal adhesions and tight/adherens junctions, abrogating two proliferative signaling pathways, Hippo/YAP and ERK1/2. Therefore, inhibition of NatB activity represents an interesting new approach to treating HCC by blocking cell proliferation and disrupting actin cytoskeleton function.

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small-sized multi-colored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin. © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA., The authors acknowledge funding from Medical Research Scotland (S.B.: 879-2015), MSCA Individual Fellowship (A.F.: 704912), OPTIMA (N.D.B.: EP/L016559/1), Wellcome Trust Sir Henry Dale Fellowship (Y.F.: 100104/Z/12/Z) and the Spanish Ministry of Science, Innovation and Universities (J.L.A, A.D.: CTQ2017-85378-R). M.V. acknowledges funds from ERC Consolidator Grant (771443), Biotechnology and Biological Sciences Research Council (BB/M025160/1) and the Royal Society (IEC\R3\170132). The authors thank the technical support from the Flow Cytometry and the Confocal Advanced Light Microscopy units at the University of Edinburgh.

International audience; Recently, we characterized the complete phase transition diagram in the phgr-Pe parameter space for two models of active brownian particles in two dimensions. These models are composed of hard disks and dumbbells, respectively, the former being isotropic and the latter anisotropic. Here, we want to outline all the most significant features of these two paradigmatic models and compare them. Remarkably, the phase diagrams of the two models are affected differently by the introduction of activity. Disks present a two-stage melting scenario from Pe=0 to about Pe=3, with a first order phase transition between liquid and hexatic and a Berezinskii-Kosterlitz-Thouless transition between hexatic and solid. At higher activities, the three phases are still observed, but the transition between liquid and hexatic becomes a BKT transitions without a distinguishable coexistence region. Dumbbells, instead, present a macroscopic coexistence between hexatically ordered regions and disordered ones, over a finite interval of packing fractions, for all activities, included Pe=0, without any observable discontinuity in the behavior upon increasing Pe.

Interaction between tight junction protein ZO-1 and integrin α5β1 reduces the resistance to force of α5β1–fibronectin bonds while simultaneously increasing their affinity. This effect is specific to subconfluent cells in which ZO-1 is displaced from its normal localization in cell–cell junctions., Fundamental processes in cell adhesion, motility, and rigidity adaptation are regulated by integrin-mediated adhesion to the extracellular matrix (ECM). The link between the ECM component fibronectin (fn) and integrin α5β1 forms a complex with ZO-1 in cells at the edge of migrating monolayers, regulating cell migration. However, how this complex affects the α5β1-fn link is unknown. Here we show that the α5β1/ZO-1 complex decreases the resistance to force of α5β1–fn adhesions located at the edge of migrating cell monolayers while also increasing α5β1 recruitment. Consistently with a molecular clutch model of adhesion, this effect of ZO-1 leads to a decrease in the density and intensity of adhesions in cells at the edge of migrating monolayers. Taken together, our results unveil a new mode of integrin regulation through modification of the mechanical properties of integrin–ECM links, which may be harnessed by cells to control adhesion and migration.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.

Pontes et al. show that plasma membrane mechanics exerts an upstream control during cell motility. Variations in plasma membrane tension orchestrate the behavior of the cell leading edge, with increase–decrease cycles in tension promoting adhesion row positioning., Cell migration is dependent on adhesion dynamics and actin cytoskeleton remodeling at the leading edge. These events may be physically constrained by the plasma membrane. Here, we show that the mechanical signal produced by an increase in plasma membrane tension triggers the positioning of new rows of adhesions at the leading edge. During protrusion, as membrane tension increases, velocity slows, and the lamellipodium buckles upward in a myosin II–independent manner. The buckling occurs between the front of the lamellipodium, where nascent adhesions are positioned in rows, and the base of the lamellipodium, where a vinculin-dependent clutch couples actin to previously positioned adhesions. As membrane tension decreases, protrusion resumes and buckling disappears, until the next cycle. We propose that the mechanical signal of membrane tension exerts upstream control in mechanotransduction by periodically compressing and relaxing the lamellipodium, leading to the positioning of adhesions at the leading edge of cells.

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV‐1)‐infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro‐mitochondrial or anti‐apoptotic strategies as in vitro cell platforms to deal with HIV‐infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt‐PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial‐nuclear encoded subunits II–IV of cytochrome‐c‐oxidase (COXII‐COXIV), respectively, as well as mitochondrial apoptotic events [voltage‐dependent‐anion‐channel‐1(VDAC‐1)‐content and caspase‐9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1‐assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC‐1 and caspase‐9 content were sharply decreased in both chronic HIV‐1‐infected promonocytic and lymphoid cell lines (

© 2015. Published by The Company of Biologists Ltd. The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension., Ministerio de Economı́a y Competitividad, Spain [grant numbers BFU2012-32532 and CONSOLIDER COAT CSD2009-00016 to M. A.A.]. G.A. was supported by the Amarouto Program for senior researchers from the Comunidad Autónoma de Madrid.

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N -acetylhexosamines are N-acetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility.

Cells are the minimum unit of life. They are born, they eat, the may grow, they may move, and, eventually, they die. By contrast, from a physicist point of view, cells are systems out of equilibrium continuously transducing between matter, energy and information. This transduction is what grants the cell their active properties. In order to perform such tasks, cells have a set of macromolecules, a machinery, which are called, Molecular Motors or Molecular Machines. The operation of molecular motors is multiple. For instance, kinesins are molecular motors able to transport cargoes along the cell, or the Bacterial Flagellar Motor works as a nanometric ionic turbine transmitting its rotation to bacterial flagella propelling the cell. The energy input of such nanometric devices have two primary sources. On one hand the hydrolysis of nucleotide derivatives, such as ATP. On the other hand, molecular motors can also be found in biological membranes obtaining energy from the natural flux of ions crossing the membrane due to mechano-chemical energetic differences at each side. The recycling of ATP molecules takes place in another molecular machine, the F0F1 ATP synthase. F0F1 is made up of two subunits that can be separated themselves in two different molecular machines. This way, the F1 motor can couple a rotatory motor with the synthesis/hydrolysis of ATP. Understanding the working of molecular motors is not straightforward. The transduction processes result from a complex set of interactions of all the molecules conforming the motor plus all the interactions with the surrounding molecules. Thus, different approaches with different levels of abstraction are necessary. In the current thesis, molecular motors are studied through the identification of the energetic transduction cycles out of the trajectory of the motor. Trajectories allow to identify the different mechanical and chemical processes driving the motor and allow to propose a spatio-temporal potential for the motor that give information of the energetic performance of the motor such as power and efficiency. This analysis is performed on the F1 motor (in its hydrolysis regime). Such analysis allowed to identify the origin of two well differentiated mechanical and chemical processes that were quantified by means of the reaction kinetics theory and the overdamped dynamics associated with the nanometric biological scale. From this analysis resulted a prediction for the average velocity of the motor with the experimental control parameters. The resulting velocity matches experimental measures of the average velocity without fitting any parameter since all the parameters needed can be extracted from alternative experimental assays. The appealing results of the average velocity lead to a proposal of motor potential for the F1 motor consisting on two linear piece-wise potentials flashing between them. Each potential presenting the experimental characteristics observed when the catalytic site of the motor is empty or occupied. The potential also hold the substepping mechanism observed experimentally. Thus, the resulting potential can be tested, together with the overdamped dynamics of the potential and the thermal fluctuations characteristic of the biological cellular scale. This results in a Langevin equation leading the dynamics of the motor. Again, the stochastic dynamics proposed are able to reproduce the velocity of the motor returning a better approximation than the deterministic approach. As happened in the previous case, there is no fitting in the parameters to test the validity of the velocity expression. Actually, the model is able to predict the measured substep angle from optimisation arguments. The mismatch between the deterministic and the stochastic results was identified as a result of a loss of ATP hydrolysis events due to thermal fluctuations that has been also properly quantified through the Fokker-Planck formalism of the corresponding Langevin equation. The motor potential proposed was also used to study experimental assays of the F1 motor working against conservative forces. The effect of a conservative torque in the working of the motor contains contributions both mechanical and chemical. Altogether, this contributions were successfully addressed presenting again an analytical and stochastic prediction for the velocity of the motor that matches the experimental observations without the need of any parameter fitting. This analysis also entailed a study of the energetic performance of the motor which is unavailable experimentally. The results show a complete divergence between the stochastic and deterministic predictions. The divergence is specially dramatical near the stall force of the motor where the determenistic analysis predicts an efficiency maximum and the stochastic analysis returns a null efficiency. This points out that the stochastic effects are very relevant to the energetic performance of the motor and can not be missed in a proper energetic study of a molecular machine. Besides the study of the F1 motor, also a rotatory device working with an ionic flux was analised. The aim of the analysis was the devise of a minimal mechanistic turbine and the study of its main working features. Such a machine is composed by a mobile piston with periodic boundary conditions at both ends of a nanometric channel separating two particle reservoirs. Hence, the turbine is able to transduce energy between the flux of ions and an external force hindering the natural motion of the piston. Again, thermal fluctuations provide a stochastic dynamic that must be studied through a Langevin equation that can be tackled analytically. This study revealed that the velocity and the flux are not coupled. Specially, two different stall forces appear for the motor. One for the velocity and one for the flux. This results in an intermediate zone where there is a continuous leakage of ions that does not allow any energetic output. This effect is originated from thermal fluctuations. Thus, when the energetic performance is evaluated, a similar behaviour than the one obtained for the F1 motor is recuperated. This minimal model was extended with more complex turbines that take into account more thoroughly the biophysics of molecular machines. All of them result in the same energetic landscape where a minimum of efficiency is obtained near the stall of the motor. Additionally, a new formalism has been developed to simplify the resulting Langevin equations (Fokker-planck white noise limit) and a new algorithm has been devised able to integrate Langevin equations with non-continuous multiplicative noise, Los Motores Moleculares son macromoléculas biológicas que se encargan de hacer las transducciones energéticas necesarias dentro de las células. Este trabajo estudia la transformación de energía de motores moleculares rotatorios reales principalmente la F1-ATPasa, el Motor Flagelar de las Bacterias y el F0. Para estudiar la dinámica del motor se han utilizado ecuaciones de Langevin sobreamortiguadas que recogen la importancia de las fluctuaciones térmicas, así como las fuerzas externas aplicadas al motor (conservativas y disipativas) y el potencial interno del motor que contiene la información físico-química de su comportamiento. Este estudio se ha aplicado a la F1-ATPasa, que se puede estudiar tanto analíticamente, obviando las fluctuaciones térmicas como desde su naturaleza estocástica mediante potenciales intermitentes. En ambos casos, el modelo es capaz de describir la dinámica del motor y su dependencia con los diferentes parámetros controlables experimentalmente: Concentración de ATP, fuerza disipativa y fuerza conservativa. En el mismo sentido se ha diseñado una turbina nanoscópica que recoge los principios básicos de la interacción mecánica entre un flujo de iones y la rotación del motor. En ambos casos, tanto en la turbina como en el F1 se observa que el ruido térmico no afecta mucho a la velocidad del motor y en cambio produce cambios enormes en parámetros energéticos como la potencia o la eficiencia. Concretamente, el escenario clásico en que un máximo de eficiencia se obtiene para la fuerza de calado desaparece obteniendo nuevos regímenes óptimos de trabajo. Adicionalmente, se ha desarrollado un formalismo para simplificar las ecuaciones de Langevin obtenidas (límite de ruido blanco) y se ha diseñado un nuevo algoritmo para integrar ecuaciones de Langevin en las cuales el ruido multiplicativo es discontinuo en el espacio.

International audience; We examine the phenomenon of hydrodynamic-induced cooperativity for pairs of flagellated micro-organism swimmers, of which spermatozoa cells are an example. We consider semiflexible swimmers, where inextensible filaments are driven by an internal intrinsic force and torque-free mechanism (intrinsic swimmers). The velocity gain for swimming cooperatively, which depends on both the geometry and the driving, develops as a result of the near-field coupling of bending and hydrodynamic stresses. We identify the regimes where hydrodynamic cooperativity is advantageous and quantify the change in efficiency. When the filaments' axes are parallel, hydrodynamic interaction induces a directional instability that causes semiflexible swimmers that profit from swimming together to move apart from each other. Biologically, this implies that flagella need to select different synchronized collective states and to compensate for directional instabilities (e. g., by binding) in order to profit from swimming together. By analyzing the cooperative motion of pairs of externally actuated filaments, we assess the impact that stress distribution along the filaments has on their collective displacements. DOI: 10.1103/PhysRevE.87.032720

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

Cells severely depleted of filamins were observed to have numerous motility-related defects, including a defect in endoplasmic spreading; smaller, more dynamic focal adhesions; and an inability to sustain high levels of traction force. The endoplasm as a separate mechanical unit spread by pulling forces is also discussed., Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB–/– mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA–/– MEFs, but not FlnB–/– MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

El citoesqueleto de actina es imprescindible para el mantenimiento de la morfología celular, así como para realizar diversas funciones como la movilidad/migración y división celular. En términos generales, este componente del citoesqueleto está constituido por los filamentos de actina o microfilamentos (MFs) y por una serie de proteínas de unión y/o relacionadas con la actina, implicadas en la organización estructural de los MFs y en la regulación de la dinámica de polimerización/despolimerización de actina. Una forma sencilla y eficaz de interferir en la dinámica de actina es mediante el uso de toxinas de actina. En este trabajo se ha realizado un estudio exhaustivo de los efectos provocados por estas toxinas a nivel de la morfología celular, arquitectura y de salida de proteínas del aparato de Golgi, bien por depolimerización de los MFs (citocalasina D, latrunculina B, micalolide B o toxina botulínica C2) o bien por su polimerización aberrante o estabilización (jasplakinolide). El análisis de los efectos provocados por las distintas toxinas revela como éstos son dependientes del tipo celular, modo de acción de la toxina así como de la concentración y tiempo de exposición a esta. Hemos observado como los cambios de la morfología de las cisternas producto de la despolimerización de los MFs se correlacionan con variaciones en la homeostasis el pH de este orgánulo. Según lo cual, pensamos que los MFs participan en el diseño y mantenimiento de la forma aplanada de las cisternas del aparato de Golgi, probablemente regulando, en parte, la maquinaria molecular implicada en el mantenimiento de la homeostasis iónica de este orgánulo. Por otro lado, hemos observado como los MFs participan de forma variable en la salida del aparato de Golgi de cargo no asociado a balsas lipídicas con destino basolateral y apical. Sin embargo, son prescindibles en la salida de cargo asociado a balsas lipídicas con destino apical. Paralelamente a estos resultados que implican a los MFs en la morfo-funcionalidad del aparato de Golgi, hemos desarrollado un modelo celular para generación de agresomas de actina filamentosa, los cuales han sido descritos en distintas enfermedades como el Alzheimer o el acoholismo crónico bajo el nombre de cuerpos de Hirano. En este sentido, el jasplakinolide es capaz de inducir la formación reversible de un agresoma de actina filamentosa similar al cuerpo de Hirano cuya degradación está mediada por los sistemas proteasomal y lisosomal/autofagia. Por último, describimos por primera vez in vitro la generación y coexistencia de dos agresomas originados por mecanismos diferentes sin que tenga lugar en ningún momento la mezcla de sus componentes moleculares.

Motility in Aeromonas caviae, in a liquid environment (in broth culture), is mediated by a single polar flagellum encoded by the fla genes. The polar flagellum filament of A. caviae is composed of two flagellin subunits, FlaA and FlaB, which undergo O-linked glycosylation with six to eight pseudaminic acid glycans linked to serine and threonine residues in their central region. The flm genetic locus in A. caviae is required for flagellin glycosylation and the addition of pseudaminic acid (Pse) onto the lipopolysaccharide (LPS) O-antigen. However, none of the flm genes appear to encode a candidate glycotransferase that might add the Pse moiety to FlaA/B. The motility-associated factors (Maf proteins) are considered as candidate transferase enzymes, largely due to their conserved proximity to flagellar biosynthesis loci in a number of pathogens. Bioinformatic analysis performed in this study indicated that the genome of A. caviae encodes a single maf gene homologue (maf1). A maf mutant was generated and phenotypic analysis showed it is both nonmotile and lacks polar flagella. In contrast to flm mutants, it had no effect on the LPS O-antigen pattern and has the ability to swarm. Analysis of flaA transcription by reverse transcriptase PCR (RT-PCR) showed that its transcription was unaltered in the maf mutant while a His-tagged version of the FlaA flagellin protein produced from a plasmid was detected in an unglycosylated intracellular form in the maf strain. Complementation of the maf strain in trans partially restored motility, but increased levels of glycosylated flagellin to above wild-type levels. Overexpression of maf inhibited motility, indicating a dominant negative effect, possibly caused by high amounts of glycosylated flagellin inhibiting assembly of the flagellum. These data provide evidence that maf1, a pseudaminyl transferase, is responsible for glycosylation of flagellin and suggest that this event occurs prior to secretion through the flagellar Type III secretion system.