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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.

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.

© 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.

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.