Distribution and chemical characterization of regular arrays in the cell walls of strains of the genus Lactobacillus

Abstract The presence of regular arrays (RAs) in the cell walls of strains of the genus Lactobacillus was examined by electron microscopy. The RAs were found in 6 species including L. bulgaricus, L. helveticus, L. acidophilus, L. fermentum, L. brevis and L. buchneri . The RAs were composed of a protein with an apparent M _r ranging from about 41000 to 55000, depending on the species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The amino acid composition of the RA proteins was shown to be acidic and hydrophobic. The antigenicity of the RA protein from L. buchneri appeared to be specific but not common among the RA proteins from the other lactobacilli.     

NSun2 promotes cell migration through methylating autotaxin mRNA

NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3′-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo. Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.     

RhoB regulates cell migration through altered focal adhesion dynamics

The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.     

Locomotion control of Caenorhabditis elegans through confinement

The model organism Caenorhabditis elegans shows two distinct locomotion patterns in laboratory situations: it swims in low viscosity liquids and it crawls on the surface of an agar gel. This provides a unique opportunity to discern the respective roles of mechanosensation (perception and proprioception) and mechanics in the regulation of locomotion and in the gait selection. Using an original device, we present what to our knowledge are new experiments where the confinement of a worm between a glass plate and a soft agar gel is controlled while recording the worm's motion. We observed that the worm continuously varied its locomotion characteristics from free swimming to slow crawling with increasing confinement so that it was not possible to discriminate between two distinct intrinsic gaits. This unicity of the gait is also proved by the fact that wild-type worms immediately adapted their motion when the imposed confinement was changed with time. We then studied locomotory deficient mutants that also exhibited one single gait and showed that the light touch response was needed for the undulation propagation and that the ciliated sensory neurons participated in the joint selection of motion period and undulation-wave velocity. Our results reveal that the control of maximum curvature, at a sensory or mechanical level, is a key ingredient of the locomotion regulation.     

Guiding cell migration through directed extension and stabilization of pseudopodia

Cell migration requires establishment of a single pseudopodium in the direction of movement. Here we highlight recent advances in our understanding of the molecular signaling mechanisms that regulate formation of pseudopodia. We discuss how signal transduction processes are spatially and temporally organized to establish cell polarity through directed extension and stabilization of dominant pseudopodia. We also highlight recent advances in technology that will further the understanding of signaling dynamics specific to pseudopodia extension and cell migration.     

JNK regulates cell migration through promotion of tyrosine phosphorylation of paxillin

The adaptor protein paxillin plays an important role in cell migration. Although the c-Jun amino-terminal kinase (JNK) phosphorylation of paxillin on Ser 178 has been found to be critical for cell migration, the precise mechanism by which JNK regulates cell migration is still not very clear. Here, the migration of human corneal epithelial (HCE) cells was used to determine which signaling pathways are involved in EGF-induced paxillin phosphorylation. Paxillin was phosphorylated on Tyr 31 and Tyr 118 after induction of migration by EGF in HCE cells. Specific inhibition of JNK activation by inhibitor SP600125 or overexpression of a dominant-negative JNK mutant not only blocked EGF-induced cell migration, but also eliminated tyrosine phosphorylation of paxillin on Tyr 31 and Tyr 118. HCE cells overexpressing paxillin-S178A mutant also exhibited lower mobility, and reduced phosphorylation of Tyr 31 and Tyr 118. However, paxillin-S178A-inhibited cell migration can be rescued by overexpression of paxillin-Y31E/Y118E mutant. Importantly, inhibition of JNK by SP600125 or overexpression of paxillin-S178A mutant prevented the association of FAK with paxillin. Taken together, these results suggest that phosphorylation of paxillin on Ser 178 by JNK is required for the association of paxillin with FAK, and subsequent tyrosine phosphorylation of paxillin.     

FGF signals for cell proliferation and migration through different pathways

FGFs are pleiotropic growth factors that control cell proliferation, migration and differentiation. However, FGF transduction studies have so far focused primarily on the mitogenic effect of this growth factor family and it has been difficult to assess if the described intracellular signaling pathways are dedicated solely to cell proliferation, or whether they are equally important for the migratory activity often seen in responsive cells. We review here papers in which the migratory effects of this growth factor family were clearly discriminated from proliferative effects. In toto, these studies suggest that cells use different signaling pathways for migration, such as Src and p38 MAP kinase, from those for proliferation, which tend to upregulate the ERKs. Which signaling pathway a cell uses for proliferation or migration appears to depend on many factors, including the structure and the quantity of available FGF trapped in the basal lamina by heparan sulfate co-factors, the disposition of cognate high affinity receptors and the general environment of the cell. Thus the density of the cell population, the state of the cell cycle, the presence of other factors or receptors will modulate the migratory response of cells to FGF.     

Evi5 promotes collective cell migration through its Rab-GAP activity

Membrane trafficking has well-defined roles during cell migration. However, its regulation is poorly characterized. In this paper, we describe the first screen for putative Rab-GTPase-activating proteins (GAPs) during collective cell migration of Drosophila melanogaster border cells (BCs), identify the uncharacterized Drosophila protein Evi5 as an essential membrane trafficking regulator, and describe the molecular mechanism by which Evi5 regulates BC migration. Evi5 requires its Rab-GAP activity to fulfill its functions during migration and acts as a GAP protein for Rab11. Both loss and gain of Evi5 function blocked BC migration by disrupting the Rab11-dependent polarization of active guidance receptors. Altogether, our findings deepen our understanding of the molecular machinery regulating endocytosis and subsequently cell signaling during migration.     

Galpha13 stimulates cell migration through cortactin-interacting protein Hax-1

Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.     

Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits.

Cell walls of Clostridium thermohydrosulfuricum and C. thermosaccharolyticum have a two-layered structure, consisting of a thin, lysozyme-sensitive murein layer and a surface (S) layer composed of hexagonally or tetragonally arranged subunits. The subunits can be removed from the murein layer by treatment of cell wall preparations, are composed of a fragile, pH-sensitive monolayer of macromolecular subunits. In both organisms the first stage of the cell division process involves only the plasma membrane and the murein layer. During the subsequent cell separation, a surplus of S-layer subunits appears at the site of division, and consequently the newly formed cell poles remain completely covered by the s layer throughout the separation process. In autolyzed cells an additional layer of subunits assembles on extended areas of the inside of the mucopeptide layer. These observations indicate that the biological function of the S layer depends on its ability to maintain a complete covering of the cell surface at all stages of cell growth and division.     

Docosahexaenoic acid suppresses migration of triple-negative breast cancer cell through targeting metastasis-related genes and microRNA under normoxic and hypoxic conditions

There is insufficient evidence with respect to the effect of the standard anticancer therapeutic agents as well as common dietary supplements on the expression of such genes and microRNAs (miRNAs). Therefore, this study was aimed to study the effect of applying linoleic acid (LA) and docosahexaenoic acid (DHA) fatty acids alone or combined with Taxol on the expression of the matrix metalloproteinase (MMP)-9, MMP-2, vimentin, and talin2 genes, tumor-suppressor miR-194 and, onco-miR-106b in triple-negative breast cancer cell line, known as MDA-MB-231. MDA-MB-231 as metastatic breast cancer cell line was cultured and treated using 0.3 μM Taxol, 100 μM DHA, and 50 μM LA for 24 hours, alone or combined with Taxol under the normoxic and hypoxic conditions. Cells were harvested, after RNA extraction and complementary DNA synthesis, analysis of the expression levels of the studied genes and miRNAs was done through the use of the quantitative real-time polymerase chain reaction (qRT-PCR). Wound healing assay and Western blot analysis were also performed for confirmation. The results of qRT-PCR showed that treating the MDA-MB-231 cells with DHA caused an increase in the miR-194 expression and a decrease in the miR-106b expression, leading to the downregulation of the MMP-2 and MMP-9, and vimentin, and upregulation of the talin2 under the normoxic and hypoxic conditions. The results of the wound healing scratch assay revealed that the administration of the DHA and the DHA-Taxol combination caused the repression of cell migration in comparison with the control groups under the normoxic and hypoxic conditions. The results of the Western blot analysis demonstrated that DHA and the DHA-Taxol combination caused an increase in the expression of the talin2 protein rather than the control cells under both normoxic and hypoxic conditions. This study showed that DHA has significant antimetastatic effects against the triple-negative breast cancer cells. DHA could serve as a promising supplementation for suppressing the breast cancer cell migration, especially under the hypoxic condition.     

Usp8 promotes tumor cell migration through activating the JNK pathway

Tumor metastasis is the most cause of high mortality for cancer patients. Identification of novel factors that modulate tumor cell migration is of great significance for therapeutic strategies. Here, we find that the ubiquitin-specific protease 8 (Usp8) promotes tumor cell migration through activating the c-Jun N-terminal kinase (JNK) pathway. Genetic epistasis analyses uncover Usp8 acts upstream of Tak1 to control the JNK pathway. Consistently, biochemical results reveal that Usp8 binds Tak1 to remove ubiquitin modification from Tak1, leading to its stabilization. In addition, human USP8 also triggers tumor cell migration and activates the JNK pathway. Finally, we show that knockdown of USP8 in human breast cancer cells suppresses cell migration. Taken together, our findings demonstrate that a conserved Usp8-Tak1-JNK axis promotes tumor cell migration, and providing USP8 as a potential therapeutic target for cancer treatment.Subject terms: Metastasis, Cell migration     

Extracellular matrix density regulates the formation of tumour spheroids through cell migration

In this work, we show how the mechanical properties of the cellular microenvironment modulate the growth of tumour spheroids. Based on the composition of the extracellular matrix, its stiffness and architecture can significantly vary, subsequently influencing cell movement and tumour growth. However, it is still unclear exactly how both of these processes are regulated by the matrix composition. Here, we present a centre-based computational model that describes how collagen density, which modulates the steric hindrance properties of the matrix, governs individual cell migration and, consequently, leads to the formation of multicellular clusters of varying size. The model was calibrated using previously published experimental data, replicating a set of experiments in which cells were seeded in collagen matrices of different collagen densities, hence producing distinct mechanical properties. At an initial stage, we tracked individual cell trajectories and speeds. Subsequently, the formation of multicellular clusters was also analysed by quantifying their size. Overall, the results showed that our model could accurately replicate what was previously seen experimentally. Specifically, we showed that cells seeded in matrices with low collagen density tended to migrate more. Accordingly, cells strayed away from their original cluster and thus promoted the formation of small structures. In contrast, we also showed that high collagen densities hindered cell migration and produced multicellular clusters with increased volume. In conclusion, this model not only establishes a relation between matrix density and individual cell migration but also showcases how migration, or its inhibition, modulates tumour growth.     

Self-assembly of rationally designed peptides under two-dimensional confinement

The rational design of interfacially confined biomolecules offers a unique opportunity to explore the cooperative relationship among self-assembly, nucleation, and growth processes. This article highlights the role of electrostatics in the self-assembly of β-sheet-forming peptides at the air-water interface. We characterize the phase behavior of a periodically sequenced sheet-forming peptide by using Langmuir techniques, Brewster angle microscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism spectroscopy. We find that peptides with an alternating binary sequence transition at high pressures from discrete circular domains to fibrous domains. The qualitative behavior is independent of surface pressure but dependent on molecular areas. In addition, thermodynamic models are employed to specifically quantify differences in electrostatics by obtaining parameters for the critical aggregation area, the limiting molecular area, and the dimensionless ratio of line tension/dipole density. Using these parameters, we are able to relate localized charge distribution to phase transitions, which will allow us to apply these molecules to examine how the dynamics of self-assembly can be directly coupled to the formation of composite nanostructures in biology.