Without epithelial–mesenchymal transitions, in which polarized epithelial cells are converted into motile cells, multicellular organisms would be incapable of getting past the blastula stage of embryonic development. However, this important developmental programme has a more sinister role in tumour progression. Epithelial–mesenchymal transition provides a new basis for understanding the progression of carcinoma towards dedifferentiated and more malignant states.

Epithelial–mesenchymal transitions in tumour progression

Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells-including fibroblasts, myocytes, neurons, and other cell types-sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

/pdf/tissue-cells-feel-and-respond-to-the-stiffness-of-their-6dx0rumap6.pdf

Tissue Cells Feel and Respond to the Stiffness of Their Substrate

Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.

Rho GTPases in cell biology.

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.

Pathogenic Escherichia coli

This review summarizes theoretical progress in the field of active matter, placing it in the context of recent experiments. This approach offers a unified framework for the mechanical and statistical properties of living matter: biofilaments and molecular motors in vitro or in vivo, collections of motile microorganisms, animal flocks, and chemical or mechanical imitations. A major goal of this review is to integrate several approaches proposed in the literature, from semimicroscopic to phenomenological. In particular, first considered are ``dry'' systems, defined as those where momentum is not conserved due to friction with a substrate or an embedding porous medium. The differences and similarities between two types of orientationally ordered states, the nematic and the polar, are clarified. Next, the active hydrodynamics of suspensions or ``wet'' systems is discussed and the relation with and difference from the dry case, as well as various large-scale instabilities of these nonequilibrium states of matter, are highlighted. Further highlighted are various large-scale instabilities of these nonequilibrium states of matter. Various semimicroscopic derivations of the continuum theory are discussed and connected, highlighting the unifying and generic nature of the continuum model. Throughout the review, the experimental relevance of these theories for describing bacterial swarms and suspensions, the cytoskeleton of living cells, and vibrated granular material is discussed. Promising extensions toward greater realism in specific contexts from cell biology to animal behavior are suggested, and remarks are given on some exotic active-matter analogs. Last, the outlook for a quantitative understanding of active matter, through the interplay of detailed theory with controlled experiments on simplified systems, with living or artificial constituents, is summarized.

Hydrodynamics of soft active matter

https://ressources.unisciel.fr/biocell/chap4/res/04_04_cell_migration_review_rottner.pdf

The lamellipodium: where motility begins

Interplay between Rac and Rho in the control of substrate contact dynamics

The Rho-GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3-mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro. However, little information is available on how these complexes function in vivo. Here we show that the specifically Rac1-associated protein-1 (Sra-1) and Nck-associated protein 1 (Nap1) interact with WAVE2 and Abi-1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra-1, Nap1, WAVE2 and Abi-1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra-1 or Nap1 by RNA interference abrogated the formation of Rac-dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra-1 and Nap1 are constitutive and essential components of a WAVE2- and Abi-1-containing complex linking Rac to site-directed actin assembly.

/pdf/sra-1-and-nap1-link-rac-to-actin-assembly-driving-1ziysrnnjg.pdf

Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation

he continuous remodelling of the actin cytoskeleton is a prerequisite for many cells to move and alter their shape. These activities are dependent on the highly regulated and site-specific formation of protein complexes that act as adaptors to link external signals with actin assembly. The members of the Ena/VASP protein family, VASP (for vasodilator-stimulated phosphoprotein), Mena and Evl, have been implicated in the temporal and spatial control of actin-filament dynamics. These proteins not only localize to sites of actin assembly, such as focal-adhesion sites, membrane ruffles and neuronal growth cones, but are also involved in platelet aggregation, axon guidance and the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes. By generating a stable melanoma cell line expressing VASP fused to green fluorescent protein (GFP), we now show that VASP not only co-localizes to adhesion sites with the adaptor proteins vinculin and zyxin (ref. 3 and data not shown), but is also recruited to the tips of lamellipodia in amounts that are directly proportional to the rate of protrusion. These data indicate that VASP may be an adaptor molecule involved in actin-based cell motility. They also raise important questions about the spatial relationships of the different components earmarked to have roles in actin-filament dynamics. In the GFP–VASP-expressing B16 melanoma cell line that we have produced, GFP–VASP was strikingly localized in a sharp line running along the tips of protruding lamellipodia (Fig. 1a and Supplementary Information). This localization was independent of the level of expression of GFP–VASP. To relate the localization of VASP to that of actin, we made intensity scans across the lamellipodia of GFP–VASPexpressing B16 cells that had been fixed and labelled with phalloidin at the end of the video sequence (Fig. 1a,b). The F-actin label showed a continuous gradient decreasing in intensity from the front to the rear of the lamellipodium (Fig. 1b, inset), as described previously for keratocyte lamellipodia. In contrast, the scan of GFP–VASP intensity showed a sharp peak at the lamellipodium front and a smaller peak at the rear. The latter peak arose from the presence of VASP in the focal complexes that accompany the base of rapidly migrating lamellipodia. The appearance of VASP in a line at the cell front was seen only in protruding, and not in retracting, lamellipodia (see Supplementary Information). Measurements (taken from the video frames) of the GFP fluorescence intensity at the tips of lamellipodia as a function of transient protrusion rate indicated that there was a linear relationship between these two variables (Fig. 1c). The peripheral localization of VASP was not dependent on cell adhesion to substrate, as VASP–GFP could be observed at the folding tips of membrane ruffles (data not shown) and was also concentrated at the tips of filopodia, which showed active lateral movements (see Supplementary Information). We also transiently transfected other cell lines with GFP–VASP; it showed the same localization, at the tips of lamellipodia and filopodia, in Swiss 3T3 cells and goldfish fibroblasts (data not shown). Parallel immunolabelling of the GFP–VASPexpressing cells with antibodies to Mena revealed that VASP and Mena co-localized (data not shown). The intensity of Mena immunolabel was inversely related to that of GFP–VASP, indicating a mutual feedback of expression levels of these two family members or competition for the same ligand. Although VASP was clearly localized in the anterior region of the lamellipodium, it was not possible to establish, by fluorescence microscopy, whether it occurred only at the front edge or in a broader band, corresponding for example to the ‘brush-like’ region described at the front of keratocyte lamellipodia. Using a polyclonal antibody to GFP, which reacted after fixation of cells with glutaraldehyde, we localized GFP–VASP in whole-mount cytoskeletons of B16 melanoma cells by immunoelectron microscopy. The results showed that VASP was confined to the anterior tip of lamellipodia, at the boundary of the actin meshwork (Fig. 2a,b). In filopodia, it was associated with electrondense material found at their tips (Fig. 2c). The localization of VASP at the membrane–actin interface at the lamellipodium front is consistent with the recent idea, developed from studies of Listeria, that VASP and its homologues act as flexible T

VASP dynamics during lamellipodia protrusion.

By co-injecting fluorescent tubulin and vinculin into fish fibroblasts we have revealed a “cross talk” between microtubules and early sites of substrate contact. This mutuality was first indicated by the targeting of vinculin-rich foci by microtubules during their growth towards the cell periphery. In addition to passing directly over contact sites, the ends of single microtubules could be observed to target several contacts in succession or the same contact repetitively, with intermittent withdrawals. Targeting sometimes involved side-stepping, or the major re-routing of a microtubule, indicative of a guided, rather than a random process. The paths that microtubules followed into contacts were unrelated to the orientation of stress fiber assemblies and targeting occurred also in mouse fibroblasts that lacked a system of intermediate filaments. Further experiments with microtubule inhibitors showed that adhesion foci can: (a) capture microtubules and stabilize them against disassembly by nocodazole; and (b), act as preferred sites of microtubule polymerization, during either early recovery from nocodazole, or brief treatment with taxol. From these and other findings we speculate that microtubules are guided into substrate contact sites and through the motor-dependent delivery of signaling molecules serve to modulate their development. It is further proposed this modulation provides the route whereby microtubules exert their influence on cell shape and polarity.

/pdf/targeting-capture-and-stabilization-of-microtubules-at-early-2t2p6zy8rb.pdf

Klemens Rottner

Papers

The lamellipodium: where motility begins

Interplay between Rac and Rho in the control of substrate contact dynamics

Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation

VASP dynamics during lamellipodia protrusion.

Targeting, Capture, and Stabilization of Microtubules at Early Focal Adhesions