Plasma Membrane Blebbing Is Controlled by Subcellular Distribution of Vimentin Intermediate Filaments.

The formation of specific cellular protrusions, plasma membrane blebs, underlies the amoeboid mode of cell motility, which is characteristic for free-living amoebae and leukocytes, and can also be adopted by stem and tumor cells to bypass unfavorable migration conditions and thus facilitate their long-distance migration. Not all cells are equally prone to bleb formation. We have previously shown that membrane blebbing can be experimentally induced in a subset of HT1080 fibrosarcoma cells, whereas other cells in the same culture under the same conditions retain non-blebbing mesenchymal morphology. Here we show that this heterogeneity is associated with the distribution of vimentin intermediate filaments (VIFs). Using different approaches to alter the VIF organization, we show that blebbing activity is biased toward cell edges lacking abundant VIFs, whereas the VIF-rich regions of the cell periphery exhibit low blebbing activity. This pattern is observed both in interphase fibroblasts, with and without experimentally induced blebbing, and during mitosis-associated blebbing. Moreover, the downregulation of vimentin expression or displacement of VIFs away from the cell periphery promotes blebbing even in cells resistant to bleb-inducing treatments. Thus, we reveal a new important function of VIFs in cell physiology that involves the regulation of non-apoptotic blebbing essential for amoeboid cell migration and mitosis.

SPICE-Met: profiling and imaging energy metabolism at the single-cell level using a fluorescent reporter mouse.

The regulation of cellular energy metabolism is central to most physiological and pathophysiological processes. However, most current methods have limited ability to functionally probe metabolic pathways in individual cells. Here, we describe SPICE-Met (Single-cell Profiling and Imaging of Cell Energy Metabolism), a method for profiling energy metabolism in single cells using flow cytometry or imaging. We generated a transgenic mouse expressing PercevalHR, a fluorescent reporter for cellular ATP:ADP ratio. Modulation of PercevalHR fluorescence with metabolic inhibitors was used to infer the dependence of energy metabolism on oxidative phosphorylation and glycolysis in defined cell populations identified by flow cytometry. We applied SPICE-Met to analyze T-cell memory development during vaccination. Finally, we used SPICE-Met in combination with real-time imaging to dissect the heterogeneity and plasticity of energy metabolism in single macrophages ex vivo and identify three distinct metabolic patterns. Functional probing of energy metabolism with single-cell resolution should greatly facilitate the study of immunometabolism at a steady state, during disease pathogenesis or in response to therapy.

Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

At the right time in the right place: How do luminal gradients position the microbiota along the gut?

The gastrointestinal system is highly compartmentalized, where individual segments perform separate tasks to achieve common physiological goals. The gut luminal content, chyme, changes its chemical and physical properties as it passes through different intestinal segments. Together, the chyme composition, mucus, pH and oxygen gradients along the gut create a variety of highly distinct ecological niches that form, maintain and reinforce the symbiosis with the particular microbiota. Hosting different microbiota members at specific locations creates one of the most complex and sophisticated gradient - gradient of the local ecosystems that live and interact with each other, providing advantages and challenges to the host and creating our microbial self. Here, we discuss how intestinal luminal gradients are created and maintained in homeostasis, their role in a correct microbiota positioning, and their change upon inflammation and cancer.

Roles of the macrophages in colon homeostasis.

The colon is primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be toxic to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows rapid quality-check of absorbed fluids to avoid intoxication of colonocytes. This mechanism relies on a population of distal colon macrophages that are equipped with "balloon-like" protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids containing fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis.

Une des principales fonctions du côlon est d'abriter la plus large proportion de microorganismes du corps humain, ainsi que d'absorber les fluides issus de la digestion. Ainsi, la muqueuse du côlon doit constamment affronter l'arrivée de produits potentiellement dangereux. Comment le système immunitaire périphérique du côlon assure-t-il la surveillance des fluides absorbés ? Il a été montré que les macrophages sont des acteurs majeurs du système immunitaire intestinal. Nous proposons que les macrophages associés à la muqueuse épithéliale participent au maintien des fonctions des régions proximales et distales du côlon. Nous avons observé que les macrophages des régions distales possèdent des « balloon-like protrusions ¼, ou BLP, qui contactent les cellules épithéliales. Notre hypothèse de travail propose que les BLPs des macrophages servent de senseurs évaluant les fluides absorbés et contrôlant le niveau d'absorption de l'épithélium intestinal, afin d'éviter que des métabolites fongiques potentiellement dangereux puissent atteindre la circulation.

Actin cytoskeleton in mesenchymal-to-amoeboid transition of cancer cells.

During development of metastasis, tumor cells migrate through different tissues and encounter different extracellular matrices. An ability of cells to adapt mechanisms of their migration to these diverse environmental conditions, called migration plasticity, gives tumor cells an advantage over normal cells for long distant dissemination. Different modes of individual cell motility-mesenchymal and amoeboid-are driven by different molecular mechanisms, which largely depend on functions of the actin cytoskeleton that can be modulated in a wide range by cellular signaling mechanisms in response to environmental conditions. Various triggers can switch one motility mode to another, but regulations of these transitions are incompletely understood. However, understanding of the mechanisms driving migration plasticity is instrumental for finding anti-cancer treatment capable to stop cancer metastasis. In this review, we discuss cytoskeletal features, which allow the individually migrating cells to switch between mesenchymal and amoeboid migrating modes, called mesenchymal-to-amoeboid transition (MAT). We briefly describe main characteristics of different cell migration modes, and then discuss the triggering factors that initiate MAT with special attention to cytoskeletal features essential for migration plasticity.

Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption.

The colon is primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be toxic to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows rapid quality check of absorbed fluids to avoid intoxication of colonocytes. This mechanism relies on a population of distal colon macrophages that are equipped with "balloon-like" protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids containing fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis. VIDEO ABSTRACT.

Transition from mesenchymal to bleb-based motility is predominantly exhibited by CD133-positive subpopulation of fibrosarcoma cells.

BACKGROUND INFORMATION: Metastatic disease is caused by the ability of cancer cells to reach distant organs and form secondary lesions at new locations. Dissemination of cancer cells depends on their migration plasticity - an ability to switch between motility modes driven by distinct molecular machineries. One of such switches is mesenchymal-to-amoeboid transition. Although mesenchymal migration of individual cells requires Arp2/3-dependent actin polymerisation, amoeboid migration is characterised by a high level of actomyosin contractility and often involves the formation of membrane blebs. The acquisition of amoeboid motility by mesenchymal cells is often associated with enhanced metastasis. RESULTS: We studied the ability of mesenchymal HT1080 fibrosarcoma cells to switch to amoeboid motility. We induced the transition from lamellipodium-rich to blebbing phenotype either by down-regulating the Arp2/3 complex, pharmacologically or by RNAi, or by decreasing substrate adhesiveness. Each of these treatments induced blebbing in a subset of fibrosarcoma cells, but not in normal subcutaneous fibroblasts. A significant fraction of HT1080 cells that switched to blebbing behaviour exhibited stem cell-like features, such as expression of the stem cell marker CD133, an increased efflux of Hoechst-33342 and positive staining for Oct4, Sox2 and Nanog. Furthermore, the isolated CD133+ cells demonstrated an increased ability to switch to bleb-rich amoeboid phenotype both under inhibitor's treatment and in 3D collagen gels. CONCLUSIONS: Together, our data show a significant correlation between the increased ability of cells to switch between migration modes and their stem-like features, suggesting that migration plasticity is an additional property of stem-like population of fibrosarcoma cells. This combination of features could facilitate both dissemination of these cells to distant locations, and their establishment self-renewal in a new microenvironment, as required for metastasis formation. SIGNIFICANCE: These data suggest that migration plasticity is a new feature of cancer stem-like cells that can significantly facilitate their dissemination to a secondary location by allowing them to adapt quickly to challenging microenvironments. Moreover, it complements their resistance to apoptosis and self-renewal potential, thus enabling them not only to disseminate efficiently, but also to survive and colonise new niches.

Time-resolved ultrastructure of the cortical actin cytoskeleton in dynamic membrane blebs.

Membrane blebbing accompanies various cellular processes, including cytokinesis, apoptosis, and cell migration, especially invasive migration of cancer cells. Blebs are extruded by intracellular pressure and are initially cytoskeleton-free, but they subsequently assemble the cytoskeleton, which can drive bleb retraction. Despite increasing appreciation of physiological significance of blebbing, the molecular and, especially, structural mechanisms controlling bleb dynamics are incompletely understood. We induced membrane blebbing in human HT1080 fibrosarcoma cells by inhibiting the Arp2/3 complex. Using correlative platinum replica electron microscopy, we characterize cytoskeletal architecture of the actin cortex in cells during initiation of blebbing and in blebs at different stages of their expansion-retraction cycle. The transition to blebbing in these conditions occurred through an intermediate filopodial stage, whereas bleb initiation was biased toward filopodial bases, where the cytoskeleton exhibited local weaknesses. Different stages of the bleb life cycle (expansion, pausing, and retraction) are characterized by specific features of cytoskeleton organization that provide implications about mechanisms of cytoskeleton assembly and bleb retraction.

Galectin-8 Favors the Presentation of Surface-Tethered Antigens by Stabilizing the B Cell Immune Synapse.

Complete activation of B cells relies on their capacity to extract tethered antigens from immune synapses by either exerting mechanical forces or promoting their proteolytic degradation through lysosome secretion. Whether antigen extraction can also be tuned by local cues originating from the lymphoid microenvironment has not been investigated. We here show that the expression of Galectin-8-a glycan-binding protein found in the extracellular milieu, which regulates interactions between cells and matrix proteins-is increased within lymph nodes under inflammatory conditions where it enhances B cell arrest phases upon antigen recognition in vivo and promotes synapse formation during BCR recognition of immobilized antigens. Galectin-8 triggers a faster recruitment and secretion of lysosomes toward the B cell-antigen contact site, resulting in efficient extraction of immobilized antigens through a proteolytic mechanism. Thus, extracellular cues can determine how B cells sense and extract tethered antigens and thereby tune B cell responses in vivo.

Author Correction: Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane.

In the original version of this Article, financial support and contributions in manuscript preparation were not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following:'M.P. and P.O. would like to thank Prof. Roderick Y.H. Lim for advice during manuscript preparation and for providing the laboratory and microscopy infrastructure.… [We also thank] the NanoteraProject, awarded to the PATLiSciII Consortium (M.P and P.O)…'.

An In Vitro System to Study the Mesenchymal-to-Amoeboid Transition.

During the last few years, significant attention has been given to the plasticity of cell migration, i.e., the ability of individual cell to switch between different motility modes, in particular between mesenchymal and amoeboid motilities. This phenomenon is called the mesenchymal-to-amoeboid transition (MAT). Such a plasticity of cell migration is a mechanism, by which cancer cells can adapt their migration mode to different microenvironments and thus it may promote tumor dissemination. It was shown that interventions at certain regulatory points of mesenchymal motility as well as alterations of environmental conditions can trigger MAT. One of the approaches to induce MAT is to mechanically confine cells and one of the simplest ways to achieve this is to cultivate cells under agarose. This method does not require any special tool, is easily reproducible and allows cell tracking by videomicroscopy. We describe here a protocol, where MAT is associated with chemotaxis.

Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane.

At the stage of carcinoma in situ, the basement membrane (BM) segregates tumor cells from the stroma. This barrier must be breached to allow dissemination of the tumor cells to adjacent tissues. Cancer cells can perforate the BM using proteolysis; however, whether stromal cells play a role in this process remains unknown. Here we show that an abundant stromal cell population, cancer-associated fibroblasts (CAFs), promote cancer cell invasion through the BM. CAFs facilitate the breaching of the BM in a matrix metalloproteinase-independent manner. Instead, CAFs pull, stretch, and soften the BM leading to the formation of gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Blocking the ability of stromal cells to exert mechanical forces on the BM could therefore represent a new therapeutic strategy against aggressive tumors.Stromal cells play various roles in tumor establishment and metastasis. Here the authors, using an ex-vivo model, show that cancer-associated fibroblasts facilitate colon cancer cells invasion in a matrix metalloproteinase-independent manner, likely by pulling and stretching the basement membrane to form gaps.

Transient microfluidic compartmentalization using actionable microfilaments for biochemical assays, cell culture and organs-on-chip.

We report here a simple yet robust transient compartmentalization system for microfluidic platforms. Cylindrical microfilaments made of commercially available fishing lines are embedded in a microfluidic chamber and employed as removable walls, dividing the chamber into several compartments. These partitions allow tight sealing for hours, and can be removed at any time by longitudinal sliding with minimal hydrodynamic perturbation. This allows the easy implementation of various functions, previously impossible or requiring more complex instrumentation. In this study, we demonstrate the applications of our strategy, firstly to trigger chemical diffusion, then to make surface co-coating or cell co-culture on a two-dimensional substrate, and finally to form multiple cell-laden hydrogel compartments for three-dimensional cell co-culture in a microfluidic device. This technology provides easy and low-cost solutions, without the use of pneumatic valves or external equipment, for constructing well-controlled microenvironments for biochemical and cellular assays.