Podosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force.

Podosomes are mechanosensitive adhesion cell structures that are capable of applying protrusive forces onto the extracellular environment. We have recently developed a method dedicated to the evaluation of the nanoscale forces that podosomes generate to protrude into the extracellular matrix. It consists in measuring by atomic force microscopy (AFM) the nanometer deformations produced by macrophages on a compliant Formvar membrane and has been called protrusion force microscopy (PFM). Here we perform time-lapse PFM experiments and investigate spatial correlations of force dynamics between podosome pairs. We use an automated procedure based on finite element simulations that extends the analysis of PFM experimental data to take into account podosome architecture and organization. We show that protrusion force varies in a synchronous manner for podosome first neighbors, a result that correlates with phase synchrony of core F-actin temporal oscillations. This dynamic spatial coordination between podosomes suggests a short-range interaction that regulates their mechanical activity.

Determining how cells generate and transduce mechanical forces at the nanoscale is a major technical challenge for the understanding of numerous physiological and pathological processes. Podosomes are submicrometer cell structures with a columnar F-actin core surrounded by a ring of adhesion proteins, which possess the singular ability to protrude into and probe the extracellular matrix. Using protrusion force microscopy, we have previously shown that single podosomes produce local nanoscale protrusions on the extracellular environment. However, how cellular forces are distributed to allow this protruding mechanism is still unknown. To investigate the molecular machinery of protrusion force generation, we performed mechanical simulations and developed quantitative image analyses of nanoscale architectural and mechanical measurements. First, in silico modeling showed that the deformations of the substrate made by podosomes require protrusion forces to be balanced by local traction forces at the immediate core periphery where the adhesion ring is located. Second, we showed that three-ring proteins are required for actin polymerization and protrusion force generation. Third, using DONALD, a 3D nanoscopy technique that provides 20 nm isotropic localization precision, we related force generation to the molecular extension of talin within the podosome ring, which requires vinculin and paxillin, indicating that the ring sustains mechanical tension. Our work demonstrates that the ring is a site of tension, balancing protrusion at the core. This local coupling of opposing forces forms the basis of protrusion and reveals the podosome as a nanoscale autonomous force generator.

Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.

Far from being passive, apoptotic cells influence their environment. For example, they promote tissue folding, myoblast fusion and modulate tumor growth. Understanding the role of apoptotic cells necessitates their efficient tracking within living tissues, a task that is currently challenging. In order to easily spot apoptotic cells in developing tissues, we generated a series of fly lines expressing different fluorescent sensors of caspase activity. We show that three of these reporters (GFP-, Cerulean- and Venus-derived molecules) are detected specifically in apoptotic cells and throughout the whole process of programmed cell death. These reporters allow the specific visualization of apoptotic cells directly within living tissues, without any post-acquisition processing. They overcome the limitations of other apoptosis detection methods developed so far and, notably, they can be combined with any kind of fluorophore.

Fly stock. Flies were grown under standard culture techniques. A Sqh-TagRFPt[9B] knock-in line was used for imaging of Myosin II homolog in Drosophila during peripodial epithelium retraction (1). A Flytrap line Vkg-GFP[G0454] was used for imaging the dynamics of the collagen (extra-cellular matrix) during leg eversion (2). The Armadillo-GFP line is from Bloomington (number 8556).Leg disc preparation. Leg discs were dissected from white pupae + 2 hours after puparium formation in Schneider's insect medium (Sigma-Aldrich) supplemented with 15% fetal calf serum, 0.5% penicillin-streptomycin and 2 µg/mL 20-hydroxyecdysone (Sigma-Aldrich, H5142). Leg discs were transferred onto a glass slide in 13.5 µL of this medium and confined between a 120 µm-deep double-sided adhesive spacer (Secure-SealTM from Sigma-Aldrich) and a glass coverslip placed on top of the spacer. Halocarbon oil was added to the sides of the spacer to prevent evaporation. To visualize cell shapes during peripodial epithelium retraction, cell membranes were labeled after dissection and before imaging via a 10-minute incubation with Far red CellMask plasma membrane stain according to manufacturer instructions (Thermo Fisher Scientific). To prevent collagen remains from interfering with peripodial epithelium curling, a collagenase treatment (0.18 units/mL) was applied for 10 minutes simultaneously with CellMask incubation.Confocal imaging of peripodial epithelium. Retraction and curling of peripodial epithelia were imaged at 24 • C on an inverted confocal laser scanning microscope (LSM-880, Zeiss) equipped with an Airyscan detector and a 40X objective (C-Apochromat, NA=1.2, Zeiss). Images were acquired at a rate of one z-stack every 2-5min and z-slices were spaced by 0.5µm. Airyscan images were then reconstructed using the Airyscan processing module of the Zen Black software (Zeiss). Movies of the retraction were then generated using Imaris (Bitplane). MDCK cell lines.MDCK-E-Cadherin-GFP cell lines (generated as described in (3)) were cultured in presence of 250ng/ml puromycin in the culture medium. MDCK NMHCIIA-GFP and MDCK NMHCIIB-GFP were generated as described in (4). Cells were then cultured in presence of G418 (1mg/ml) in the culture medium.

Cell shape affects proliferation and differentiation, which are processes known to depend on integrin-based focal adhesion (FA) signaling. Because shape results from force balance and FAs are mechanosensitive complexes transmitting tension from the cell structure to its mechanical environment, we investigated the interplay between 3D cell shape, traction forces generated through the cell body, and FA growth during early spreading. Combining measurements of cell-scale normal traction forces with FA monitoring, we show that the cell body contact angle controls the onset of force generation and, subsequently, the initiation of FA growth at the leading edge of the lamella. This suggests that, when the cell body switches from convex to concave, tension in the apical cortex is transmitted to the lamella where force-sensitive FAs start to grow. Along this line, increasing the stiffness resisting cell body contraction led to a decrease of the lag time between force generation and FA growth, indicating mechanical continuity of the cell structure and force transmission from the cell body to the leading edge. Remarkably, the overall normal force per unit area of FA increased with stiffness, and its values were similar to those reported for local tangential forces acting on individual FAs. These results reveal how the 3D cell shape feeds back on its internal organization and how it may control cell fate through FA-based signaling.mechanosensing | cell spreading | cortical tension | cell mechanics W hen cells are cultured on flat substrates, their functions and fate can be modulated by the size and shape of the surface they are allowed to spread on (1-6). In particular, the cell spread area was shown to control the balance between proliferation and apoptosis (1), DNA synthesis (3), and histone acetylation (6). Although the mechanisms behind these phenomena remain to be defined, focal adhesions (FAs) and actomyosin-dependent contractility are clearly involved. For instance, constitutively active focal adhesion kinase (FAK) restores proliferation in nonadherent cells (7,8). Indeed, FAs are assemblies of multiple proteins among which many are part of fundamental networks of regulation of cell functions (9, 10). Moreover, FAs are mechanosensitive anchors that resist the tension developed in the cell architecture. They are known to grow under the application of an external force, and to shrink when internal tension is decreased (11-13). Thus, many recent studies investigated the correlation between local traction forces and maturation of individual FAs using 2D deformable substrates (14-18).However, to understand how the cell spread area could control cell fate, a description of the link between the overall cell shape and FA dynamics is still missing. Noteworthily, cell traction forces (19,20) and cell signaling (1, 3, 6) were shown to be nonlinear functions of the cell spread area. In other words, the magnitude of traction forces, the fraction of apoptotic cells, or the level of histone acetylation present a switch from a low to...

Epithelial−mesenchymal transition (EMT) is an essential process both in physiological and pathological contexts. Intriguingly, EMT is often associated with tissue invagination during development; however, the impact of EMT on tissue remodeling remain unexplored. Here, we show that at the initiation of the EMT process, cells produce an apico-basal force, orthogonal to the surface of the epithelium, that constitutes an important driving force for tissue invagination in Drosophila . When EMT is ectopically induced, cells starting their delamination generate an orthogonal force and induce ectopic folding. Similarly, during mesoderm invagination, cells undergoing EMT generate an apico-basal force through the formation of apico-basal structures of myosin II. Using both laser microdissection and in silico physical modelling, we show that mesoderm invagination does not proceed if apico-basal forces are impaired, indicating that they constitute driving forces in the folding process. Altogether, these data reveal the mechanical impact of EMT on morphogenesis.