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Biophysical properties of podosomes in living macrophages. (A) AFM deflection and topographical images of podosomes on fibrinogen spots. Dotted lines delineate fibrinogen spots underneath the cell, and blue arrowheads show podosomes. (Scale bars, 5 μm.) (B) Podosome height values from AFM topographical images of living macrophages on micropatterned fibrinogen exhibited a Gaussian distribution (red curve), with a mean of 578 ± 209 nm (n = 125 podosomes in 19 cells from seven donors). (C ) The distribution of Young's modulus values was Gaussian (red curve), with a mean of 43.8 ± 9.3 kPa (n = 39 podosomes in 17 cells from four donors). (D) Young's modulus vs. indentation depth of podosomes. Representative curves of different podosomes are displayed (n = 10 podosomes in 10 cells from five donors). Dashed line shows the average indentation depth above which the Young's modulus reaches a plateau. (E) Transmission electron micrograph of a podosome at the cell periphery: a, dorsal membrane and cortical actin; b, podosome core. (Scale bar, 500 nm.) (F ) Upper: AFM deflection of an unroofed macrophage plated on fibrinogen spots (Left) and overlay of AFM deflection combined to fluorescence image (Right). Lower: Correlative microscopy of AFM deflection and fluorescence images of F-actin and vinculin of an unroofed macrophage. (Scale bars, 2 μm.) (G) Quantification of podosome height of control and unroofed macrophages; mean values are 392.5 ± 110 nm and 328 ± 94 nm, respectively. Measurements were performed on at least 392 podosomes in 26 cells from at least four donors for each condition. (H) Height and Young's modulus podosomes in macrophages plated for 2 h on fibrinogen spots or nonpatterned gelatin and fibrinogen. Height and Young's modulus were measured on at least 125 podosomes in eight cells and on 17 podosomes in four cells, respectively, from at least two donors for each condition. One-way ANOVA shows that variance is not significant over the tested conditions.
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Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), a...
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... using this approach we measured the height of hundreds of podosomes (Fig. 1C), validated by correlative fluorescence mi- croscopy ( Fig. 1B and Fig. S2A). The distribution of podosome height values was Gaussian, with an average of 591 ± 125 nm, and was correlated to their relative F-actin content (r 2 ≈ 0.7; Fig. S2B). When plated on nonpatterned fibrinogen, some macrophages re- organized their podosomes as rosettes. The height of podosome ro- settes did not significantly differ from that of isolated podosomes, with an average of 572 ± 119 nm (Fig. S2C), suggesting that podo- somes have steady height independent of their ...
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... Gaussian, with an average of 591 ± 125 nm, and was correlated to their relative F-actin content (r 2 ≈ 0.7; Fig. S2B). When plated on nonpatterned fibrinogen, some macrophages re- organized their podosomes as rosettes. The height of podosome ro- settes did not significantly differ from that of isolated podosomes, with an average of 572 ± 119 nm (Fig. S2C), suggesting that podo- somes have steady height independent of their ...
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... further insight into the structure/function relationships of podosomes, we investigated podosome rheological properties by using AFM as a nanoscale indenter. Force volume mapping of macrophages plated on fibrinogen patterns revealed that podo- some-induced membrane bumps in the landscape correlated with pixels of higher stiffness (Fig. S2D). Although this approach offers a good overview of podosome stiffness, we chose to improve our analysis by using a force-distance curve approach, which allows a greater accuracy and a faster acquisition for podosome Young's modulus determination. The values of Young's modulus showed a Gaussian distribution, with a mean of 201.2 ± 69.8 ...
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... Podosome Physical Properties in Living Macrophages. Be- cause AFM can be operated in near physiological conditions, podosome biophysical properties in living macrophages were investigated on the basis of our work on fixed cells. Time series of AFM topological images on living macrophages ( Fig. 2A and Fig. S3A) showed that podosome height values were homogeneous, with a Gaussian distribution and a mean value of 578 ± 209 nm, which was comparable to fixed cells (Fig. 2B). Moreover, the sequence of AFM images suggested that podosome formation and dynamics (lifespan ≈4 min) are amenable to AFM in living macrophages without disturbing the cells ...
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... podosome biophysical properties in living macrophages were investigated on the basis of our work on fixed cells. Time series of AFM topological images on living macrophages ( Fig. 2A and Fig. S3A) showed that podosome height values were homogeneous, with a Gaussian distribution and a mean value of 578 ± 209 nm, which was comparable to fixed cells (Fig. 2B). Moreover, the sequence of AFM images suggested that podosome formation and dynamics (lifespan ≈4 min) are amenable to AFM in living macrophages without disturbing the cells when compared with time-lapse fluorescence imaging ( Fig. S3 and Movie S1). Podosome average Young's modulus in living macrophages was 43.8 ± 9.3 kPa ( Fig. 2C), ...
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... fixed cells (Fig. 2B). Moreover, the sequence of AFM images suggested that podosome formation and dynamics (lifespan ≈4 min) are amenable to AFM in living macrophages without disturbing the cells when compared with time-lapse fluorescence imaging ( Fig. S3 and Movie S1). Podosome average Young's modulus in living macrophages was 43.8 ± 9.3 kPa ( Fig. 2C), which was fivefold higher than that of podosome-free regions (8 ± 1.7 kPa) and lower than in fixed macrophages in which formaldehyde increases biological sample stiffness (17). This value of podosome stiffness was two-to threefold higher than that of stress fibers measured by AFM in other work (12), suggesting that podosome stiffness ...
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... would reach a plateau when the AFM tip is indenting the F-actin polymer (12). Analysis of multiple force-distance curves showed that the stiffness plateau was reach after the first 80 nm of indentation in all cases, indicating that the thickness of superficial layers crossed by the AFM tip before indenting the podosome F-actin core is ≈80 nm (Fig. 2D). To verify the accuracy of this estimation, two complementary approaches were used. First, transmission electron microscopy was performed on trans- versal sections of macrophages, and second, dorsal membrane was removed by unroofing to gain direct access to podosome cores. Transmission electron microscopy images of the cell periphery ...
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... performed on trans- versal sections of macrophages, and second, dorsal membrane was removed by unroofing to gain direct access to podosome cores. Transmission electron microscopy images of the cell periphery showed the proximity between the dorsal plasma membrane and the electron-dense F-actin core of podosomes anchored into the ventral membrane (Fig. 2E). The dorsal plasma membrane is supported by a thin cortex of F-actin, with a total thickness (membrane and F-actin cortex) ranging from 60 nm to 80 nm. Thus, the fluid phase of the cytoplasm probably flows out of the region when the AFM tip applies pressure on the plasma mem- brane. Direct measurement of F-actin core by AFM after cell ...
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... of the region when the AFM tip applies pressure on the plasma mem- brane. Direct measurement of F-actin core by AFM after cell unroofing showed a difference of 64 ± 7 nm when compared with intact cells. This value corresponded to the thickness of the dorsal membrane and its associated actin cortex that were removed during the unroofing process (Fig. 2 F and G). In these experi- ments, intact and unroofed cells were fixed with glutaraldehyde instead of formaldehyde, and this could explain why podosome height values were lower than those measured before. Altogether the three approaches indicated that the AFM tip reaches the top of the podosome F-actin after the first 80 nm of indentation. In ...
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... podosome formation was modulated differently de- pending on the nature of the ECM protein (Fig. S1), we won- dered whether their physical properties could be influenced by the underlying ECM protein. Podosome height and stiffness on nonpatterned gelatin were compared with fibrinogen patterned or not (Fig. 2H), and no significant differences were observed. These results suggest that podosomes have constant structural properties independent of the nature and the organization of the ...
Citations
... AFM has been proven to be a powerful tool for investigating cell structures at nanometer-scale resolution [76]. In the early stages, Labernadie et al. [77] combined AFM with correlative fluorescence microscopy and patterned substrates to characterize the biophysical properties of podosomes, such as height, hardness, and rheological properties. However, AFM cannot probe the basal tip of the podosome in contact with the substrate and is therefore unable to measure the protrusive forces of podosomes. ...
The mechanical forces exerted by cells on their surrounding microenvironment are known as cellular traction forces. These forces play crucial roles in various biological processes, such as tissue development, wound healing and cell functions. However, it is hard for traditional techniques to measure cellular traction forces accurately because their magnitude (from pN to nN) and the length scales over which they occur (from nm to μm) are extremely small. In order to fully understand mechanotransduction, highly sensitive tools for measuring cellular forces are needed. Current powerful techniques for measuring traction forces include traction force microscopy (TFM) and fluorescent molecular force sensors (FMFS). In this review, we elucidate the force imaging principles of TFM and FMFS. Then we highlight the application of FMFS in a variety of biological processes and offer our perspectives and insights into the potential applications of FMFS.
... For individual podosomes, our simulations predict that higher substrate stiffness increases the steady-state protrusive force F ps = F p0 k s =ðk s + k c Þ 1 À V d =V pms , while reducing the substrate displacements l 1s = F ps =k s (Supplementary Fig. 11A), in line with experimental evidence 7,12 . Microscopically, this increased protrusive force on stiffer substrates is due to the stronger and denser F-actin networks in the podosome core 3,8 . This mechanosensitive structural difference affects the cluster dynamics because a denser core actin (i.e., more actin filament number N a ) on stiffer substrates requires more G-actin to assemble and releases more G-actin upon disassembly (Fig. 5B). ...
Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.
... We then determined the impact of different actin-disturbing drugs on cell fusion by pre-treating MDMs with Jasplakinolide, Cytochalasin D, and the Arp2/3 inhibitor CK666. All these drugs were used at non-cytotoxic concentrations but capable of disrupting the formation or organization of podosomes, the main F-actin adhesion structures in macrophages (Fig. S6 B; Labernadie et al., 2010;Wiesner et al., 2014). They also significantly reduced MDM infection by fusion with infected T cells , attesting that actin cytoskeleton is critical for this heterotypic cell fusion process. ...
... To assess the role of myosin activity in the HIV-1-induced heterotypic cell fusion process, MDMs were first pre-treated with either the myosin II inhibitor Blebbistatin or a broad phosphatase inhibitor (Calyculin A) before the co-culture. As expected (Kolega, 2006;Labernadie et al., 2010), upon Blebbistatin and Calyculin A treatments, MDMs showed an altered actomyosin cytoskeleton without any effect on cell viability (as the cell density was comparable with the control condition) or podosome formation ( Together, these findings demonstrate that actin dynamics and moderate myosin activity inside macrophages act in concert to favor their infection through heterotypic fusion with infected T lymphocytes. ...
Macrophages are essential for HIV-1 pathogenesis and represent major viral reservoirs. Therefore, it is critical to understand macrophage infection, especially in tissue macrophages, which are widely infected in vivo, but poorly permissive to cell-free infection. Although cell-to-cell transmission of HIV-1 is a determinant mode of macrophage infection in vivo, how HIV-1 transfers toward macrophages remains elusive. Here, we demonstrate that fusion of infected CD4⁺ T lymphocytes with human macrophages leads to their efficient and productive infection. Importantly, several tissue macrophage populations undergo this heterotypic cell fusion, including synovial, placental, lung alveolar, and tonsil macrophages. We also find that this mode of infection is modulated by the macrophage polarization state. This fusion process engages a specific short-lived adhesion structure and is controlled by the CD81 tetraspanin, which activates RhoA/ROCK-dependent actomyosin contractility in macrophages. Our study provides important insights into the mechanisms underlying infection of tissue-resident macrophages, and establishment of persistent cellular reservoirs in patients.
... Micropatterning allows standardization of the ECM and cell shape, leading to an in-depth understanding in the regulatory mechanisms of cell cytoskeleton, interaction between organelles and cell fate from the perspective of cell morphology [20]. On the other hand, micropatterning creates specific conditions to induce the formation of subcellular structures, such as stress fibers [21] and podosomes [22,23], providing convenience of associated studies. ...
Actin cytoskeleton plays crucial roles in various cellular functions. Extracellular matrix (ECM) can modulate cell morphology by remodeling the internal cytoskeleton. To define how geometry of ECM regulates the organization of actin cytoskeleton, we plated individual NIH 3T3 cells on micropatterned substrates with distinct shapes and sizes. It was found that the stress fibers could form along the nonadhesive edges of T-shaped pattern, but were absent from the opening edge of V-shaped pattern, indicating that the organization of actin cytoskeleton was dependent on the mechanical environment. Furthermore, a secondary actin ring was observed on 50μm circular pattern while did not appear on 30μm and 40μm pattern, showing a size-dependent organization of actin cytoskeleton. Finally, osteoblasts, MDCK and A549 cells exhibited distinct organization of actin cytoskeleton on T-shaped pattern, suggesting a cell-type specificity in arrangement of actin cytoskeleton. Together, our findings brought novel insight into the organization of actin cytoskeleton on micropatterned environments.
... Podosomes have been described as actin cones 7,30,32 , ranging from 400-700 nm in height 6,7,33 . We instead observed an hourglass shape that, to our knowledge, has not been previously described. ...
Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15–20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal an hourglass shape of the podosome actin core, a protruding knob at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin are characterized.
... Podosomes have been described as actin cones 7,30,32 , ranging from 400 -700 nm in height 6,7,33 . We instead observed an hourglass shape that, to our knowledge, has not been previously described. ...
Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an "hourglass" shape of the podosome actin core, a protruding "knob" at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin were characterized.
... In addition to the mechanical probing of the substratum, podosomes also show internal cycles of stiffness and periodic fluctuations of their actin content, both of which show a periodicity of 30-40 s, which led to the concept of an oscillatory behaviour of podosomes. (Labernadie et al., 2014(Labernadie et al., , 2010Proag et al., 2015Proag et al., , 2016van den Dries et al., 2013a). Of note, podosome oscillations, and also podosome protrusion, as well as vinculin recruitment, depend on actin polymerization, as these phenomena can be blocked by low doses (2 µM) of cytochalasin D, which leads to reduced actin polymerization (Herzog et al., 2020;Labernadie et al., 2014;van den Dries et al., 2013a). ...
... Moreover, while the lateral actin filaments contain myosin II, most of the myosin II pool seems to be associated with the podosome-connecting cables (van den Dries et al., 2019b) (Fig. 3 B). Myosin II is important for podosome oscillation, as its inhibition reduces oscillation of actin intensity (Panzer et al., 2016;van den Dries et al., 2013a) and of podosome stiffness (Labernadie et al., 2010). Moreover, also generation of the protrusive force of podosomes involves myosin II activity, in addition to actin polymerization (Labernadie et al., 2014). ...
Podosomes are highly dynamic actin-rich structures in a variety of cell types, especially monocytic cells. They fulfill multiple functions such as adhesion, mechanosensing, or extracellular matrix degradation, thus allowing cells to detect and respond to a changing environment. These abilities are based on an intricate architecture that enables podosomes to sense mechanical properties of their substratum and to transduce them intracellularly in order to generate an appropriate cellular response. These processes are enabled through the tightly orchestrated interplay of more than 300 different components that are dynamically recruited during podosome formation and turnover. In this review, we discuss the different phases of the podosome life cycle and the current knowledge on regulatory factors that impact on the genesis, activity, dissolution and reemergence of podosomes. We also highlight mechanoregulatory processes that become important during these different stages, on the level of individual podosomes, and also at podosome sub- and superstructures.
... Labernadie et al. measured the podosome mechanics within the living macrophage using AFM methodology and observed that the podosome stiffness is 43.8 ± 9.5 kPa (reported as mean ± s.e.m.). This specialized cellular structure is crucial in assisting the motility of macrophages through ECM degradation and tissue invasion (Labernadie et al., 2010). Integrin-controlled immune cell processes mentioned here and in Table 2 support the role of mechanotransducing integrin in inflammatory Note. ...
The pathophysiology of autoimmune disorders is multifactorial, where immune cell migration, adhesion, and lymphocyte activation play crucial roles in its progression. These immune processes are majorly regulated by adhesion molecules at cell–extracellular matrix (ECM) and cell–cell junctions. Integrin, a transmembrane focal adhesion protein, plays an indispensable role in these immune cell mechanisms. Notably, integrin is regulated by mechanical force and exhibit bidirectional force transmission from both the ECM and cytosol, regulating the immune processes. Recently, integrin mechanosensitivity has been reported in different immune cell processes; however, the underlying mechanics of these integrin-mediated mechanical processes in autoimmunity still remains elusive. In this review, we have discussed how integrin-mediated mechanotransduction could be a linchpin factor in the causation and progression of autoimmune disorders. We have provided an insight into how tissue stiffness exhibits a positive correlation with the autoimmune diseases’ prevalence. This provides a plausible connection between mechanical load and autoimmunity. Overall, gaining insight into the role of mechanical force in diverse immune cell processes and their dysregulation during autoimmune disorders will open a new horizon to understand this physiological anomaly.
... Although invadopodia and podosomes are ultimately protrusive, it is possible that they are initiated in response to inward membrane deformations. Indeed, invadopodia concentrate their actin polymerization and matrix degradation at collagen fibers that indent the cell and impede cell movement (Ferrari et al., 2019), and podosomes only become exvaginations upon actomyosin contraction (Labernadie et al., 2010) and may be initiated through other membrane deformations. In dendritic cells, WASP stimulates focal actin-based protrusions in response to local compression (Gaertner et al., 2021 Preprint). ...
To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP’s front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.
... The formation of podosomes depends not only on the structure and composition of the underlying substrate and the presence and distribution of specific integrin ligands, but podosomes also act as mechanosensors [250,322]. A higher matrix stiffness extends the lifespan of podosomes and results in a closer spacing between them [323]. ...
Cancer progression with uncontrolled tumor growth, local invasion, and metastasis depends largely on the proteolytic activity of numerous matrix metalloproteinases (MMPs), which affect tissue integrity, immune cell recruitment, and tissue turnover by degrading extracellular matrix (ECM) components and by releasing matrikines, cell surface-bound cytokines, growth factors, or their receptors. Among the MMPs, MMP-14 is the driving force behind extracellular matrix and tissue destruction during cancer invasion and metastasis. MMP-14 also influences both intercellular as well as cell–matrix communication by regulating the activity of many plasma membrane-anchored and extracellular proteins. Cancer cells and other cells of the tumor stroma, embedded in a common extracellular matrix, interact with their matrix by means of various adhesive structures, of which particularly invadopodia are capable to remodel the matrix through spatially and temporally finely tuned proteolysis. As a deeper understanding of the underlying functional mechanisms is beneficial for the development of new prognostic and predictive markers and for targeted therapies, this review examined the current knowledge of the interplay of the various MMPs in the cancer context on the protein, subcellular, and cellular level with a focus on MMP14.
