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The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity.

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The actin cortex, commonly described as a thin 2-dimensional layer of actin filaments beneath the plasma membrane, is beginning to be recognized as part of a more dynamic and three-dimensional composite material. In this review, we focus on the elements that contribute to the three-dimensional architecture of the actin cortex. We also argue that actin-rich structures such as filopodia and stress fibers can be viewed as specialized integral parts of the 3D actin cortex. This broadens our definition of the cortex, shifting from its simplified characterization as a thin, two-dimensional layer of actin filaments.

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The actin cortex is an essential element of the cytoskeleton allowing cells to control and modify their shape. It is involved in cell division and migration. However, probing precisely the physical properties of the actin cortex has proved to be challenging: it is a thin and dynamic material, and its location in the cell-directly under the plasma membrane-makes it difficult to study with standard light microscopy and cell mechanics techniques. In this chapter, we present a novel protocol to probe dynamically the thickness of the cortex and its fluctuations using superparamagnetic microbeads in a uniform magnetic field. A bead ingested by the cell and another outside the cell attract each other due to dipolar forces. By tracking their position with nanometer precision, one can measure the thickness of the cortex pinched between two beads and monitor its evolution in time. We first present the set of elements necessary to realize this protocol: a magnetic field generator adapted to a specific imaging setup and the aforementioned superparamagnetic microbeads. Then we detail the different steps of a protocol that can be used on diverse cell types, adherent or not.

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In the wild, bacteria are most frequently found in the form of multicellular structures called biofilms. Biofilms grow at the surface of abiotic and living materials with wide-ranging mechanical properties. The opportunistic pathogen Pseudomonas aeruginosa forms biofilms on indwelling medical devices and on soft tissues, including burn wounds and the airway mucosa. Despite the critical role of substrates in the foundation of biofilms, we still lack a clear understanding of how material mechanics regulate their architecture and the physiology of resident bacteria. Here, we demonstrate that physical properties of hydrogel material substrates define P. aeruginosa biofilm architecture. We show that hydrogel mesh size regulates twitching motility, a surface exploration mechanism priming biofilms, ultimately controlling the organization of single cells in the multicellular community. The resulting architectural transitions increase P. aeruginosa's tolerance to colistin, a last-resort antibiotic. In addition, mechanical regulation of twitching motility affects P. aeruginosa clonal lineages, so that biofilms are more mixed on relatively denser materials. Our results thereby establish material properties as a factor that dramatically affects biofilm architecture, antibiotic efficacy, and evolution of the resident population. IMPORTANCE The biofilm lifestyle is the most widespread survival strategy in the bacterial world. Pseudomonas aeruginosa biofilms cause chronic infections and are highly recalcitrant to antimicrobials. The genetic requirements allowing P. aeruginosa to grow into biofilms are known, but not the physical stimuli that regulate their formation. Despite colonizing biological tissues, investigations of biofilms on soft materials are limited. In this work, we show that biofilms take unexpected forms when growing on soft substrates. The physical properties of the material shape P. aeruginosa biofilms by regulating surface-specific twitching motility. Physical control of biofilm morphogenesis ultimately influences the resilience of biofilms to antimicrobials, linking physical environment with tolerance to treatment. Altogether, our work established that the physical properties of a surface are a critical environmental regulator of biofilm biogenesis and evolution.

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