Cells are the fundamental unit of life. All living matter is made of cells: from the small systems imperceptible to our eye, like bacteria or archaea; to bigger systems, like plants or magnificent trees, fungi, and animals - including ourselves: the humans. All these systems vary in size yet they are all alive. And the common element of these systems is that they are all composed of cells. Cells are therefore fundamental, but also very complex systems. One may say, broadly, that cells are a cocktail of subsystems that, in combination and in the right balance, can become this basic unit of life. Understanding cells, and their diverse mechanisms, would therefore imply that, eventually, curious scientists (like the author herself ) may eventually be able to (better) understand life.
From a physics perspective, cells are fascinating because they constantly endure mechanical stresses and strains that challenge their survival, yet they also actively deform themselves. Our own cells exhibit remarkable deformability in response to external forces such as blood flow or muscle contraction. They also actively alter their own shape. During wound healing, cells in the skin for instance move as coherent cell sheets to heal wounds and renew tissue. And upon cellular division and differentiation, cells experience considerable shape transformations and therefore endure big deformations. Cell deformability is also an important factor in many diseases. During cancer metastasis, tumor cells for instance squeeze themselves through tissues and vessel and lymph node walls. All in all: cells are often pushed to deform, yet they somehow manage to endure those changes. So we may ask ourselves: How do they do this? To address this question, we can take a closer look at the units that forma cell....@en

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called ‘ACTIF’ that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells.

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Cell migration is a fundamental process for life and is highly dependent on the dynamical and mechanical properties of the cytoskeleton. Intensive physical and biochemical crosstalk among actin, microtubules, and intermediate filaments ensures their coordination to facilitate and enable migration. In this review, we discuss the different mechanical aspects that govern cell migration and provide, for each mechanical aspect, a novel perspective by juxtaposing two complementary approaches to the biophysical study of cytoskeletal crosstalk: live-cell studies (often referred to as top-down studies) and cell-free studies (often referred to as bottom-up studies). We summarize the main findings from both experimental approaches, and we provide our perspective on bridging the two perspectives to address the open questions of how cytoskeletal crosstalk governs cell migration and makes cells move.

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Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal biopolymers may lead to a host of active and adaptable materials. However, engineering such materials with precisely tuned properties requires measuring how the dynamics depend on the network composition and synthesis methods. Quantifying such dynamics is challenged by variations across the time, space, and formulation space of composite networks. The protocol here describes how the Fourier analysis technique, differential dynamic microscopy (DDM), can quantify the dynamics of biopolymer networks and is particularly well suited for studies of cytoskeleton networks. DDM works on time sequences of images acquired using a range of microscopy modalities, including laser-scanning confocal, widefield fluorescence, and brightfield imaging. From such image sequences, one can extract characteristic decorrelation times of density fluctuations across a span of wave vectors. A user-friendly, open-source Python package to perform DDM analysis is also developed. With this package, one can measure the dynamics of labeled cytoskeleton components or of embedded tracer particles, as demonstrated here with data of intermediate filament (vimentin) networks and active actin-microtubule networks. Users with no prior programming or image processing experience will be able to perform DDM using this software package and associated documentation.

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