Generation and Analysis of Biosensors to Measure Mechanical Forces Within Cells

Austen, Katharina; Kluger, Carleen; Freikamp, Andrea; Chrostek-Grashoff, Anna; Grashoff, Carsten

doi:10.1007/978-1-62703-604-7_15

Cited by 25 publications

(29 citation statements)

References 16 publications

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“…We have previously generated a probe (called TSMod), in which an elastic peptide is flanked by two fluorophores allowing the measurement of molecular forces between 1–6 pN using Förster resonance energy transfer (FRET) 12 , 17 – 19 . Yet individual myosin motors can generate single pN forces 20 and forces across distinct integrin receptors were recently shown to be significantly higher 21 , 22 .…”

Section: Resultsmentioning

confidence: 99%

Extracellular rigidity sensing by talin isoform-specific mechanical linkages

et al. 2015

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The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule–calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages upon cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton (pN) forces and bear, on average, 7–10 pN during cell adhesion depending on their association with f-actin and vinculin. Disruption of talin’s mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform-specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing.

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Section: Resultsmentioning

confidence: 99%

Extracellular rigidity sensing by talin isoform-specific mechanical linkages

et al. 2015

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“…3 a), for which protocols have been described before. 2 To evaluate the biosensor’s biological functionality, these constructs should be expressed in cells depleted of the endogenous protein, which has several advantages (Fig. 3 d).…”

Section: A Guide To Evaluating Genetically-encoded Fret-based Tensionmentioning

confidence: 99%

How to Measure Molecular Forces in Cells: A Guide to Evaluating Genetically-Encoded FRET-Based Tension Sensors

et al. 2014

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The ability of cells to sense and respond to mechanical forces is central to a wide range of biological processes and plays an important role in numerous pathologies. The molecular mechanisms underlying cellular mechanotransduction, however, have remained largely elusive because suitable methods to investigate subcellular force propagation were missing. Here, we review recent advances in the development of biosensors that allow molecular force measurements. We describe the underlying principle of currently available techniques and propose a strategy to systematically evaluate new Förster resonance energy transfer (FRET)-based biosensors.

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“…The cultivation of cells on a planar glass surface, as conventionally used for microscopic observations, does not force cells to exercise their capabilities of recognizing surface structures and responding to spatial cues. To explore these capacities, structured hydrogels (Zorlutuna et al, 2012) or elastic networks of extracellular matrices (Austen et al, 2013) have been developed, and the behavior of cells in these 3D environments has been monitored. On a substrate surface patterned by ridges, cells show responses that are relevant to tissue engineering: the orientation, shape, or positioning of the cells vary depending on the width, depth, or profile of the ridges (Driscoll et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

et al. 2016

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In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments.

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Generation and Analysis of Biosensors to Measure Mechanical Forces Within Cells

Cited by 25 publications

References 16 publications

Extracellular rigidity sensing by talin isoform-specific mechanical linkages

Extracellular rigidity sensing by talin isoform-specific mechanical linkages

How to Measure Molecular Forces in Cells: A Guide to Evaluating Genetically-Encoded FRET-Based Tension Sensors

Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

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