Viola Vogel

Local force and geometry sensing regulate cell functions

The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms.

Extracellular-matrix tethering regulates stem-cell fate

To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

Bacterial Adhesion to Target Cells Enhanced by Shear Force

Surface adhesion of bacteria generally occurs in the presence of shear stress, and the lifetime of receptor bonds is expected to be shortened in the presence of external force. However, by using Escherichia coli expressing the lectin-like adhesin FimH and guinea pig erythrocytes in flow chamber experiments, we show that bacterial attachment to target cells switches from loose to firm upon a 10-fold increase in shear stress applied. Steered molecular dynamics simulations of tertiary structure of the FimH receptor binding domain and subsequent site-directed mutagenesis studies indicate that shear-enhancement of the FimH-receptor interactions involves extension of the interdomain linker chain under mechanical force. The ability of FimH to function as a force sensor provides a molecular mechanism for discrimination between surface-exposed and soluble receptor molecules.

Force-induced unfolding of fibronectin in the extracellular matrix of living cells.

Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes.

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fns soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII7 and FnIII15 with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed.

Cell fate regulation by coupling mechanical cycles to biochemical signaling pathways

Many aspects of cellular motility and mechanics are cyclic in nature such as the extension and retraction of lamellipodia or filopodia. Inherent to the cycles of extension and retraction that test the environment is the production of mechano-chemical signals that can alter long-term cell behavior, transcription patterns, and cell fate. We are just starting to define such cycles in several aspects of cell motility, including periodic contractions, integrin cycles of binding and release as well as the normal oscillations in motile activity. Cycles of local cell contraction and release are directly coupled to cycles of stressing and releasing extracellular contacts (matrix or cells) as well as cytoplasmic mechanotransducers. Stretching can alter external physical properties or sites exposed by matrix molecules as well as internal networks; thus, cell contractions can cause a secondary wave of mechano-regulated outside-in and internal cell signal changes. In some cases, the integration of both external and internal signals in space and time can stimulate a change in cell state from quiescence to growth or differentiation. In this review we will develop the basic concept of the mechano-chemical cycles and the ways in which they can be described and understood.

Local surface potentials and electric dipole moments of lipid monolayers: Contributions of the water/lipid and the lipid/air interfaces

Abstract The water/air interface is split up into two interfaces by the formation of a lipid monolayer, i.e., the water/lipid and the lipid/air interface. The absolute contribution of the lipid/air interface to the total surface potential is determined by partial dipole compensation in a specially designed molecular assembly presented in this paper. The dipole moment per terminal CH3-group of aligned hydrocarbon chains within a close-packed monolayer is +0.35 D, directed from the monolayer (−) to the air (+). The local and effective dipole moments of hydrated lipid head groups can now be calculated from surface potential measurements since the contribution of the lipid/air interface is known. Such data are given for various charged, zwitterionic, and unchanged lipids.

Harnessing biological motors to engineer systems for nanoscale transport and assembly

Living systems use biological nanomotors to build lifes essential molecules--such as DNA and proteins--as well as to transport cargo inside cells with both spatial and temporal precision. Each motor is highly specialized and carries out a distinct function within the cell. Some have even evolved sophisticated mechanisms to ensure quality control during nanomanufacturing processes, whether to correct errors in biosynthesis or to detect and permit the repair of damaged transport highways. In general, these nanomotors consume chemical energy in order to undergo a series of shape changes that let them interact sequentially with other molecules. Here we review some of the many tasks that biomotors perform and analyse their underlying design principles from an engineering perspective. We also discuss experiments and strategies to integrate biomotors into synthetic environments for applications such as sensing, transport and assembly.

Biophysics of Catch Bonds

Receptor-ligand bonds strengthened by tensile mechanical force are referred to as catch bonds. This review examines experimental data and biophysical theory to analyze why mechanical force prolongs the lifetime of these bonds rather than shortens the lifetime by pulling the ligand out of the binding pocket. Although many mathematical models can explain catch bonds, experiments using structural variants have been more helpful in determining how catch bonds work. The underlying mechanism has been worked out so far only for the bacterial adhesive protein FimH. This protein forms catch bonds because it is allosterically activated when mechanical force pulls an inhibitory domain away from the ligand-binding domain. Other catch bond-forming proteins, including blood cell adhesion proteins called selectins and the motor protein myosin, show evidence of allosteric regulation between two domains, but it remains unclear if this is related to their catch bond behavior.

Shear‐dependent ‘stick‐and‐roll’ adhesion of type 1 fimbriated Escherichia coli

It is generally assumed that bacteria are washed off surfaces as fluid flow increases because they adhere through ‘slip‐bonds’ that weaken under mechanical force. However, we show here that the opposite is true for Escherichia coli attachment to monomannose‐coated surfaces via the type 1 fimbrial adhesive subunit, FimH. Raising the shear stress (within the physiologically relevant range) increased accumulation of type 1 fimbriated bacteria on monomannose surfaces by up to two orders of magnitude, and reducing the shear stress caused them to detach. In contrast, bacterial binding to anti‐FimH antibody‐coated surfaces showed essentially the opposite behaviour, detaching when the shear stress was increased. These results can be explained if FimH is force‐activated; that is, that FimH mediates ‘catch‐bonds’ with mannose that are strengthened by tensile mechanical force. As a result, on monomannose‐coated surfaces, bacteria displayed a complex ‘stick‐and‐roll’ adhesion in which they tended to roll over the surface at low shear but increasingly halted to stick firmly as the shear was increased. Mutations in FimH that were predicted earlier to increase or decrease force‐induced conformational changes in FimH were furthermore shown here to increase or decrease the probability that bacteria exhibited the stationary versus the rolling mode of adhesion. This ‘stick‐and‐roll’ adhesion could allow type 1 fimbriated bacteria to move along mannosylated surfaces under relatively low flow conditions and to accumulate preferentially in high shear regions.