Lecture 10 13-12

• Possible mechanosensors: whole cell, cytoskeleton, adhesion complexes, focal adhesion, adhesion/adaptor proteins, integrins
• Three theories as to how cells can integrate all those mechanosensitive signals

  • Stress-driven FA mechanosensing
  • Strain-driven FA mechanosensing
  • Thermodynamically-driven FA mechanosensing
• Stress-driven FA mechanosensing

  • In FA there are molecules that can act as molecular switches
  • FA contains molecular switches: switch-like model
  • Inactive protein switches becomes active when stressed above a critical stress
  • Examples of such sensors: fibronectin, integrin, talin, vinculin, Src, FAK, p130Cas
• Strain-driven FA mechanosensing

  • Actin myosin apply some force to the adhesion plaque > that area is strained such that the lower layer is mechanically compressed > molecular distance between players within the adhesion plaque becomes smaller > higher affinity of the proteins, for example talin, to bond with each other > maturation of FA
  • Triggered by local elastic strain (extension or compression)
  • No protein conformational change
  • In short: upper FA layer is strained > lower layer is compressed > increased affinity of plaque proteins > FA enlargement
• Thermodynamically-driven FA mechanosensing

  • No protein switch
  • Maturation of FA thermodynamically driven
  • Elastic stress generated within plaque by stress fibers causes FA self-assembly and growth in pulling force direction
  • Reducing force leads to disassembly
  • Addition of new molecules relaxes stress and reduces elastic energy
  • FA is elastic and is itself a mechanosensory
• Positive mechanosensing feedback loop

  • External mechanical cues give outside-in signaling for the cell
  • Cell can recognize and sense these cues and can activate a verity of mechanical signaling
  • Such as Rho > leads to contractility > can stimulate maturation and formation of FA > senses mechanical cues from outside
  • Therefore, positive feedback loop: cells sense external mechanical cues > develop more tools that can be used to recognize or sense more mechanical cues and so on
• Mechanosensing and mechanoresponse

  • After mechanosensing, mechanotransduction takes place: cells translate mechanical cues that they sense into a chemical signal that cells can recognize. Then cells respond to these cues.
  • Mechanosensing happens quickly
  • Mechanoresponse is slower
• Extracellular cues lead to cellular response through cell and matrix proteins.

  Passive and active cues that, through cell-matrix interaction, lead up to a certain cellular response.
• Cell shape

  • The same cells assume different shapes depending on the environment
  • Cell shape can thus be quite different, and cell shape also influences how cells behave
  • Since this is the case, is it then possible to manipulate the geometry and shape of the cell?
• Cell geometry affects actin organization

  • One way to try to manipulate cell geometry: protein patterning
  • Create a substrate on which you can pattern ECM proteins in a specific shape
  • Intracellular cytoskeletal organization can be manipulated by manipulating the micropattern
  • So intracellular cytoskeleton organization sensitivity depends on the spatial distribution of extracellular ligands
  • The cell shape is all similar (all traingle), but the stress fibers are formed at different places
• Cell shape and orientation

  Why cell shape manipulation useful?

  • Cell shape and orientation are crucial for proper functioning of tissues
  • Example: in heart muscle, cells have to be oriented in a very specific way in order for the heart muscle to contract and beat properly
  • Usually guided by tissue/ECM architecture
• Contact guidance

  What kind of cues can control the shape and size of a cell?

  • Most well-known cue: contact guidance
  • = The ability of cells to align with the anisotropy of the microenvironment
  • Once a cell is aligned in a certain direction, the downstream functions is then also done in that direction
• 3 proposed mechanoresponse mechanisms

  In order to make cells align, it is possible to use microgrooves and ridges, but it is not this simple. Many experimental parameters still have to be optimized.

  Microgrooves and ridges: groove width, ridge width and groove height are all variables that can be changed. These parameters can influence the behavior of the cells.
• If groove width is most important: has something to do with the ability or inability of cells to bridge multiple ridges. So if groove width is too large, they cannot bridge the grooves.
• If ridge width is most important: if ridge width is large enough, they are not compelled to make a bridge. But if it is narrow then they are compelled to make a bridge.
• If groove height is most imporant: if height is too low, cells will sink into the gap. If the height is too high, then cells won't sink in.
• Contact guidance is one way to control cell shape and orientation, another way is strain avoidance.
• Strain avoidance

  The ability of cells to align against the direction of imposed cyclic strain (so if cyclic strain is in vertical direction, stress fibers will align in horizontal direction).

  SF regulation in strain avoidance response

  • With rock inhibitor, the SF in the cells are gone, only the peripheral stress fibers remained. The orientation of cells are changed, more randomly orientated and more in direction of applied strain.
  • With myosin inhibitor orientation changes same way as with rock inhibitor, in here only central stress fibers maintain.
• Stress fiber follows static stretch direction

  • Strain avoidance response only happens when you apply cyclic stretch. When you apply static stretch, the stress fiber follows the direction of the stretch.
  • In contrast to strain avoidance in response to cyclic stretch, cells (and stress fibers) align to static stretch direction
• Differential response of actin cap and basal fibers

  Two layers of stress fibers: SF that run on top of nucleus (= actin cap) and SF that run underneath the nucleus (= basal actin). The direction of the basal actin always follows contact guidance cues. The direction of the actin cap always follow direction of strain avoidance. So different sets of actin filaments within the same cells can be regulated by different mechanical cues from the environment.
• Cell migration - mechanotaxis

  • Cell migration can be directed by mechanical cues (mechanotaxis)
  • Taxis: movement in response to stimulus
  • Most common and well-studied cues that can influence migration direction:
  • Durotaxis (migration of cell directed by stiffness of substrate)
  • Haptotaxis (adhesion sites)
  • Flow/shear stress
• Durotaxis

  • Migration up rigidity gradient, so towards higher stiffness
  • Experiment was done where half substrate was soft other half was stiff
  • If cell started off at soft side it eventually migrated to stiff side
  • If cell started off at stiff side it explores around, but remains on stiff side
• Sensing for durotaxis: role of FA

  • FA-mediated force transmission is necessary for durotaxis
  • When substrate stiffness is low, and cells apply force to it, there is a lot of displacement
  • When substrate stiffness is high, then low displacement when force is applied > increased tension > calcium influx that reinforces formation of FA
  • Paxillin (Pxn): one of proteins in FA
• Durotaxis or durokinesis?

  • Even without taxis, when there is no directional migration preference, there can still be durotaxis like behavior if there is durokinesis
  • When cells on soft substrate: migrating in random behavior
  • When cells on stiff substrate: migration trajectory a lot straighter, a lot more persistence in migration direction
  • > when substrate from soft to stiff, then you see durotaxis-like behavior as the migration behavior differs on these types of substrate
• Haptotaxis

  • Chemotaxis: migration direction of cells depend on chemical gradient, cells migrate towards source of chemical gradient
  • Haptotaxis: similar to chemotaxis. The chemoattractant (which triggers migration of cells) is bound to substrate
  • Haptotaxis is migration up a gradient of cellular adhesion sites or substrate-bound chemoattractants
  • Particularly relevant in cancer metastasis, wound healing, tissue morphogenesis
• Flow-directed migration

  • Cells will migrate in the direction of the flow
  • Important to consider for cardiovascular systems as these cells are always subjected to some kind of flow: diseases, cell therapy, regenerative medicine
• Deformation-directed migration

  • Local substrate deformation directs migration direction
  • Microneedle was poked in substrate which contained cell (but cell itself was not touched)
  • Microneedle was poked away from cell > cell migrates slightly closer to microneedle
  • Microneedle was poked towards cell > cell migrates away from microneedle
  • Cells can pick up local deformation of substrate and make a decision to migrate away or towards source of deformation
• Cell differentiation

  • Huge potential for medicine
  • Crucial for regenerative medicine
  • It is chemosensitive and also mechanosensitive
• Concluding remarks

  • Cell-matrix interactions define cell behavior
  • Cell-matrix interactions rely on mechanosensing and mechanotransduction
  • Some mechanisms have been elucidated; many still unknown