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3.3 Actin Force Assays
In vitro experiments in minimum motility media suggested that force genera-tion and motility of Listeria type mogenera-tion is merely due to the polymerizagenera-tion of actin filaments against the motile objects. Although hypothesized [53], for ARP2/3 mediated actin polymerization so far no molecular motors based on conformation-change were discovered. In case of formin based actin polymeri-zation, force generation due to conformation changes of the formin molecule is more likely. The structure and processivity of the formin motor suggest that conformation changes take place during actin polymerization. However, a direct experimental prove is yet to be found [22].
From basic motility assays, the characteristics of actin based force generation remains rather puzzling and even inconsistent. For example, velocity measure-ments of actin propelled colloids in media with varying viscosity suggested a self-strengthening response of the actin network as the drag force is increased.
This rendered the velocity of propulsion largely independent of the drag force [48]. However, similar measurements performed by another group of researches indicated that the velocities depended on the viscous drag force [54]. Others ob-served that Listeria appeared to advance in discrete steps of 5.5 nm, similar to the size of an actin monomer [42]. These steps could suggest some intrinsic mo-lecular scale mechanism at the interface between filaments and the surface, which is also yet to be proven. The next logic step in further understanding actin based motion would be a direct measurement of the polymerization force on a growing actin gel. Several techniques have been successfully used. Their work-ing principles and the results will be briefly explained in the followwork-ing.
Micropipettes: Marcy et al. used a micromanipulation approach [55]. Here a Listeria like comet grows at a bead attached to a thin glass fiber. The force is measured by recoding the deflection of the glass fiber using optical microscopy.
To apply forces, the comet was pushed or pulled by a micropipette, while re-cording the growth speed of the comet. By pushing (positive) or pulling (nega-tive) forces on the order of -1.7 to 4.3 nN were applied and the force–velocity relation was established. Marcy et al. found linear force-velocity regimes for both pulling and pushing forces, which decays more rapidly for pulling forces.
Furthermore, by pulling the actin tail away from the bead at high speed, the elastic modulus of the gel and the force necessary to detach the tail from the bead were estimated. Also thickening of the gel was observed upon pushing
forces, which could explain the self-strengthening of the actin network upon compression.
Friction forces in the actin network have been measured in the same group utilizing a very similar setup [56]. With the micropipette the comet was pulled 2-3 times faster than its natural growth speed, which resulted in an oscillating behavior of force and velocity. This result suggests a stick slip phenomenon where smooth movement occurs when an average number of filaments remain attached to the bead, whereas stick-slip motion occurs when a cooperative breaking happens. This work suggests that both, actin polymerization and con-nection of actin filaments to the surface, is controlled by the N-WASP|ARP2/3 complex.
Atomic Force Microscopy (AFM): A modified AFM was used to study the force generation and load dependence of actin polymerization by Parekh et al.
[57]. The AFM measurement technique was optimized to account for the unpre-dictable drift in z-direction which becomes problematic in long term measure-ments at constant piezo positions [58]. Here actin was polymerized in cell ex-tracts at the apex of a standard contact mode imaging cantilever. Parekh ob-tained force–velocity curves of growing actin networks until network elongation ceased at the stall force. The growth velocity was found to be load-independent over a wide range of forces before stalling, which could be due to self-strengthening of the actin network. When decreasing the forces on the growing network, the velocity increased to a value greater than the previous velocity, similar to the results found by Marcy et al. [55, 56]. Among other differences to the AFM experiments shown in this work (see section 5.2), the measurements by Parekh et al. involve a flat force probe geometry. Also the actin network grows in a cytoplasmic extract, whereas we use a completely reconstructed me-dium comprised of pure proteins. This gives us the opportunity to control the properties of the actin gel and to test various the gel compositions. One aim of our approach is to learn about the role of regulatory proteins in the generation of force.
The same group also performed AFM based microrheology assays on den-dritic actin networks and reported stress stiffening followed by a regime of re-versible stress softening at increasing loads [59]. Stress stiffening is attributed to entropic elasticity of individual filaments, while the softening behavior can be explained by elastic buckling of individual filaments under compression.
3.3 Actin Force Assays
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Lipid vesicles/droplets: To probe the polymerization forces quantitatively in a reconstructed in vitro assay, two similar experimental systems have been in-troduced at about the same time. Lipid vesicles [50] and lipid droplets [60] were coated with ActA to form a dendritic actin comet in a suitable ARP2/3 contain-ing medium. Unlike hard plastic beads (see section 5.1) the “soft” vesicles and droplets deform as the dendritic actin network evolves at their surface. The rea-son is buildup of elastic tension due to insertion of monomers at curved surfaces (see section 3.5). Both groups analyze the shape of the soft colloidal objects and deduce the compression forces associated with actin polymerization. According to Giardini et al. [60] the forces are on the order of 0.4-4 nN for a droplet with a spherical radius of 1.45 µm. The forces determined by Upadhyaya et al. [50] are on the order of 3-8 nN/µm2. An example of a deformed lipid vesicle is shown in Figure 3-7C (p. 33).
Optical tweezers: Force measurements on a small number (approximately eight) of parallel filaments were performed by Footer et al. [61]. The micro fa-bricated setup mimics the geometry of filopodial of crawling cells protrusions.
The unparalleled sensitivity of optical tweezers was required to the measure force which was on the order of 1 pN. This relatively small value was attributed to the fact that only one filament at a time is in contact with the force probe.
This is consistent with the theoretical load required to stall the elongation of a single filament. The results imply that living cells must use actin-associated fac-tors to enhance the force generation ability of small filopodia-like actin bundles.