Single Filaments to Reveal the Multiple Flavors of Actin Antoine Jégou, Guillaume Romet-Lemonne  Biophysical Journal  Volume 110, Issue 10, Pages 2138-2146 (May 2016) DOI: 10.1016/j.bpj.2016.04.025 Copyright © 2016 Biophysical Society Terms and Conditions

Figure 1 Some of the main experimental configurations for the live observation and manipulation of single actin filaments and regulatory proteins. (A) In the canonical setup, TIRF illuminates fluorophores in a shallow region above the coverslips, and labeled filaments are anchored to the surface. (B) Microfluidics takes advantage of fluid friction (viscous drag) to align filaments with the flow, close to the coverslip, with only one end anchored to the surface. (C) Tweezer techniques (here, optical tweezers) manipulate filaments by anchoring their ends, or portions near their ends, to microbeads. (D) The background fluorescent signal from solution can be reduced further using convex lens-induced confinement or zero mode waveguides. (E) A low background enables the observation of individual molecules (here, the Arp2/3 complex). (F) High speed AFM can be used to monitor filament dynamics live. (G) Surface patterning techniques have mostly been applied to large populations of actin filaments, but can also be used to study individual filaments. To see this figure in color, go online. Biophysical Journal 2016 110, 2138-2146DOI: (10.1016/j.bpj.2016.04.025) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 2 Different flavors of actin filaments, and how they affect the binding of ADF/cofilin. Starting at the top, and going clockwise. ADF/cofilin binds preferably to ADP-actin rather than ADP-Pi-actin, Its binding and fragmentation activity is delayed if the filament is under mechanical tension. Fragmentation by ADF/cofilin is more efficient if filaments are bundled by fascin (though the opposite has been reported on other types of bundles). Specific nucleators and elongators, like formins, generate different filament structures and may affect ADF/cofilin binding as they affect the binding of other ABPs, such as tropomyosins (Tpm3.1 and Tpm1.12 shown here), which in turn regulate ADF/cofilin binding. Posttranslational modifications, especially those targeting ADF/cofilin binding sites on subdomain2 (SD2) are likely to have an impact. The binding of ADF/cofilin is more cooperative on cytoplasmic actin than α-skeletal actin filaments. To see this figure in color, go online. Biophysical Journal 2016 110, 2138-2146DOI: (10.1016/j.bpj.2016.04.025) Copyright © 2016 Biophysical Society Terms and Conditions

Figure 3 Where does the barbed end begin? (A) Schematic representation of an actin filament, where we speculate that subunits near the barbed end have a greater freedom to change conformations. (B) A formin FH2 dimer tracking an elongating barbed end takes advantage of this conformational freedom to locally impose its preferred filament structure. (C) CP, in contrast, imposes a filament structure that is compatible with the standard F-actin structure. (D) When both CP and a formin coexist at the barbed end (29,37), they compete through the conflicting conformations they each want to impose to the barbed end region. Eventually, the formin (or CP) will detach from the filament, or the formin will diffuse along the filament (29), away from the barbed end. To see this figure in color, go online. Biophysical Journal 2016 110, 2138-2146DOI: (10.1016/j.bpj.2016.04.025) Copyright © 2016 Biophysical Society Terms and Conditions