75 nM is significantly higher than that reported to be optimum by Andrianantoandro and Pollard, 2006 (10 nM), and therefore, we observed severing in the presence of lower concentrations, down to 10 nM.
Cofilin is an actin filament severing protein (reviewed by Pollard (2000)), and although our experiments were performed at pH 6.8, which should suppress severing activity (Yonezawa et al., 1985; Hawkins et al., 1993; Pavlov et al., 2006), we observed frequent severing of the filaments near or within short cofilin clusters.
"In me you may observe fit punishment," the severed head helpfully points out.
In the presence of 650 nM cofilin, many of the filaments were fully decorated with cofilin when observations started, and severing was observed exclusively near the ends of the long clusters (Video 12 and Figure 6 figure supplement 4).
More recently, barbel amputation was attempted as a mark-recapture technique for juvenile shortnose sturgeon (Acipenser brevirostrum); however, it was observed that many of the severed barbels completely or partially regrew, making this approach inappropriate for long-term studies [35], [36].
Moreover, we observed a myosin-II-null cell severing a budded yeast, making it unlikely that myosin-II plays a role in generating the force required for severing.
SETD2 RNAi-depleted ccRCC cells phenocopied the severed p53 cell cycle checkpoint observed in SETD2 mutant ccRCC cells.
But the characters in the film, directed by Ms. Jaoui from a screenplay she wrote with her husband, Jean-Pierre Bacri (they both star in the movie), are so precisely observed that the forging and severing of these connections feels intuitively right.
Severing was often observed between 40 (Video 12 and Figure 6 figure supplement 4) and 200 nM cofilin.
Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries.
We also observed a stress shielded region close to the severed tendon ends, which may impair collagen fibre realignment during the remodelling stage of repair due to the lack of tensile stress.
