We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation.

The COVID-19 pandemic ushered in an unprecedented period of both crisis and innovation in higher education. The shift to an online learning environment was particularly problematic for courses in which students learn disciplinary practices. Scientific practice requires hands-on training and collaborative engagement with instructors and peers, dimensions of the learning environment that were challenging to recreate online. Here, we describe the resulting instructional innovations and challenges experienced in shifting multiple undergraduate- and graduate-level molecular bioscience labs, including Genetics, Cell Biology, Bioinformatics, and Advanced Microscopy, to an online learning environment. Instructors pursued novel approaches, techniques, and at-home lab tools with varying success. Many innovations were retained after the transition back to an in-person learning environment because they uniquely supported previously overlooked aspects of student learning. Consistent with other reports, we found that marginalized students pursuing science were disproportionately burdened by COVID-19 and the shift to an online learning environment. A description of what worked for online learning, what didn’t, and what is worth holding onto in the future is valuable for constructing learning environments that effectively support learners in their disciplinary practice.

Student evaluations of teaching (SETs) are a ubiquitous feature of higher education. However, scholars have presented numerous challenges to the accuracy, validity, reliability and objectivity of SETs as a measure of teaching effectiveness. Given the potential for bias, the use of SETs in professional review may constitute a form of institutional discrimination. Therefore, institutions of higher learning need to develop, adopt and refine better methods for collecting and using student feedback. This paper describes the steps taken by a mid-sized comprehensive university in the USA over a three-year period to do that. We describe the work of our committee dealing with this issue, how we collaborated with the rest of the university to enact change, and the Learning Environment Survey (LENS) system that the university eventually selected and modified. We also report findings from a pilot study of the new instrument, which was favorably received by both students and faculty, and make recommendations for other institutions of higher education.

Muscle aging is accompanied by blunted muscle regeneration in response to injury and disuse. Oxidative stress likely underlies this diminished response, but muscle redox sensors that act in regeneration have not yet been characterized. Calmodulin contains multiple redox sensitive methionines whose oxidation alters the regulation of numerous cellular targets. We have used the CRISPR-Cas9 system to introduce a single amino acid substitution M109Q that mimics oxidation of methionine to methionine sulfoxide in one or both alleles of the CALM1 gene, one of three genes encoding the muscle regulatory protein calmodulin, in C2C12 mouse myoblasts. When signaled to undergo myogenesis, mutated myoblasts failed to differentiate into myotubes. Although early myogenic regulatory factors were present, cells with the CALM1 M109Q mutation in one or both alleles were unable to withdraw from the cell cycle and failed to express late myogenic factors. We have shown that a single oxidative modification to a redox-sensitive muscle regulatory protein can halt myogenesis, suggesting a molecular target for mitigating the impact of oxidative stress in age-related muscle degeneration.