A domain-swapped CaMKII conformation facilitates linker-mediated allosteric regulation.

Ca2+ signaling plays a key role in physiological processes such as memory formation and cardiac function. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the primary kinase that responds to Ca2+ inputs in these cells. There are four CaMKII paralogs in mammals which are alternatively spliced in the variable linker region to create upwards of 70 different variants. In this study, we systematically studied different linker regions and determined that the position of charged residues within the linker region modulates the Ca2+/CaM sensitivity of the holoenzyme. We present an X-ray crystal structure of full-length CaMKIIδ that shows a domain-swapped conformation of the subunits within the dodecameric holoenzyme. In this structure, the kinase domain of one subunit is docked onto the hub domain of a different subunit, providing an additional interface within the holoenzyme. Mutations at the equatorial and lateral interfaces revealed that the kinase-hub interaction dissociates as the hub-hub interfaces are disturbed, which led alterations in the stoichiometry of CaMKII holoenzyme and Ca2+/CaM sensitivity. Molecular dynamics simulations of linker-containing domain-swapped and non-domain-swapped CaMKIIs reveal that the domain-swapped configuration facilitates an interaction between the calmodulin binding domain and the variable linker region, such that dynamic electrostatic forces between charges on these segments can modulate the equilibrium between the compact and extended conformational states of the holoenzyme. Small angle X-ray scattering data confirms that a negatively charged linker CaMKII holoenzyme adopts a more compact conformation compared to a positively charged linker. These data support a model where patches of charged linker residues interact with the calmodulin binding domain to allosterically regulate sensitivity to Ca2+/CaM. Our findings provide a new framework for understanding CaMKII structure and allosteric regulation by the variable linker region in Ca2+-sensitive cells.

Transcription inhibition suppresses nuclear blebbing and rupture independently of nuclear rigidity.

Chromatin plays an essential role in the nuclear mechanical response and determining nuclear shape, which maintain nuclear compartmentalization and function. However, major genomic functions, such as transcription activity, might also impact cell nuclear shape via blebbing and rupture through their effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with several RNA polymerase II inhibitors in wild-type cells and perturbed cells that presented increased nuclear blebbing. Transcription inhibition suppressed nuclear blebbing for several cell types, nuclear perturbations and transcription inhibitors. Furthermore, transcription inhibition suppressed nuclear bleb formation, bleb stabilization and bleb-based nuclear ruptures. Interestingly, transcription inhibition did not alter the histone H3 lysine 9 (H3K9) modification state, nuclear rigidity, and actin compression and contraction, which typically control nuclear blebbing. Polymer simulations suggested that RNA polymerase II motor activity within chromatin could drive chromatin motions that deform the nuclear periphery. Our data provide evidence that transcription inhibition suppresses nuclear blebbing and rupture, in a manner separate and distinct from chromatin rigidity.

The environmental context of inducible HSP70 expression in Eastern Brook Trout.

Much research has focused on the population-level effects of climate change on Eastern Brook Trout (Salvelinus fontinalis). While some studies have considered here sub-lethal stress caused by warming waters, the role of multiple, interacting stressors remains largely unexplored. We used inducible heat shock protein 70 (HSP70) as a molecular biomarker to assess in situ response of Eastern Brook Trout in headwater streams to multiple potential stressors, including temperature. Over 7 sampling events during 2018 and 2019, we sampled 141 fish and found that HSP70 expression and 3-day mean water temperature exhibited a quadratic relationship (R 2-adj = 0.68). Further analyses showed that HSP70 expression was explained by temperature, relative water level and their interaction (R 2-adj = 0.75), while fish size and capture location were not factors. We observed a significant increase in HSP70 expression during periods of low relative water level with warm temperatures (~18°C) and also during high relative water level with cold temperatures (~8°C). Our results suggest that temperatures at the edges of the preferred range coupled with relative water level might act together to trigger the cellular stress response in Eastern Brook Trout and that there is greater variation in response at colder temperatures. These findings reinforce the need to consider complex, interactive stressors in influencing the health and persistence of Eastern Brook Trout populations into the future.