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Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the per s phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.

Visualizations are useful tools for helping students to understand chemistry concepts at the particulate level. A classroom activity was developed based on learning theory and evidence-based practices, combining protein visualization with thermodynamic parameters from differential scanning fluorimetry (DSF) data analysis. Coding of student responses showed that many students were able to establish the desired connections among protein structure, thermodynamic parameters, and experimental data analysis, while a few did not recognize all the differences between the folded and unfolded forms of the protein. The activity elicits student prior knowledge through the pre-class activity, has the students examine the interactions within a protein molecule through the PyMOL activity, introduces DSF analysis using the learning cycle through the Guided Inquiry activity, and tests student learning through the post-class activity. Upon completing the activity, the majority of students successfully met the learning goals.

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Cdc25 phosphatase primes entry to mitosis by removing the inhibitory phosphate that is transferred to mitosis promoting factor (MPF) by Weel related kinases. A positive feedback loop then boosts Cdc25 and represses Wee1 activities to drive full-scale MPF activation and commitment to mitosis. Dominant mutations in the Schizosaccharomyces pombe spindle pole body (SPB) component Cut12 enable cdc25.22 mutants to overcome a G2 arrest at 36[degrees] and enter mitosis. The recessive temperature-sensitive cur12.1 mutation results in the formation of monopolar spindles in which the spindle pole marker Sad1 is enriched on the nonfunctional SPB at 36[degrees]. We identified mutations at five loci that suppressed the lethality of the recessive cut12.1 mutation at 36[degrees] and conferred lethality at 20[degrees]. Three of the five mutations led to the formation of monopolar spindles at restrictive temperatures, affected cell size at commitment to mitosis, and generated multiple Sad1 foci at nuclear periphery. The five loci, tfb2.rt1, tfb5.rt5, pla1.rt3, rp14301.rt4, and rot2.1, and multicopy suppressors, including tfb[1.sup.+] and dbp[10.sup.+], are involved in transcription, translation, or RNA processing, prompting us to establish that elevating Cdc25 levels with the dominant cdc25.d1 allele, suppressed cur12.1. Thus, rot mutants provide a further link between protein production and cell-cycle progression.

Abstract The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of [...]