PERIOD phosphoclusters control temperature compensation of the Drosophila circadian clock

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. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines (S44, 45 and 47) belonging to the PER phosphodegron, the functional homolog of mammalian PER2’s S487 phosphodegron, which impacts temperature compensation. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused decreased temperature compensation, 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 decreased thermal compensation, consistent with its inhibitory role on S47 phosphorylation. Interestingly,the S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A caused excessive temperature compensation of phosphorylation-dependent PER degradation. Thus, we show a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work also reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.

Download Full-text

Technological Advances in Peritoneal Dialysis Research Peritoneal Cell Culture: Fibroblasts

Peritoneal Dialysis International ◽

10.1177/089686080602600302 ◽

2006 ◽

Vol 26 (3) ◽

pp. 292-299 ◽

Cited By ~ 20

Author(s):

Janusz Witowski ◽

Achim Jörres

Keyword(s):

Cell Culture ◽

Peritoneal Dialysis ◽

Primary Cultures ◽

Cell Types ◽

Anatomical Location ◽

Functional Heterogeneity ◽

Technological Advances ◽

Structural Lattice ◽

Made In

Fibroblasts have been traditionally viewed as providing little more than a structural lattice for other cell types. However, recent data indicate that fibroblasts play a key and early role in many pathophysiological processes, including inflammation, fibrosis, and neoplasia. Moreover, depending on the anatomical location, fibroblasts display significant functional heterogeneity. Therefore, it is important to study the subpopulation of fibroblasts derived exactly from the organ of interest rather than to extrapolate the observations made in other fibroblast subsets. Cell culture provides a powerful tool for studying the role of fibroblasts in various contexts. In this review, we describe procedures for establishing and identifying primary cultures of human peritoneal fibroblasts. We also briefly discuss the potential involvement of peritoneal fibroblasts in peritoneal pathology.

Download Full-text

Circadian Clock-Specific Roles for the Light Response Protein WHITE COLLAR-2

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2619-2628.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2619-2628 ◽

Cited By ~ 31

Author(s):

Michael A. Collett ◽

Jay C. Dunlap ◽

Jennifer J. Loros

Keyword(s):

Circadian Clock ◽

Temperature Compensation ◽

Light Response ◽

White Collar ◽

Normal Activity ◽

Relative Increase ◽

Period Length ◽

Mrna Levels ◽

Temperature Dependent ◽

Protein Levels

ABSTRACT To understand the role of white collar-2 in theNeurospora circadian clock, we examined alleles ofwc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2(ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator offrq, is critical for establishing period length and temperature compensation in this circadian system.

Download Full-text

Temperature compensation and temperature sensation in the circadian clock

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1511215112 ◽

2015 ◽

Vol 112 (46) ◽

pp. E6284-E6292 ◽

Cited By ~ 31

Author(s):

Philip B. Kidd ◽

Michael W. Young ◽

Eric D. Siggia

Keyword(s):

Circadian Clock ◽

Temperature Compensation ◽

Temperature Oscillation ◽

Circadian Clocks ◽

Luciferase Reporter ◽

Alternative Theory ◽

Temperature Dependent ◽

Western Blots ◽

Temperature Changes ◽

Clock Component

All known circadian clocks have an endogenous period that is remarkably insensitive to temperature, a property known as temperature compensation, while at the same time being readily entrained by a diurnal temperature oscillation. Although temperature compensation and entrainment are defining features of circadian clocks, their mechanisms remain poorly understood. Most models presume that multiple steps in the circadian cycle are temperature-dependent, thus facilitating temperature entrainment, but then insist that the effect of changes around the cycle sums to zero to enforce temperature compensation. An alternative theory proposes that the circadian oscillator evolved from an adaptive temperature sensor: a gene circuit that responds only to temperature changes. This theory implies that temperature changes should linearly rescale the amplitudes of clock component oscillations but leave phase relationships and shapes unchanged. We show using timeless luciferase reporter measurements and Western blots against TIMELESS protein that this prediction is satisfied by the Drosophila circadian clock. We also review evidence for pathways that couple temperature to the circadian clock, and show previously unidentified evidence for coupling between the Drosophila clock and the heat-shock pathway.

Download Full-text

Poly(2-oxazoline) Matrices with Temperature-Dependent Solubility—Interactions with Water and Use for Cell Culture

Materials ◽

10.3390/ma13122702 ◽

2020 ◽

Vol 13 (12) ◽

pp. 2702

Author(s):

Natalia Oleszko-Torbus ◽

Marcelina Bochenek ◽

Alicja Utrata-Wesołek ◽

Agnieszka Kowalczuk ◽

Andrzej Marcinkowski ◽

...

Keyword(s):

Cell Culture ◽

Cell Separation ◽

Polymer Materials ◽

Temperature Dependent ◽

Shape Stability ◽

Gradient Copolymers ◽

Cloud Point Temperature ◽

The Stability

In this work, we studied the stability of matrices with temperature-dependent solubility and their interactions with water at physiological temperature for their application in cell culture in vitro. Gradient copolymers of 2-isopropyl- with 2-n-propyl-2-oxazoline (P(iPrOx-nPrOx)) were used to prepare the matrices. The comonomer ratio during polymerization was chosen such that the cloud point temperature (TCP) of the copolymer was below 37 °C while the glass transition (Tg) was above 37 °C. The role of the support for matrices in the context of their stability in aqueous solution was examined. Therefore, matrices in the form of both self-supported bulk polymer materials (fibrillar mats and molds) and polymer films supported on the silica slides were examined. All of the matrices remained undissolved when incubated in water at a temperature above TCP. For the self-supported mats and molds, we observed the loss of shape stability, but, in the case of films supported on silica slides, only slight changes in morphology were observed. For a more in-depth investigation of the origin of the shape deformation of self-supported matrices, we analyzed the wettability, thickness, and water uptake of films on silica support because the matrices remained undeformed under these conditions. It was found that, above the TCP of P(iPrOx-nPrOx), the wettability of the films decreased, but at the same time the films absorbed water and swelled. We examined how this specific behavior of the supported films influenced the culture of fibroblasts. The temperature-dependent solubility of the matrices and the possibility of noninvasive cell separation were also examined.

Download Full-text

On the nature of the circadian clock in mammals

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1993.264.5.r821 ◽

1993 ◽

Vol 264 (5) ◽

pp. R821-R832 ◽

Cited By ~ 3

Author(s):

J. D. Miller

Keyword(s):

Cell Culture ◽

Functional Analysis ◽

Circadian Clock ◽

Suprachiasmatic Nucleus ◽

Response Curve ◽

Essential Role ◽

Phase Response Curve ◽

Coupling Model ◽

Clock Mechanism

The evidence for the essential role of the suprachiasmatic nucleus (SCN) for the generation and maintenance of circadian rhythms in mammals is briefly reviewed. The pharmacology of the phase-response curve is considered and a new circadian measure, the phase-dose-response surface (PDRS), is introduced. The role of neurotransmission, ion fluxes, and non-neuronal cellular elements in the generation and maintenance of circadian rhythmicity is considered. Cell culture of the SCN is proposed as a tool for the functional analysis of clock mechanism. The critical contribution of coupling and synchronization of clock elements is reviewed in the context of the explicit predictions generated by a strong coupling model of the circadian clock. Finally, the nature of the circadian output signal is analyzed from a phylogenetic viewpoint.

Download Full-text