Dynamics of the circadian clock protein PERIOD2 in living cells

Abstract

In mammals, circadian rhythms are generated by delayed negative feedback, in which period (PER1-PER3) and cryptochrome (CRY1, CRY2) proteins gradually accumulate in the nucleus to suppress the transcription of their own genes. Although the importance of nuclear import and export signals for the subcellular localization of clock proteins is well established, little is known about the dynamics of these processes as well as their importance for the generation of circadian rhythms. We show by pharmacological perturbations of oscillating cells that nuclear import and export are of crucial importance for the circadian period. Live-cell fluorescence microscopy revealed that nuclear import of the key circadian protein PER2 is fast and further accelerated by CRY1. Moreover, PER2 nuclear import is crucially dependent on a specific nuclearreceptor-binding motif in PER2 that also mediates nuclear immobility. Nuclear export, however, is relatively slow, supporting a model of PER2 nuclear accumulation by rapid import, slow export and substantial nuclear degradation.

Differential Effects of SUMO1/2 on Circadian Protein PER2 Stability and Function

“…PER2 nuclear entry is facilitated by dimerization with CRY1 (15,(45)(46)(47) and interaction of the PER2/CRY/CKI complex with GAPVD1, a Rab GTPase guanine nucleotide exchange factor involved in endocytosis and cytoplasmic trafficking (48). Consistent with these previous observations, PER2 K736R had significantly decreased CRY1 and GAPVD1 interaction ( Fig 4E, 4F).…”

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells

“…Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>6 times) compared to PER2 questioning the current model of the circadian oscillator.Our knock-in strategy will allow the generation of additional single, double or triple knock-in cells for circadian clock proteins, which should greatly advance our understanding about the cell biology of circadian clocks.Recent biochemical studies with mouse liver lysates suggest that during the repressive phase, essentially all nuclear PER and CRY proteins are coordinated together in one large repressive complex, with only a minor amount of CRY1 monomers detectable (17). Again, double knock-out of either Per1/2 or Cry1/2 completely prevented the formation of this repressive complex.Most of the current knowledge of PER and CRY protein dynamics resulted either from biochemical data with mixed lysates of many thousand cells, or from single-cell imaging of over-expressed fluorescent tagged fusion proteins (12,13,17,18). Both approaches have clear limitations: Population samplinge.g.…”

Dynamics of the circadian clock protein PERIOD2 in living cells

“…Bioluminescence recording U2-OS cells (human, ATCC HTB-96) stably expressing firefly luciferase from a Bmal1 promoter fragment (Maier et al, 2009) or Per2 promoter fragment (Liu et al, 2008) were seeded onto a white 96-well plate (20610 4 cells/well). After 24 hours, cells were synchronized with dexamethasone (1 mM) for 30 minutes, washed with PBS and cultured in 150 ml of Phenol-Red-free DMEM containing 10% fetal bovine serum, antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) and 250 mM D-luciferin (Biothema, Darmstadt, Germany).…”

“…To this end, we treated two different U2-OS reporter cell lines expressing firefly luciferase either from a Bmal1 promoter fragment (Maier et al, 2009) or from the Per2 promoter (Liu et al, 2008) with ivermectin, a specific inhibitor of importin a/bdependent nuclear import (Wagstaff et al, 2012) or leptomycin B, an efficient and selective inhibitor of CRM1/exportin-1-mediated nuclear export (Wolff et al, 1997). Both inhibitors dosedependently dampened circadian rhythms and lengthened the circadian period by up to 3-4 hours ( Fig.…”