Journal of Cell Science RESEARCH ARTICLE Dynamics of the circadian clock protein PERIOD2 in living cells Rupert O ¨ llinger 1 , Sandra Korge 1 , Thomas Korte 2 , Barbara Koller 1 , Andreas Herrmann 2 and Achim Kramer 1, * 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 nuclear- receptor-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. KEY WORDS: Circadian rhythms, Period2, Nuclear localization INTRODUCTION Circadian clocks have evolved in a multitude of organisms, allowing the prediction of and preparation for daily recurring environmental events. In almost all eukaryotes, molecular circadian rhythms are generated by cell-autonomous gene regulatory networks within nearly every cell of an organism. In mammals, period (PER1–PER3) and cryptochrome (CRY1 and CRY2) proteins are integral components of the circadian oscillator in that they inhibit their own expression by repressing the activity of the transcription factor heterodimer CLOCK– BMAL1 (the latter of which is also known as ARNTL) (Buhr and Takahashi, 2013). Importantly, this negative feedback is delayed, allowing the generation of oscillations in PER and CRY transcript and protein levels. In recent years, many details about the mechanisms of transcriptional activation by CLOCK–BMAL1 and inhibition by PER–CRY-containing complexes have been uncovered; surprisingly, however, little is known about the processes that create the delay in transcriptional auto-inhibition, although a precise timing of the negative feedback is crucial for a correct circadian period. Because the localization of PER–CRY- containing protein complexes gradually shifts from cytosolic to nuclear over the course of a circadian cycle, it is believed that appropriately timed nuclear accumulation of PER and CRY proteins contributes to the temporal delay between transcriptional activation and repression, although this has not been directly tested so far. The shuttling of macromolecules between cytoplasm and nucleus occurs through the nuclear pore with well-characterized nuclear import and export mechanisms involving receptor-based recognition of nuclear localization signals (NLS) or nuclear export signals (NES) on protein cargoes (Ullman et al., 1997). Specialized import and export receptors, such as importin b or heterodimers of importin-a or importin-b and exportin-1 (also known as CRM1) bind to NLS and NES sequences, respectively, and together the complexes move through the nuclear pore. Subsequently, importin and exportin proteins are released from the cargo, which involves the small GTP-binding protein Ran. For many clock proteins (including PERs and CRYs), classical nuclear import and export signals have been described (for a review, see Tamanini et al., 2005). Factors that influence subcellular localization – possibly by modulating the accessibility of NLS and/or NES sequences – are phosphorylation and dephosphorylation, as well as complex formation with other clock proteins. For example, phosphorylation of PER2 by CK2 and GSK3b has been reported to modulate its subcellular localization (Iitaka et al., 2005; Maier et al., 2009). In addition, although PER and CRY proteins can localize to the nucleus even in the absence of one another, binding to CRY promotes nuclear accumulation and stabilization of PER proteins (Kume et al., 1999; Miyazaki et al., 2001; Yagita et al., 2002). Moreover, the CoRNR site of PER2, a binding motif for nuclear receptors, and the promyelotic leukemia protein PML have both been reported to be important for nuclear entry (Albrecht et al., 2007; Miki et al., 2012). Despite these insights into the domains and factors that contribute to the nucleocytoplasmic distribution of clock proteins, the dynamics of nuclear import and export of negative clock factors are essentially unknown for the mammalian circadian system. In addition, the impact of altering import or export rates on circadian rhythms has not been tested. In two other eukaryotic clock systems, the dynamics of nuclear/cytoplasmic localization seems to be largely distinct: in Drosophila melanogaster an interval timer retains the negative clock elements PER and TIM in the cytoplasm for several hours before they dissociate and shuttle to the nucleus (Meyer et al., 2006). The Neurospora crassa negative clock protein FRQ, however, is rapidly (within minutes) shuttled between cytoplasm and nucleus, and its nucleocytoplasmic localization is gradually shifted by phosphorylation during the course of a circadian cycle (Diernfellner et al., 2009). Here, we show that both nuclear import and nuclear export are essential for normal circadian rhythms. Pharmacological inhibition of both pathways lengthens the circadian period in U2-OS reporter cells by up to 4 hours. We use live-cell fluorescence microscopy to analyze the kinetics of nuclear import and export of PER2 as one of the scaffold proteins of the negative feedback complex. Nuclear import of PER2 is fast and further accelerated by CRY1; however, 1 Laboratory of Chronobiology, Charite ´ – Universita ¨ tsmedizin Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany. 2 Department of Biology, Molecular Biophysics, Humboldt-University Berlin, Invalidenstrasse 43, 10115 Berlin, Germany. *Author for correspondence (achim.kramer@charite.de) Received 15 May 2014; Accepted 13 July 2014 ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4322–4328 doi:10.1242/jcs.156612 4322