Centrioles are ancient organelles that build centrosomes, the major microtubule-organizing centers of animal cells. Extra centrosomes are a common feature of cancer cells. To investigate the importance of centrosomes in the proliferation of normal and cancer cells, we developed centrinone, a reversible inhibitor of Polo-like kinase 4 (Plk4), a serine-threonine protein kinase that initiates centriole assembly. Centrinone treatment caused centrosome depletion in human and other vertebrate cells. Centrosome loss irreversibly arrested normal cells in a senescence-like G1 state by a p53-dependent mechanism that was independent of DNA damage, stress, Hippo signaling, extended mitotic duration, or segregation errors. In contrast, cancer cell lines with normal or amplified centrosome numbers could proliferate indefinitely after centrosome loss. Upon centrinone washout, each cancer cell line returned to an intrinsic centrosome number “set point.” Thus, cells with cancer-associated mutations fundamentally differ from normal cells in their response to centrosome loss.

Light is a prominent stimulus that synchronizes endogenous circadian rhythmicity to environmental light/ dark cycles. Nocturnal light elevates mRNA of the Period1 (Per1) gene and induces long term state changes, expressed as phase shifts of circadian rhythms. The cellular mechanism for Per1 elevation and light-induced phase advance in the suprachiasmatic nucleus (SCN), a process initiated primarily by glutamatergic neurotransmission from the retinohypothalamic tract, was examined. Glutamate (GLU)-induced phase advances in the rat SCN were blocked by antisense oligodeoxynucleotide (ODN) against Per1 and Ca 2؉ /cAMP response element (CRE)-decoy ODN. CRE-decoy ODN also blocked light-induced phase advances in vivo. Furthermore, the CRE-decoy blocked GLU-induced accumulation of Per1 mRNA. Thus, Ca 2؉ /cAMP response element-binding protein (CREB) and Per1 are integral components of the pathway transducing light-stimulated GLU neurotransmission into phase advance of the circadian clock.Mammalian circadian rhythmicity is generated by endogenous alternations in transcription/translation of putative clock genes within the suprachiasmatic nucleus (SCN) 1 of the basal hypothalamus. As a projection site of the retinohypothalamic tract, the SCN is poised to respond to retinal light information, mediated primarily by glutamatergic (GLU) neurotransmission, to assure time-of-day congruence between the endogenous pacemaker and the external environment. The mechanisms by which the SCN decodes and processes light information are complex and change as the biochemical clock states progress through their 24-h cycle (1). Light resets the clock throughout the night via glutamatergic-N-methyl-D-aspartate receptor-mediated Ca 2ϩ influx, which activates nitric-oxide synthase to liberate nitric oxide (NO) (2). At this point, the light signaling pathway diverges. In the early night, the light-induced state change, which delays subsequent rhythms, proceeds through NO-dependent activation of a neuronal ryanodine receptor. Light-induced state changes in the late night are independent of ryanodine receptor activation, but require activation of protein kinase G (PKG) (3-5). The discovery of several specific genes associated with circadian rhythmicity, including Period (Per) and Timeless (Tim) (for review, see Ref. 6), raises questions regarding the mechanisms that interface nocturnal light signals with the molecular clockwork. Throughout the night, light stimuli sufficient to cause long term state changes, or phase shifts, of circadian rhythms of rodent wheel running correlate with increased phosphorylation of the transcription factor, Ca 2ϩ /cAMP response element-binding protein (CREB) (7, 8), activation of Ca 2ϩ /cAMP response element (CRE)-mediated transcription (9), and a rise in Per1 mRNA (10 -15). This investigation was undertaken to determine whether CRE-mediated activation of Per1 is required for light/GLU-induced phase resetting of the SCN clock. We hypothesized that the GLU-induced phase advance requires activation of CRE and elevation ...

Summary The ability of cells to faithfully duplicate their two centrioles once per cell cycle is critical for proper mitotic progression and chromosome segregation. Multi-ciliated cells represent an interesting variation of centriole duplication in that these cells generate greater than 100 centrioles, which form the basal bodies of their motile cilia. This centriole amplification is proposed to require a structure termed the deuterosome, thought to be capable of promoting de novo centriole biogenesis. Here, we begin to molecularly characterize the deuterosome and identify it as a site for the localization of Cep152, Plk4, and SAS6. Additionally we identify CCDC78 as a centriole-associated and deuterosome protein that is essential for centriole amplification. Overexpression of Cep152, but not Plk4, SAS6 or CCDC78, drives over-amplification of centrioles. However, in CCDC78 morphants, Cep152 fails to localize to the deuterosome and centriole biogenesis is impaired, indicating CCDC78-mediated recruitment of Cep152 is required for deuterosome-mediated centriole biogenesis.

The suprachiasmatic nucleus (SCN) contains a biological clock that generates timing signals that drive daily rhythms in behaviors and homeostatic functions. In addition to this pacemaker function, the SCN gates its own sensitivity to incoming signals, which permits appropriate temporal adjustment to achieve synchrony with environmental and organismic states. A series of time-domains, in which the SCN restricts its own sensitivity to a limited set of stimuli that adjust clock phase, can be distinguished. Pituitary adenylyl cyclase-activating peptide (PACAP) and cAMP directly reset clock phase during the daytime domain; both cause phase advances only during the clock's day-time domain, but are without effect at night. In contrast, acetylcholine and cGMP analogs phase advance the clock only when applied during the night. Sensitivity to light and glutamate arises concomitant with sensitivity to acetylcholine and cGMP. Light and glutamate cause phase delays in the early night, by elevating intracellular Ca(2+) via neuronal ryanodine receptors. In late night, light and glutamate utilize a cGMP-mediated mechanism to induce phase advances. Nocturnal responses of SCN primed by light or glutamate can be modulated by effectors of phase-resetting in daytime, namely, PACAP and cAMP. Finally, the dusk and dawn domains are characterized by sensitivity to the pineal hormone, melatonin, acting through protein kinase C. These changing patterns of sensitivities demonstrate that the circadian clock controls multiple intracellular gates, which ensures that they can be opened selectively only at specific points in the circadian cycle. Discerning the molecular bases of these changes is fundamental to understanding integrative and regulatory mechanisms in the circadian system.

Despite a central circadian role in Drosophila for the transcriptional regulator Timeless (dTim), the relevance of mammalian Timeless (mTim) remains equivocal. Conditional knockdown of mTim protein expression in the rat suprachiasmatic nucleus (SCN) disrupted SCN neuronal activity rhythms, and altered levels of known core clock elements. Full-length mTim protein (mTIM-fl) exhibited a 24-hour oscillation, where as a truncated isoform (mTIM-s) was constitutively expressed. mTIM-fl associated with the mammalian clock Period proteins (mPERs) in oscillating SCN cells. These data suggest that mTim is required for rhythmicity and is a functional homolog of dTim on the negative-feedback arm of the mammalian molecular clockwork.