Precise timing of CONSTANS (CO) gene expression is necessary for day-length discrimination for photoperiodic flowering. The FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and GIGANTEA (GI) proteins regulate CO transcription in Arabidopsis. We demonstrate that FKF1and GI proteins form a complex in a blue-light dependent manner. The timing of this interaction regulates the timing of daytime CO expression. FKF1 function is dependent on GI, which interacts with a CO repressor, CYCLING DOF FACTOR 1 (CDF1), and controls CDF1 stability. GI, FKF1, and CDF1 proteins associate with CO chromatin. Thus, the FKF1-GI complex forms on the CO promoter in late afternoon to regulate CO expression, providing a mechanistic view of how the coincidence of light with circadian timing regulates photoperiodic flowering.Many plants monitor seasonal changes in day-length to regulate flowering time for successful reproduction (1). In Arabidopsis, regulation of daytime CO expression is the primary process of time measurement in the photoperiodic flowering pathway (2, 3). FKF1 and GI proteins positively regulate CO transcription (4, 5). FKF1 and GI gene expression has similar diurnal patterns (5, 6), implying that these proteins may interact to regulate CO.We tested their direct interaction in yeast and found that FKF1 interacts with GI (Fig. 1A). Our results obtained using truncated FKF1 proteins suggests that this interaction occurs through the FKF1 LOV (Light, Oxygen, or Voltage) domain (Fig. 1A). In addition, the GI N-terminus was sufficient to interact with FKF1 ( fig. S1).To assess whether this interaction occurs in vivo, and whether it is modulated by photoperiod or light conditions, we generated transgenic plants constitutively expressing both haemagglutinin (HA)-tagged FKF1 (HA-FKF1) and tandem affinity purification (TAP)-tagged GI (GI-TAP) proteins [35S∷HA-FKF1 35S∷GI-TAP lines (7)] for coimmunoprecipitation experiments. In the 35S∷HA-FKF1 35S∷GI-TAP #18 / fkf1 line, a similar amount of GI-TAP protein was precipitated at every time point in both long-day (16 hours light / 8 hours dark) and short-day (8 hours light / 16 hours dark) conditions (Fig. 1, B and C). HA-FKF1 protein was coimmunoprecipitated with GI-TAP protein (Fig. 1, B and C), demonstrating that GI-TAP and HA-FKF1 proteins form a complex in vivo. In both daylength conditions, the amount of coimmunoprecipitated HA-FKF1 protein increased until 4 hours after light onset, remained constant for the rest of day, and declined in the dark (Fig. 1, B and C), suggesting that light or the circadian clock modulate the FKF1 and GI interaction.We therefore analyzed the interaction in dark-grown samples. A minimal amount of HA-FKF1 was coimmunoprecipitated with GI-TAP protein in the dark (Fig. 1D) Next we analyzed how light quality (wavelength) affects this interaction. Similar amounts of FKF1 and GI interacted in blue-light irradiated samples (Fig. 1E) compared to white-light grown samples, but little interaction was observed in red-light irradiated samples ( Fig. 1E), indicati...

Modulating the Clock Because of the close association of the circadian clock with a wide range of physiological processes, identification of clock-modulating small molecules may prove useful for the treatment of circadian-related disorders, which include circadian sleep disorders, cardiovascular disease, cancer, and metabolic disease. Hirota et al. (p. 1094 , published online 12 July) screened for chemical compounds that affected the period of the circadian clock in a human osteosarcoma cell line. A carbazole derivative named KL001 appeared to act by inhibiting proteolytic degradation of the cryptochrome proteins, which in turn caused a lengthening of the circadian period. KL001 also inhibited glucagon-induced gluconeogenesis in primary cultures of mouse hepatocytes.

R e s e a R c h a R t i c l e1 8 1 6jci.org Volume 126Number 5 May 2016 26, 54, 55). We assayed the level of GTP-Rab5 in brains of 12-monthold Ts65Dn and 2N mice following a published protocol (56). As previously reported (28), the level of full-length APP in Ts65Dn was approximately 1.5-fold than in 2N samples ( We next tested whether the increase in App gene dose in Ts65Dn BFCNs was responsible for enlargement of Rab5 + endosomes (Figure 1). By immunoblotting, the APP siRNA caused an approximately 30% reduction in the level of full-length APP, as compared with control siRNA ( Figure 2E). Rab5 + puncta in BFCNs treated with either the APP siRNA or control siRNA were analyzed (Figure 2, F and G) as in Figure 1. Large, sometimes lobulated Rab5 + puncta were seen in cultures treated with the control siRNA, whereas these structures were typically smaller and rounded in cultures treated with the APP siRNA ( Figure 2G). Treatment with the APP siRNA significantly reduced the size of Rab5 + puncta in Ts65Dn neurons to a value equivalent to that in 2N neurons ( Figure 2F). Thus, increased App gene dose is necessary for increased Rab5 activation and for early endosome enlargement in Ts65Dn neurons.Full-length APP and β-CTF caused enlargement of early endosomes in PC12 cells. To determine how increased APP expression caused an increase in Rab5 activation, we asked which APP product(s) were responsible (Supplemental Figure 1A). We transfected PC12M cells with full-length APP-GFP, C99-GFP (β-CTF), C83-GFP (α-CTF), or AICD-GFP and examined endosomes by live cell imaging (Supplemental Figure 1B). Bright foci of GFP + intracellular structures were present in PC12M cells that overexpressed APP-GFP or C99-GFP. In contrast, cells expressing C83-GFP or AICD-GFP showed diffuse, hazy signals for GFP, with occasional foci in C83-GFP cells. In APP-GFP and C99-GFP cells, the GFP + intracellular structures were, on average, approximately 2 μm 2 (Supplemental Figure 1E). GFP signals in C83-GFP or AICD-GFP cells contained speckled small puncta within the haze, as well as a small number of larger bright puncta, as seen in cells expressing C99-GFP and APP-GFP (Supplemental Figure 1B). However, the average puncta size in C83-GFP and AICD-GFP cells was approximately 1.2 and 1.3 μm 2 , respectively. Thus, overexpressing APP and β-CTF, but not α-CTF or AICD, routinely induced formation of enlarged, bright intracellular structures. We also tested two APP mutants: APP M596V and APP SWE . APP M596V , which abolishes β-secretase cleavage, prevents production of β-CTF (57); APP SWE enhances β-secretase cleavage to increase the level of β-CTF (57). Both induced the formation of enlarged intracellular structures (Supplemental Figure 1C).We examined colocalization of APP or C99 with Rab5 in cotransfection experiments; APP-mCherry with GFP-Rab5 WT ( Figure 3B); C99-GFP with mCherry-Rab5 WT ( Figure 3C); and Rab5 + endosomes (26, 28, 37) was correlated with reduced endosomal trafficking and signaling of nerve growth factor (NGF), leading to degeneration...

Plants perceive environmental signals such as day length and temperature to determine optimal timing for the transition from vegetative to floral stages. Arabidopsis flowers under long-day conditions through the CONSTANS (CO)-FLOWERING LOCUS T (FT) regulatory module. It is thought that the environmental cues for photoperiodic control of flowering are initially perceived in the leaves. We have previously shown that GIGANTEA (GI) regulates the timing of CO expression, together with FLAVIN-BINDING, KELCH REPEAT, F BOX protein 1. Normally, CO and FT are expressed exclusively in vascular bundles, whereas GI is expressed in various tissues. To better elucidate the role of tissue-specific expression of GI in the flowering pathway, we established transgenic lines in which GI is expressed exclusively in mesophyll, vascular bundles, epidermis, shoot apical meristem, or root. We found that GI expressed in either mesophyll or vascular bundles rescues the late-flowering phenotype of the gi-2 loss-of-function mutant under both short-day and long-day conditions. Interestingly, GI expressed in mesophyll or vascular tissues increases FT expression without up-regulating CO expression under short-day conditions. Furthermore, we examined the interaction between GI and FT repressors in mesophyll. We found that GI can bind to three FT repressors: SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO (TEM)1, and TEM2. Finally, our chromatin immunoprecipitation experiments showed that GI binds to FT promoter regions that are near the SVP binding sites. Taken together, our data further elucidate the multiple roles of GI in the regulation of flowering time. S uccessful reproduction in higher plants depends on appropriate timing of flowering. Understanding the mechanisms underlying flowering time pathways can provide insight into the networks mediating the effects of environmental cues on developmental programs, and has important implications for crop production.Plants use multiple environmental cues to determine the timing of flowering, such as temperature, quality and quantity of light, and day-length changes. Among these signals, day-length change is the most reliable because it occurs in regular and predictable cycles year after year. Photoperiodism refers to the rhythms of biological processes that are based on day-length changes and is found in many species including insects, birds, and mammals. Although the molecular mechanism for photoperiodism is not well-understood, biologists have identified several key elements governing this phenomenon in plants.In the model plant Arabidopsis thaliana, flowering is accelerated when the length of daylight is prolonged compared with darkness (16 h of light and 8 h of darkness, designated as long day; LD). Time measurement in the photoperiodic flowering pathway is regulated by daytime expression of CONSTANS (CO) (1, 2). Under LD, CO expression coincides with light. CO protein is stabilized by light (a component of a process referred to as external coincidence) and it activates a downstream factor, FLOWERING...

Migration of cells through the reorganization of the actin cytoskeleton is essential for morphogenesis of multicellular animals. In a cell culture system, the actin-related protein (Arp) 2/3 complex functions as a nucleation core for actin polymerization when activated by the members of the WASP(Wiskott-Aldrich syndrome protein) family. However, the regulation of cell motility in vivo remains poorly understood. Here we report that homologues of the mammalian Arp2/3 complex and N-WASP in Caenorhabditis elegansplay an important role in hypodermal cell migration during morphogenesis, a process known as ventral enclosure. In the absence of one of any of the C. elegans Arp2/3 complex subunits (ARX-1, ARX-2, ARX-4, ARX-5, ARX-6 or ARX-7) or of N-WASP (WSP-1), hypodermal cell migration led by actin-rich filopodia formation is inhibited during ventral enclosure owing to the reduction of filamentous actin formation. However, there is no effect on differentiation of hypodermal cells and dorsal intercalation. Disruption of the function of ARX-1 and WSP-1 in hypodermal cells also resulted in hypodermal cell arrest during ventral enclosure, suggesting that their function is cell autonomous. WSP-1 protein activated Arp2/3-mediated actin polymerization in vitro. Consistent with these results, the Arp2/3 complex and WSP-1 colocalized at the leading edge of migrating hypodermal cells. The stable localization of WSP-1 was dependent on the presence of Arp2/3 complex,suggesting an interaction between the Arp2/3 complex and WSP-1 in vivo.