Nuclear target search at the single molecule level: protein interactions define the exploration landscape

Gene regulation relies on highly mobile transcription factors (TFs) exploring the nucleoplasm in search of their targets. Our view of the nucleus has evolved from that of an isotropic and homogenous reactor to that of a highly organized yet very dynamic organelle. However important questions remain on how these regulatory factors explore the nuclear environment in search of their DNA or protein targets, and how their exploration strategy affects the kinetics of transcriptional regulation. We implemented a single-molecule tracking assay to determine the TFs dynamics using photoactivatable tags in human cells. We investigated the mobility of several nuclear proteins, including the transcription factor c-Myc and the elongation factor P-TEFb. We found that, while their diffusion speed was comparable, these proteins largely differed in terms of their exploration geometry. We discovered that c-Myc is a global explorer diffusing in the nucleus without spatial constraints. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment, constrained by a fractal nuclear architecture. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. We also measured the mobility of a P-TEFb mutant in which the interaction with the CTD of the RNA Pol II was truncated. In this case, the single-molecule experiments suggested a global exploration of the P-TEFb mutant, consistent with free diffusion. Our observations are in line with a model in which the exploration geometry of TFs is constrained by their interactions and not by exclusion properties. Our findings have strong implications on how proteins react in the nucleus and how their function can be regulated in space and time

Chemical Composition and Larvicidal Activities of the Himalayan Cedar, Cedrus deodara Essential Oil and Its Fractions Against the Diamondback Moth, Plutella xylostella

Plants and plant-derived materials play an extremely important role in pest management programs. Essential oil from wood chips of Himalayan Cedar, Cedrus deodara (Roxburgh) Don (Pinales: Pinaceae), was obtained by hydrodistillation and fractionated to pentane and acetonitrile from which himachalenes and atlantones enriched fractions were isolated. A total of forty compounds were identified from these fractions using GC and GC-MS analyses. Essential oils and fractions were evaluated for insecticidal activities against second instars of the diamondback moth, Plutella xylostella L. (Lepidoptera: Yponomeutidae), using a leaf dip method. All samples showed promising larvicidal activity against larvae of P. xylostella. The pentane fraction was the most toxic with a LC50 value of 287 µg/ml. The himachalenes enriched fraction was more toxic (LC50 = 362 µg/ml) than the atlantones enriched fraction (LC50 = 365 µg/ml). LC50 of crude oil was 425 µg/ml and acetonitrile fraction was LC50 = 815 µg/ml. The major constituents, himachalenes and atlantones, likely accounted for the insecticidal action. Present bioassay results revealed the potential for essential oil and different constituents of C. deodara as botanical larvicides for their use in pest management