To understand how commitment to cell division in late G1 phase (Start) is controlled by growth and nutrients in budding yeast, we determined the absolute concentrations of the G1/S transcription factors SBF (composed of Swi4 and Swi6) and MBF (composed of Mbp1 and Swi6), the transcriptional repressor Whi5, and the G1 cyclins, Cln1 and Cln2, in single live yeast cells using scanning number and brightness (sN&B) microscopy. In rich medium, Whi5, Mbp1, and Swi6 concentrations were independent of cell size, whereas Swi4 concentration doubled in G1 phase, leading to a size-dependent decrease in the Whi5/Swi4 ratio. In small cells, SBF and MBF copy numbers were insufficient to saturate target G1/S promoters, but this restriction diminished as cells grew in size. In poor medium, SBF and MBF subunits, as well as Cln1, were elevated, consistent with a smaller cell size at Start. A mathematical model constrained by sN&B data suggested that size- and nutrient-dependent occupancy of G1/S promoters by SBF/MBF helps set the cell size threshold for Start activation.
We have developed a versatile, one-step melt synthesis of water-soluble, highly emissive silicon nanoparticles using bi-functional, low-melting solids (such as glutaric acid) as reaction media. Characterization through transmission electron microscopy, selected area electron diffraction, X-ray photoelectron spectroscopy, and Raman spectroscopy shows that the one-step melt synthesis produces nanoscale Si cores surrounded by a silicon oxide shell. Analysis of the nanoparticle surface using FT-IR, zeta potential, and gel electrophoresis indicates that the bi-functional ligand used in the one-step synthesis is grafted onto the nanoparticle, which allows for tuning of the particle surface charge, solubility, and functionality. Photoluminescence spectra of the as-prepared glutaric acid-synthesized silicon nanoparticles show an intense blue-green emission with a short (ns) lifetime suitable for biological imaging. These nanoparticles are found to be stable in biological media and have been used to examine cellular uptake and distribution in live N2a cells.
Luminescent, mono-diimine, ruthenium complexes, [(H)Ru(CO)(PPh3)2(dcbpy)][PF6] (1, dcbpy = 4,4′-dicarboxy bipyridyl) and [(H)Ru(CO)(dppene)(5-amino-1,10-phen)][PF6] (2, dppene = bis diphenylphosphino-ethylene, phen = 9,10-phenanthroline), have been conjugated with 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE) and with cholesterol in the case of 2. Compound 1 gives the bis-lipid derivative [(H)Ru(CO)(PPh3)2(dcbpy-N-DPPE2)][PF6] (3), while 2 provides the mono-lipid conjugate [(H)Ru(CO)(dppene)(1,10-phen-5-NHC(S)-N-DPPE)][ PF6] (4), and the cholesterol derivative [(H)Ru(CO)(dppene)(1,10-phen-5-NHC(O)OChol)][PF6] (5, Chol = cholesteryl), using standard conjugation techniques. These compounds were characterized by spectroscopic methods, and their photophysical properties were measured in organic solvents. The luminescence of lipid conjugates 3 and is quenched in organic solvents while compound 4 a weak, short-lived, blue-shifted emission in solution. The cholesterol conjugate shows the long-lived, microsecond-timescale emission associated with triplet metal-to-ligand charge-transfer (3MLCT) excited states. Incorporation of conjugate 3 in lipid bilayer vesicles restores the luminescence, but with blue shifts (~80 nm) accompanied by nanosecond-timescale lifetimes. In the vesicles conjugate 4 shows a similar short-lived and blue-shifted emission to that observed in solution but with increased intensity. Conjugation of the complex [(H)Ru(CO)(PhP2C2H4C(O)O-N-succinimidyl)2(bpy)][PF6] (6”) with DPPE gives the phosphine-conjugated complex [(H)Ru(CO)(PhP2C2H4C(O)-N-DPPE)2(bpy)][PF6] (7). Complex 7 also exhibits a short-lived and blue-shifted emission in solution and in vesicles as observed for 3 and 4. We have also conjugated the complex [Ru(bpy)2(5-amino-1,10-phenanthroline)][PF6]2 (8) with both cholesterol (9) and DPPE (10). Neither 9 nor the previously reported 10 exhibited the blue shifts observed for 3 and 4 when incorporated into LUVs. The anisotropies of the emissions of 3, 4 and 7 were also measured in LUVs and of 5 in both glycerol and LUVs. High fundamental anisotropies were observed for 3 and 4 and 7.
[1] Laboratory measurements of the quenching of CO 2 (n 2 ) by O atoms are presented over the 142-490 K temperature range relevant to the 75-120 km altitude region of the terrestrial atmosphere. The primary cooling mechanism in this region occurs when CO 2 is efficiently excited through collisions with ambient O atoms, populating the bending vibrational (n 2 ) modes. A significant fraction of the vibrationally excited CO 2 relaxes through spontaneous 15-mm emission that escapes into space, thereby removing kinetic energy from this region of the atmosphere and generating a local cooling effect. The rate coefficient for the vibrational relaxation of CO 2 (n 2 ) by O atoms, k O (n 2 ), is measured using transient diode laser absorption spectroscopy. A slight negative temperature dependence is observed for k O (n 2 ), with values ranging from 2.7 (AE0.4) Â 10 À12 cm 3 s À1 at 142 K to 1.3 (AE0.2) Â 10 À12 cm 3 s À1 at 490 K.
In budding yeast, the transcription factors SBF and MBF activate a large program of gene expression in late G1 phase that underlies commitment to cell division, termed Start. SBF/MBF are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the questions of how SBF/MBF are dynamically distributed across the G1/S regulon and how this impacts the Start transition. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits in fixed cells revealed each transcription factor was organized into discrete clusters containing approximately eight copies regardless of cell size and that the total number of clusters increased as cells grew through G1 phase. Stochastic modeling using reasonable biophysical parameters recapitulated growth-dependent SBF/MBF clustering and predicted TF dynamics that were confirmed in live cell PALM experiments. This spatio-temporal organization of SBF/MBF may help coordinate activation of G1/S regulon and the Start transition.
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