Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations of qdots have farreaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting, and diagnostics.

/pdf/quantum-dots-for-live-cells-in-vivo-imaging-and-diagnostics-3y9ayecoh8.pdf

Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics

Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.

http://science.sciencemag.org/content/sci/312/5771/217.full.pdf

The fluorescent toolbox for assessing protein location and function

The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

/pdf/bioorthogonal-chemistry-fishing-for-selectivity-in-a-sea-of-125vhdfza6.pdf

Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

TRP channels are the vanguard of our sensory systems, responding to temperature, touch, pain, osmolarity, pheromones, taste and other stimuli. But their role is much broader than classical sensory transduction. They are an ancient sensory apparatus for the cell, not just the multicellular organism, and they have been adapted to respond to all manner of stimuli, from both within and outside the cell.

TRP channels as cellular sensors

In electrically nonexcitable cells, Ca2+ influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and prolifera...

Store-operated calcium channels.

We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269−272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565−578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants (∼10-11 M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed α-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable a...

/pdf/new-biarsenical-ligands-and-tetracysteine-motifs-for-protein-41yc7m6ztf.pdf

New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.

Activation of Store-Operated Ca2+ Current in Xenopus Oocytes Requires SNAP-25 but Not a Diffusible Messenger

We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515-1527). In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 microg CALNUC/mg Golgi protein, 2.5 x 10(5) CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 microM, binding capacity = 1.1 micromol Ca2+/micromol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand alpha helix from CALNUC completely abolished its Ca2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, alpha-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store.

/pdf/overexpression-of-calnuc-nucleobindin-increases-agonist-and-2qmfpwza08.pdf

Overexpression of CALNUC (nucleobindin) increases agonist and thapsigargin releasable Ca2+ storage in the Golgi.

Ca(2+) currents activated by depletion of Ca(2+) stores in Xenopus oocytes were studied with a two-electrode voltage clamp Buffering of cytosolic Ca(2+) with EGTA and MeBAPTA abolished I(Cl(Ca)) and unmasked a current in oocytes that was activated by InsP(3) or ionomycin in minutes and by thapsigargin or the chelators themselves over hours At -60 mV in 10 mM extracellular CaCl(2), the current was typically around -90 or -160 nA in oocytes loaded with EGTA or MeBAPTA, respectively This current was judged to be a Ca(2+)-selective current for the following reasons: (a) it was inwardly rectifying and reversed at membrane potentials usually more positive than +40 mV; (b) it was dependent on extracellular [CaCl(2)] with K(m) = 115 mM; (c) it was highly selective for Ca(2+) against monovalent cations Na(+) and K(+), because replacing Na(+) and K(+) by N-methyl-d-glucammonium did not reduce the amplitude or voltage dependence of the current significantly; and (d) Ca(2+), Sr(2+), and Ba(2+) currents had similar instantaneous conductances, but Sr(2+) and Ba(2+) currents appeared to inactivate more strongly than Ca(2+) This Ca(2+) current was blocked by metal ions with the following potency sequence: Mg(2+) << Ni(2+) approximately Co(2+) approximately Mn(2+) < Cd(2+) << Zn(2+) << La(3+) It was also inhibited by niflumic acid, which is commonly used to block I(Cl(Ca)) PMA partially inhibited the Ca(2+) current, and this effect was mostly abolished by calphostin C, indicating that the Ca(2+) current is sensitive to protein kinase C These results are the first detailed electrophysiological characterization of depletion-activated Ca(2+) current in nondialyzed cells Because exogenous molecules and channels are easy to introduce into oocytes and the distortions in measuring I(Cl(Ca)) can now be bypassed, oocytes are now a superior system in which to analyze the activation mechanisms of capacitative Ca(2+) influx

/pdf/calcium-current-activated-by-depletion-of-calcium-stores-in-2q6cltt9ye.pdf

Yong Yao

Papers

New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications.

Activation of Store-Operated Ca2+ Current in Xenopus Oocytes Requires SNAP-25 but Not a Diffusible Messenger

Overexpression of CALNUC (nucleobindin) increases agonist and thapsigargin releasable Ca2+ storage in the Golgi.

Calcium Current Activated by Depletion of Calcium Stores in Xenopus Oocytes