Biomedical subjects

Sally A Kim

Publications and source records attributed to Sally A Kim.

4 recordsLinked to original sources

PubMed2006

Fluorescence cross-correlation spectroscopy in living cells.

Cell biologists strive to characterize molecular interactions directly in the intracellular environment. The intrinsic resolution of optical microscopy, however, allows visualization of only coarse subcellular localization. By extracting information from molecular dynamics, fluorescence cross-correlation spectroscopy (FCCS) grants access to processes on a molecular scale, such as diffusion, binding, enzymatic reactions and codiffusion, and has become a valuable tool for studies in living cells. Here we review basic principles of FCCS and focus on seminal applications, including examples of intracellular signaling and trafficking. We consider FCCS in the context of fluorescence resonance energy transfer and multicolor imaging techniques and discuss application strategies and recent technical advances.

Algorithms↗

PubMed2005

Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry.

We successfully demonstrate the effectiveness of two-photon fluorescence cross-correlation spectroscopy (TPCCS) to study the complex binding stoichiometry of calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaMKII). Practical considerations are made for developing an intracellular cross-correlation assay, including characterization of the fluorescent molecules involved, calibration procedures of the setup, and optimal measurement conditions. Potential pitfalls and artifacts are discussed, and the complex stoichiometry of the molecular system is accounted for by a new experimental and theoretical framework for TPCCS. Our tailored model accommodates up to 12 red-labeled CaMs binding to a single green-labeled dodecameric CaMKII holoenzyme and accounts for the probability distributions of bound ligand as well as the respective changes in fluorescence emission upon binding. The model was experimentally demonstrated both in solution and in living cells by analyzing the binding of Alexa 633(C2)CaM to eGFP-CaMKII under different biochemical conditions known to induce the basal, activated, and autophosphorylated forms of the enzyme. Key binding parameters, such as binding degree, concentrations of reactants, and binding affinities, were determined under varying conditions with certain assumptions. TPCCS thus offers the unique ability to test our biochemical understanding of protein dynamics in the intracellular milieu.

Biophysical Phenomena↗

PubMed2003

Intracellular calmodulin availability accessed with two-photon cross-correlation.

The availability and interactions of signaling proteins are tightly regulated in time and space to produce specific and localized effects. For calmodulin (CaM), a key transducer of intracellular Ca(2+) signaling, binding to its variety of targets initiates signaling cascades and regulates its subcellular localization, thereby making it unavailable for subsequent binding interactions. Among CaM's numerous targets, Ca(2+)/CaM-dependent protein kinase II is one of the most striking due to its unique ability to increase its affinity for CaM by autophosphorylation and to translocate when bound to Ca(2+)/CaM. Two-photon fluorescence correlation spectroscopy and cross-correlation spectroscopy were utilized to compare mobility and molecular interactions between CaM and Ca(2+)/CaM-dependent protein kinase II in solution and in living cells. These techniques revealed that CaM availability in cells could be altered by a change in intracellular conditions. Two-photon fluorescence cross-correlation spectroscopy exemplifies a generally applicable approach for studying protein-protein interactions in living cells that allows access to the behavior of signaling molecules within their native environment to probe for heterogeneities in signaling pathways in different cellular compartments.

Animals↗

PubMed2003

Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience.

Based on time-averaging fluctuation analysis of small fluorescent molecular ensembles in equilibrium, fluorescence correlation spectroscopy has recently been applied to investigate processes in the intracellular milieu. The exquisite sensitivity of fluorescence correlation spectroscopy provides access to a multitude of measurement parameters (rates of diffusion, local concentration, states of aggregation and molecular interactions) in real time with fast temporal and high spatial resolution. The introduction of dual-color cross-correlation, imaging, two-photon excitation, and coincidence analysis coupled with fluorescence correlation spectroscopy has expanded the utility of the technique to encompass a wide range of promising applications in living cells that may provide unprecedented insight into understanding the molecular mechanisms of intracellular neurobiological processes.

Animals↗