Biomedical subjects

M Neal Waxham

Publications and source records attributed to M Neal Waxham.

10 recordsLinked to original sources

PubMed2006

Multiple diffusion mechanisms due to nanostructuring in crowded environments.

One of the key questions regarding intracellular diffusion is how the environment affects molecular mobility. Mostly, intracellular diffusion has been described as hindered, and the physical reasons for this behavior are: immobile barriers, molecular crowding, and binding interactions with immobile or mobile molecules. Using results from multi-photon fluorescence correlation spectroscopy, we describe how immobile barriers and crowding agents affect translational mobility. To study the hindrance produced by immobile barriers, we used sol-gels (silica nanostructures) that consist of a continuous solid phase and aqueous phase in which fluorescently tagged molecules diffuse. In the case of molecular crowding, translational mobility was assessed in increasing concentrations of 500 kDa dextran solutions. Diffusion of fluorescent tracers in both sol-gels and dextran solutions shows clear evidence of anomalous subdiffusion. In addition, data from the autocorrelation function were analyzed using the maximum entropy method as adapted to fluorescence correlation spectroscopy data and compared with the standard model that incorporates anomalous diffusion. The maximum entropy method revealed evidence of different diffusion mechanisms that had not been revealed using the anomalous diffusion model. These mechanisms likely correspond to nanostructuring in crowded environments and to the relative dimensions of the crowding agent with respect to the tracer molecule. Analysis with the maximum entropy method also revealed information about the degree of heterogeneity in the environment as reported by the behavior of diffusive molecules.

Biophysics↗

PubMed2006

The endosome-associated protein Hrs is hexameric and controls cargo sorting as a "master molecule".

The structure of the endosomal-associated protein, Hrs, has been determined with cryo-electron microscopy. Hrs interacts with a number of proteins, including SNAP-25 and STAM1, forming a complex that binds ubiquitin moieties. Analytical ultracentrifugation studies revealed that Hrs exists as a hexamer. The symmetry and the structure of the hexameric form of Hrs were determined with the single-particle reconstruction method. Hrs comprises three antiparallel dimers with a central core and distinct caps on either end. Crystal structures of VHS and FYVE domains fit into the Hrs end caps in the EM density map. Thus, the location of domains that interact with the endosomal membrane, the VHS, FYVE, and C-terminal domains, facilitates the anchorage of Hrs to the membrane, initiating the functional processes of Hrs on the endosome. Based on our model, the Hrs hexamer interacts with the membrane and acts as a "master molecule" that presents multiple sites for protein binding.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Kinetics of calmodulin binding to calcineurin.

Calcineurin (CaN) binds Ca(2+)-saturated calmodulin (CaM) with relatively high affinity; however, an accurate steady-state K(d) value has not been determined. In this report, we describe, using steady-state and stopped-flow fluorescence techniques, the rates of association and dissociation of Ca(2+)-saturated CaM from CaN heterodimer (CaNA/CaNB) and CaNA only. The rate of Ca(2+)/CaM association was determined to be 4.6 x 10(7) M(-1)s(-1). The rate of Ca(2+)/CaM dissociation from CaN was slower than previously reported and was approximately 0.0012 s(-1). In preparations of CaNA alone (no regulatory CaNB subunit), the dissociation rate was slowed further to 0.00026 s(-1). From these data we calculate a K(d) for binding of Ca(2+)-saturated CaM to CaN of 28 pM. This K(d) is significantly lower than previously reported estimates of approximately 1 nM and indicates that CaN is one of the highest affinity CaM-binding proteins identified to date.

Calcineurin↗

PubMed2005

A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association.

The activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays an integral role in regulating synaptic development and plasticity. We designed a live-cell-imaging approach to monitor an activity-dependent clustering of green fluorescent protein (GFP)-CaMKII holoenzymes, termed self-association, a process that we hypothesize contributes to the translocation of CaMKII to synaptic and nonsynaptic sites in activated neurons. We show that GFP-CaMKII self-association in human embryonic kidney 293 (HEK293) cells requires a catalytic domain and multimeric structure, requires Ca2+ stimulation and a functional Ca2+/CaM-binding domain, is regulated by cellular pH and Thr286 autophosphorylation, and has variable rates of dissociation depending on Ca2+ levels. Furthermore, we show that the same rules that govern CaMKII self-association in HEK293 cells apply for extrasynaptic and postsynaptic translocation of GFP-CaMKII in hippocampal neurons. Our data support a novel mechanism for targeting CaMKII to postsynaptic sites after neuronal activation. As such, CaMKII may form a scaffold that, in combination with other synaptic proteins, recruits and localizes additional proteins to the postsynaptic density. We discuss the potential function of CaMKII self-association as a tag of synaptic activity.

Animals↗

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↗

PubMed2004

Calmodulin and Munc13 form a Ca2+ sensor/effector complex that controls short-term synaptic plasticity.

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is a key determinant of network properties; is involved in many physiological processes such as motor control, sound localization, or sensory adaptation; and is critically dependent on cytosolic [Ca2+]. However, the underlying molecular mechanisms and the identity of the Ca2+ sensor/effector complexes involved are unclear. We now identify a conserved calmodulin binding site in UNC-13/Munc13s, which are essential regulators of synaptic vesicle priming and synaptic efficacy. Ca2+ sensor/effector complexes consisting of calmodulin and Munc13s regulate synaptic vesicle priming and synaptic efficacy in response to a residual [Ca2+] signal and thus shape short-term plasticity characteristics during periods of sustained synaptic activity.

Animals↗

PubMed2004

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium.

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.

Animals↗

PubMed2004

Comparative analyses of the three-dimensional structures and enzymatic properties of alpha, beta, gamma and delta isoforms of Ca2+-calmodulin-dependent protein kinase II.

Ca(2+)-calmodulin-dependent protein kinase II (CaM-kinase II) is a ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four genes (alpha, beta, gamma, and delta) in mammalian cells. We have documented the three-dimensional structures and the biophysical and enzymatic properties of the four gene products. Biophysical analyses showed that each isoform assembles into oligomeric forms and their three-dimensional structures at 21-25 A revealed that all four isoforms were dodecamers with similar but highly unusual architecture. A gear-shaped core comprising the association domain has the catalytic domains tethered on appendages, six of which extend from both ends of the core. At this level of resolution, we can discern no isoform-dependent differences in ultrastructure of the holoenzymes. Enzymatic analyses showed that the isoforms were similar in their K(m) for ATP and the peptide substrate syntide, but showed significant differences in their interactions with Ca(2+)-calmodulin as assessed by binding, substrate phosphorylation, and autophosphorylation. Interestingly, the rank order of CaM binding affinity (gamma > beta > delta > alpha) does not directly correlate with the rank order of their CaM dependence for autophosphorylation (beta > gamma > delta > alpha). Simulations utilizing this data revealed that the measured differences in CaM binding affinities play a minor role in the autophosphorylation of the enzyme, which is largely dictated by the rate of autophosphorylation for each isoform.

Adenosine Triphosphate↗

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

A new role for IQ motif proteins in regulating calmodulin function.

IQ motifs are found in diverse families of calmodulin (CaM)-binding proteins. Some of these, like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood. We hypothesized that these IQ motif proteins might have unique effects on the Ca2+ binding properties of CaM, since they bind to CaM in the presence or absence of Ca2+. Here we show that PEP-19 accelerates by 40 to 50-fold both the slow association and dissociation of Ca2+ from the C-domain of free CaM, and we identify the sites of interaction between CaM and PEP-19 using NMR. Importantly, we demonstrate that PEP-19 can also increase the rate of dissociation of Ca2+ from CaM when bound to intact CaM-dependent protein kinase II. Thus, PEP-19, and presumably similar members of the IQ family of proteins, has the potential to alter the Ca2+-binding dynamics of free CaM and CaM that is bound to other target proteins. Since Ca2+ binding to the C-domain of CaM is the rate-limiting step for activation of CaM-dependent enzymes, the data reveal a new concept of importance in understanding the temporal dynamics of Ca2+-dependent cell signaling.

Amino Acid Motifs↗