Biomedical subjects

Jennifer J Linderman

Publications and source records attributed to Jennifer J Linderman.

8 recordsLinked to original sources

PubMed2004

Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins.

A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G(beta2) in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10(6). These methods were coupled with immunodetection methods and an adenosine 5(')-diphosphate-ribosylation assay to measure the amount of membrane-associated G(ialpha) proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.

Biomarkers↗

PubMed2003

Self organization of membrane proteins via dimerization.

Protein-protein dimerization is ubiquitous in biology, but its role in self-organization remains unexplored. Here we use Monte Carlo simulations to demonstrate that under diffusion-limited conditions, reversible dimerization alone can cause membrane proteins to cluster into oligomer-like structures. When multiple distinct protein species are able to form dimers, then heterodimerization and homodimerization can organize proteins into structured clusters that can affect cellular physiology. As an example, we demonstrate how receptor dimerization could provide a physical mechanism for regulating information flow by controlling receptor-receptor cross talk. These results are physically realistic for some membrane proteins, including members of the G-protein coupled receptor family, and may provide a physiological reason as to why many proteins dimerize.

Dimerization↗

PubMed2003

Priming-induced localization of G(ialpha2) in high density membrane microdomains.

Subcellular fractionation of human neutrophils on linear sucrose density gradients was utilized to test the hypothesis that priming regulates the subcellular and sub-plasma membrane distribution of neutrophil G-protein subunits, G(ialpha2) and G(ialpha3), N-formyl peptide receptor, Lyn kinase and phospholipase C(beta2). G(ialpha2), but not G(ialpha3), moved from a lighter to a higher density plasma membrane fraction. Unoccupied N-formyl peptide receptors were found throughout the plasma membrane fractions and this distribution did not change with priming. In unprimed cells G(ialpha2) and its effector, phospholipase C(beta2), were segregated in different membrane compartments; priming caused G(ialpha2) to move to the compartment in which phospholipase C(beta2) resided. Thus, an important component of the mechanism of priming may involve regulation of the location of G-proteins and effector molecules in plasma membrane compartments where their abilities to couple may be enhanced.

Alkaline Phosphatase↗

PubMed2003

Untangling ligand induced activation and desensitization of G-protein-coupled receptors.

Long-term treatment with a drug to a G-protein-coupled receptor (GPCR) often leads to receptor-mediated desensitization, limiting the therapeutic lifetime of the drug. To better understand how this therapeutic window might be controlled, we created a mechanistic Monte Carlo model of the early steps in GPCR signaling and desensitization. Using this model we found that the rates of G-protein activation and receptor phosphorylation can be partially decoupled by varying the drug-receptor dissociation rate constant, k(off), and the drug's efficacy, alpha. The maximum ratio of G-protein activation to receptor phosphorylation (GARP) was found for drugs with an intermediate k(off) value and small alpha-value. Changes to the cellular environment, such as changes in the diffusivity of membrane molecules and the G-protein inactivation rate constant, affected the GARP value of a drug but did not change the characteristic shape of the GARP curve. These model results are examined in light of experimental data for a number of GPCRs and are found to be in good agreement, lending support to the idea that the desensitization properties of a drug might be tailored to suit a specific application.

Animals↗

PubMed2003

Micro- and nanotechnologies for studying cellular function.

The study of complex biological systems requires methods to perturb the system in complex yet controlled ways to elucidate mechanisms and dynamic interactions, and to recreate in vivo conditions in flexible in vitro set-ups. This paper reviews recent advances in the use of micro- and nanotechnologies in the study of complex biological systems and the advantages they provide in these two areas. Particularly useful for controlling the chemical and mechanical microenvironments of cells is a set of techniques called soft lithography, whereby elastomeric materials are used to transfer and generate micro- and nanoscale patterns. Examples of some of the capabilities of soft lithography include the use of elastomeric stamps to generate micropatterns of protein and the use of elastomeric channels to localize chemicals with subcellular spatial resolutions. These types of biological micro- and nanotechnologies combined with mathematical modeling will propel our understandings of cellular and subcellular physiology to new heights.

Animals↗

PubMed2002

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

Animals↗

PubMed2002

Quantification of fibronectin adsorption to silicone-rubber cell culture substrates.

As the role of mechanical force in cellular signaling gained recognition, investigators designed a number of devices to deliver controlled regimens of mechanical force to cultured cells. One type of device uses thin silicone-rubber membranes to support monolayer cell adhesion and to transmit mechanical force in the form of biaxial strain. We have observed that cell attachment and spreading are impaired on these membranes compared to polystyrene, even when both are passively coated with identical amounts of extracellular matrix. The purpose of these studies was to quantify the efficiency and stability of passive matrix adsorption onto commercially available elastic culture substrates. A theoretically saturating density (1 microg/cm2) of fibronectin was added to each well, and the initial efficiency of adsorption to the walls and elastic membranes was found to be 31 +/- 2% of the protein added. Strikingly, when the protein adsorbed specifically to the membranes was quantified after seven days, only 10-26 ng/cm2 fibronectin were present, revealing that most of the adsorption is to the sides of the wells. These results indicate that the adsorption of matrix proteins to silicone-rubber substrates is relatively inefficient and that investigators who use these systems must be aware of this fact and design their experiments accordingly.

Adsorption↗

PubMedDate not supplied

Externally applied cyclic strain regulates localization of focal contact components in cultured smooth muscle cells.

Mechanical signals are critical regulators of cellular gene expression, yet little is understood of the mechanism whereby cells sense mechanical forces. In this study we have tested the hypothesis that mechanical strain applied to populations of cells via their adhesion substrate rapidly alters the cellular distribution of focal contact proteins. Focal contact-associated components (vinculin, a-actinin, paxillin) were assayed by immunofluorescence microscopy and quantitative western blotting. Application of a single step increase in strain in multiple experiments caused overall a small change in focal contact-associated vinculin. In contrast, cyclic strain induced a large and very reproducible increase in detergent-insoluble vinculin (52% relative to static) after just 1 min of strain. Insoluble paxillin was transiently enriched with a similar time course, whereas insoluble a-actinin did not change significantly in response to cyclic strain. Rhodamine-labeled chicken vinculin added to permeabilized cells preferentially localized to focal contacts in response to cyclic strain, but not a single step increase in strain. These findings establish that insoluble levels of focal contact components are altered rapidly following application of an appropriate number of mechanical perturbations, and suggest that at least one component of the mechanism does not involve soluble intermediates.

Actinin↗