Charles A. Day

Tracking microdomain dynamics in cell membranes

Studies of the diffusion of proteins and lipids in the plasma membrane of cells have long pointed to the presence of membrane domains. A major challenge in the field of membrane biology has been to characterize the various cellular structures and mechanisms that impede free diffusion in cell membranes and determine the consequences that membrane compartmentalization has on cellular biology. In this review, we will provide a brief summary of the classes of domains that have been characterized to date, focusing on recent efforts to identify the properties of lipid rafts in cells through measurements of protein and lipid diffusion.

Active Metabolite of Atorvastatin Inhibits Membrane Cholesterol Domain Formation by an Antioxidant Mechanism

The advanced atherosclerotic lesion is characterized by the formation of microscopic cholesterol crystals that contribute to mechanisms of inflammation and apoptotic cell death. These crystals develop from membrane cholesterol domains, a process that is accelerated under conditions of hyperlipidemia and oxidative stress. In this study, the comparative effects of hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) on oxidative stress-induced cholesterol domain formation were tested in model membranes containing physiologic levels of cholesterol using small angle x-ray diffraction approaches. In the absence of HMG-CoA reductase, only the atorvastatin active o-hydroxy metabolite (ATM) blocked membrane cholesterol domain formation as a function of oxidative stress. This effect of ATM is attributed to electron donation and proton stabilization mechanisms associated with its phenoxy group located in the membrane hydrocarbon core. ATM inhibited lipid peroxidation in human low density lipoprotein and phospholipid vesicles in a dose-dependent manner, unlike its parent and other statins (pravastatin, rosuvastatin, simvastatin). These findings indicate an atheroprotective effect of ATM on membrane lipid organization through a potent antioxidant mechanism.

A Generalization of Theory for Two-Dimensional Fluorescence Recovery after Photobleaching Applicable to Confocal Laser Scanning Microscopes

Fluorescence recovery after photobleaching (FRAP) using confocal laser scanning microscopes (confocal FRAP) has become a valuable technique for studying the diffusion of biomolecules in cells. However, two-dimensional confocal FRAP sometimes yields results that vary with experimental setups, such as different bleaching protocols and bleaching spot sizes. In addition, when confocal FRAP is used to measure diffusion coefficients (D) for fast diffusing molecules, it often yields D-values that are one or two orders-of-magnitude smaller than that predicted theoretically or measured by alternative methods such as fluorescence correlation spectroscopy. Recently, it was demonstrated that this underestimation of D can be corrected by taking diffusion during photobleaching into consideration. However, there is currently no consensus on confocal FRAP theory, and no efforts have been made to unify theories on conventional and confocal FRAP. To this end, we generalized conventional FRAP theory to incorporate diffusion during photobleaching so that analysis by conventional FRAP theory for a circular region of interest is easily applicable to confocal FRAP. Finally, we demonstrate the accuracy of these new (to our knowledge) formulae by measuring D for soluble enhanced green fluorescent protein in aqueous glycerol solution and in the cytoplasm and nucleus of COS7 cells.

Simplified equation to extract diffusion coefficients from confocal FRAP data.

Quantitative measurements of diffusion can provide important information about how proteins and lipids interact with their environment within the cell and the effective size of the diffusing species. Confocal fluorescence recovery after photobleaching (FRAP) is one of the most widely accessible approaches to measure protein and lipid diffusion in living cells. However, straightforward approaches to quantify confocal FRAP measurements in terms of absolute diffusion coefficients are currently lacking. Here, we report a simplified equation that can be used to extract diffusion coefficients from confocal FRAP data using the half time of recovery and effective bleach radius for a circular bleach region, and validate this equation for a series of fluorescently labeled soluble and membrane‐bound proteins and lipids. We show that using this approach, diffusion coefficients ranging over three orders of magnitude can be obtained from confocal FRAP measurements performed under standard imaging conditions, highlighting its broad applicability.

Rofecoxib increases susceptibility of human LDL and membrane lipids to oxidative damage: A mechanism of cardiotoxicity

Clinical investigations have demonstrated a relationship between the extended use of rofecoxib and the increased risk for atherothrombotic events. This has led to the removal of rofecoxib from the market and concern over the cardiovascular safety of other cyclooxygenase (COX)-2 selective agents. Experimental findings from independent laboratories now indicate that the cardiotoxicity of rofecoxib may not be a class effect but because of its intrinsic chemical properties. Specifically, rofecoxib has been shown to increase the susceptibility of human low-density lipoprotein and cellular membrane lipids to oxidative modification, a contributing factor to plaque instability and thrombus formation. Independently of COX-2 inhibition, rofecoxib also promoted the nonenzymatic formation of isoprostanes and reactive aldehydes from biologic lipids. The basis for these observations is that rofecoxib alters lipid structure and readily forms a reactive maleic anhydride in the presence of oxygen. By contrast, other selective (celecoxib, valdecoxib) and nonselective (naproxen, diclofenac) inhibitors did not influence rates of low-density lipoprotein and membrane lipid oxidation. We have now further confirmed these findings by demonstrating that the prooxidant activity of rofecoxib can be blocked by the potent antioxidant astaxanthin in homochiral form (all-trans 3S, 3′S). These findings provide a mechanistic rationale for differences in cardiovascular risk among COX-selective inhibitors because of their intrinsic physicochemical properties.

Mechanisms underlying the confined diffusion of cholera toxin B-subunit in intact cell membranes.

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes.

Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation

Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed.

Analysis of Protein and Lipid Dynamics Using Confocal Fluorescence Recovery After Photobleaching (FRAP)

Fluorescence recovery after photobleaching (FRAP) is a powerful, versatile, and widely accessible tool to monitor molecular dynamics in living cells that can be performed using modern confocal microscopes. Although the basic principles of FRAP are simple, quantitative FRAP analysis requires careful experimental design, data collection, and analysis. In this unit, we discuss the theoretical basis for confocal FRAP, followed by step‐by‐step protocols for FRAP data acquisition using a laser‐scanning confocal microscope for (1) measuring the diffusion of a membrane protein, (2) measuring the diffusion of a soluble protein, and (3) analysis of intracellular trafficking. Finally, data analysis procedures are discussed, and an equation for determining the diffusion coefficient of a molecular species undergoing pure diffusion is presented. Curr. Protoc. Cytom. 62:2.19.1‐2.19.29.

Microtubule Motors Power Plasma Membrane Tubulation in Clathrin-Independent Endocytosis

How the plasma membrane is bent to accommodate clathrin‐independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin‐independent carriers by binding and cross‐linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin‐induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B‐subunit into surface‐derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin‐independent endocytosis.

Functions of cholera toxin B-subunit as a raft cross-linker

Lipid rafts are putative complexes of lipids and proteins in cellular membranes that are proposed to function in trafficking and signalling events. CTxB (cholera toxin B-subunit) has emerged as one of the most studied examples of a raft-associated protein. Consisting of the membrane-binding domain of cholera toxin, CTxB binds up to five copies of its lipid receptor on the plasma membrane of the host cell. This multivalency of binding gives the toxin the ability to reorganize underlying membrane structure by cross-linking otherwise small and transient lipid rafts. CTxB thus serves as a useful model for understanding the properties and functions of protein-stabilized domains. In the present chapter, we summarize current evidence that CTxB associates with and cross-links lipid rafts, discuss how CTxB binding modulates the architecture and dynamics of membrane domains, and describe the functional consequences of this cross-linking behaviour on toxin uptake into cells via endocytosis.