Phospholipids undergo hop diffusion in compartmentalized cell membrane

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5–100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 μs. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (φ) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-φ compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 μm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids

Nanoclustering as a dominant feature of plasma membrane organization

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent.Postprint (published version

Fluorescence Recovery After Photobleaching Techniques to Measure Translational Mobility in Microscopic Samples

The scope of photobleaching applications and the method itself are briefly reviewed. Two current applications in this laboratory are then outlined. First, the use of spatial Fourier transforms to analyze video photobleaching measurements is presented. This method extracts diffusion coefficients using all the image data and it does not require that the initial condition created by photobleaching be known. Second, the use of genetic engineering methods coupled with photobleaching analysis is discussed as means to uncover the structural determinants of membrane protein lateral mobility

Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamide substrates for cell mechanotransduction assays

Increasing numbers of cell mechanotransduction studies are currently utilizing elastic substrates fabricated from polyacrylamide in the form of thin gels. Their versatility depends on the ability to ensure the appropriate gel stiffness and control the uniformity and geometry of extracellular matrix protein coating of the gel. Beginning with a brief quantitative emphasis on the elastic properties of polyacrylamide gels, we present an inexpensive and highly reproducible method for uniform coating with a wide variety of extracellular matrix proteins. We used a reducing agent, hydrazine hydrate, to modify nonreactive amide groups in polyacrylamide to highly reactive hydrazide groups that can form covalent bonds with aldehyde or ketone groups in oxidized proteins. This simple conjugation method overcomes the limitations of previously used photoactivatable cross-linkers: nonuniform coating due to nonuniformity of irradiation and technically challenging procedures for micropatterning. As demonstrated in our study of cell polarity during constrained migration, this conjugation method is especially effective in gel micropatterning by manual microcontact printing of protein patterns as small as 5 microm and enables numerous studies of constrained cell attachment and migration that were previously unfeasible due to high cost or difficulty in controlling the protein coating

Understanding lipid rafts and other related membrane domains

Evidence in support of the classical lipid raft hypothesis has remained elusive. Data suggests that transmembrane proteins and the actin-containing cortical cytoskeleton can organize lipids into short-lived nanoscale assemblies that can be assembled into larger domains under certain conditions. This supports an evolving view in which interactions between lipids, cholesterol, and proteins create and maintain lateral heterogeneity in the cell membrane

Direct measurement of the lamellipodial protrusive force in a migrating cell

There has been a great deal of interest in the mechanism of lamellipodial protrusion (Pollard, T., and G. Borisy. 2003. Cell. 112:453–465). However, one of this mechanism's endpoints, the force of protrusion, has never been directly measured. We place an atomic force microscopy cantilever in the path of a migrating keratocyte. The deflection of the cantilever, which occurs over a period of ∼10 s, provides a direct measure of the force exerted by the lamellipodial leading edge. Stall forces are consistent with ∼100 polymerizing actin filaments per micrometer of the leading edge, each working as an elastic Brownian ratchet and generating a force of several piconewtons. However, the force-velocity curves obtained from this measurement, in which velocity drops sharply under very small loads, is not sensitive to low loading forces, and finally stalls rapidly at large loads, are not consistent with current theoretical models for the actin polymerization force. Rather, the curves indicate that the protrusive force generation is a complex multiphase process involving actin and adhesion dynamics

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)–induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 → Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization

Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation

Detection of protein-protein interactions in cells is crucial for understanding the biological functions of proteins, including their roles in signal transduction. However, current methods require specific antibodies both for immunoprecipitation and detection, making them expensive and sometimes unreliable. Here we describe protocols for protein-protein interaction assays that use nonimmune IgG-conjugated Sepharose to precipitate the IgG binding domain (ZZ) fused to the bait protein; the interaction partner is fused to Avitag and biotinylated by BirA so that it can be detected by a one-step blot with Dylight 680 streptavidin to detect the Avitag fusion protein. Since this method does not require specific antibodies and is inexpensive, sensitive, and reliable, it should be useful for detecting protein-protein interactions in cells

In Silico Generation of Alternative Hypotheses Using Causal Mapping (CMAP)

Previously, we introduced causal mapping (CMAP) as an easy to use systems biology tool for studying the behavior of biological processes that occur at the cellular and molecular level. CMAP is a coarse-grained graphical modeling approach in which the system of interest is modeled as an interaction map between functional elements of the system, in a manner similar to portrayals of signaling pathways commonly used by molecular cell biologists. CMAP describes details of the interactions while maintaining the simplicity of other qualitative methods (e.g., Boolean networks)

Transient anchorage of cross-linked glycosyl-phosphatidylinositol–anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides

How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol–anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle–induced clusters of both Thy-1 and CD73, a 5′ exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton