The alpha(L)beta(2) integrin (leukocyte function-associated antigen-1 [LFA-1]) is regulated to engage and maintain T cell adhesion. Conformational changes in the receptor are associated with changes in receptor-ligand affinity and are necessary for firm adhesion. Less well understood is the relationship between receptor conformation and the regulation of its lateral mobility. We have used fluorescence photobleaching recovery and single-particle tracking to measure the lateral mobility of specific conformations of LFA-1. These measurements show that different receptor conformations have distinct diffusion profiles and that these profiles vary according to the activation state of the cell. Notably, a high-affinity conformation of LFA-1 is mobile on resting cells but immobile on phorbol-12-myristate-13-acetate-activated cells. This activation-induced immobilization is prevented by a calpain inhibitor and by an allosteric LFA-1 inhibitor. Our results suggest that current models of LFA-1 regulation are incomplete and that LFA-1 confinement by cytoskeletal attachment regulates cell adhesion both negatively and positively.

Merlin and Ezrin are central to a mechanism whereby mechanical forces transduced across the apical actomyosin cytoskeleton from cell junctions control the mobility and internalization of EGFR, providing novel insight into how cells inhibit mitogenic signaling in response to cell contact.

Many DNA-interacting proteins diffuse on DNA to perform their biochemical functions. Processivity factors diffuse on DNA to permit unimpeded elongation by their associated DNA polymerases, but little is known regarding their rates and mechanisms of diffusion. The processivity factor of herpes simplex virus DNA polymerase, UL42, unlike ''sliding clamp'' processivity factors that normally form rings around DNA, binds DNA directly and tightly as a monomer, but can still diffuse on DNA. To investigate the mechanism of UL42 diffusion on DNA, we examined the effects of salt concentration on diffusion coefficient. Ensemble studies, employing electrophoretic mobility shift assays on relatively short DNAs, showed that off-rates of UL42 from DNA depended on DNA length at higher but not lower salt concentrations, consistent with the diffusion coefficient being salt-dependent. Direct assays of the motion of single fluorescently labeled UL42 molecules along DNA revealed increased diffusion at higher salt concentrations. Remarkably, the diffusion coefficients observed in these assays were Ϸ10 4 -fold higher than those calculated from ensemble experiments. Discrepancies between the single-molecule and ensemble results were resolved by the observation, in single-molecule experiments, that UL42 releases relatively slowly from the ends of DNA in a salt-dependent manner. The results indicate that UL42 ''hops'' rather than ''slides,'' i.e., it microscopically dissociates from and reassociates with DNA as it diffuses rather than remaining so intimately associated with DNA that cation condensation on the phosphate backbone does not affect its motion. These findings may be relevant to mechanisms of other processivity factors and DNA-binding proteins.herpes simplex virus ͉ linear diffusion D NA polymerases are central to DNA replication. Most replicative DNA polymerases include accessory subunits that promote replication of long stretches of DNA without dissociating from the template. The best known of these processivity factors are the ''sliding clamps'' (reviewed in ref. 1), which include polymerase subunits of bacteria, eukaryotes, and archaea that form multimeric rings around DNA with the aid of ATP-dependent clamp-loaders. These rings then tether their cognate catalytic subunits to DNA, permitting processive DNA synthesis.A variety of cellular and viral polymerases include processivity subunits that do not use ATP or other proteins for loading onto DNA. Of these, herpes simplex virus (HSV) UL42 is one of the best characterized. This protein's structure resembles that of a monomer of the sliding clamp proliferating cell nuclear antigen (2), yet UL42 binds directly to DNA as a monomer with relatively high affinity (apparent dissociation constant (K d ) in the nanomolar range) (3-5). This direct binding of DNA by UL42 tethers the catalytic subunit of HSV DNA polymerase (Pol) to DNA, thereby enabling processivity (3,(5)(6)(7).An important attribute of processivity factors is their ability to diffuse on DNA. Such diffusion permits teth...

SummaryLaminin-binding integrins (a3b1, a6b1, a6b4, a7b1) are almost always expressed together with tetraspanin CD151. In every coexpressing cell analyzed to date, CD151 makes a fundamental contribution to integrin-dependent motility, invasion, morphology, adhesion and/or signaling. However, there has been minimal mechanistic insight into how CD151 affects integrin functions. In MDA-MB-231 mammary cells, tetraspanin CD151 knockdown impairs a6 integrin clustering and functions without decreasing a6 integrin expression or activation. Furthermore, CD151 knockdown minimally affects the magnitude of a6 integrin diffusion, as measured using single particle tracking. Instead, CD151 knockdown has a novel and unexpected dysregulating effect on the mode of a6 integrin diffusion. In control cells a6 integrin shows mostly random-confined diffusion (RCD) and some directed motion (DMO). In sharp contrast, in CD151-knockdown cells a6 integrin shows mostly DMO. In control cells a6 diffusion mode is sensitive to actin disruption, talin knockdown and phorbol ester stimulation. By contrast, CD151 knockdown cell a6 integrin is sensitive to actin disruption but desensitized to talin knockdown or phorbol ester stimulation, indicating dysregulation. Both phorbol ester and EGF stimulate cell spreading and promote a6 RCD in control cells. By contrast, CD151-ablated cells retain EGF effects but lose phorbol-ester-stimulated spreading and a6 RCD. For a6 integrins, physical association with CD151 promotes a6 RCD, in support of a6-mediated cable formation and adhesion. By comparison, for integrins not associated with CD151 (e.g. av integrins), CD151 affects neither diffusion mode nor av function. Hence, CD151 support of a6 RCD is specific and functionally relevant, and probably underlies diverse CD151 functions in skin, kidney and cancer cells.

Decay-accelerating factor (DAF, also known as CD55), a glycosylphosphatidylinositol-linked (GPI-linked) plasma membrane protein, protects autologous cells from complement-mediated damage by inhibiting complement component 3 (C3) activation. An important physical property of GPI-anchored complement regulatory proteins such as DAF is their ability to translate laterally in the plasma membrane. Here, we used singleparticle tracking and tether-pulling experiments to measure DAF lateral diffusion, lateral confinement, and membrane skeletal associations in human erythrocyte membranes. In native membranes, most DAF molecules exhibited Brownian lateral diffusion. Fluid-phase complement activation caused deposition of C3b, one of the products of C3 cleavage, onto erythrocyte glycophorin A (GPA). We then determined that DAF, C3b, GPA, and band 3 molecules were laterally immobilized in the membranes of complement-treated cells, and GPA was physically associated with the membrane skeleton. Mass spectrometry analysis further showed that band 3, α-spectrin, β-spectrin, and ankyrin were present in a complex with C3b and GPA in complement-treated cells. C3b deposition was also associated with a substantial increase in erythrocyte membrane stiffness and/or viscosity. We therefore suggest that complement activation stimulates the formation of a membrane skeletonlinked DAF-C3b-GPA-band 3 complex on the erythrocyte surface. This complex may promote the removal of senescent erythrocytes from the circulation. IntroductionThe complement system is a major effector component of the innate immune response (1). The complement cascade, which involves sequential activation of serum complement proteins, leads to diverse inflammatory effects and, in some cases, lysis of the target. Activation of complement can occur through the classical, alternative, and lectin pathways. All 3 pathways lead to the formation of complement component 3 (C3) convertase, a central enzymatic complement complex that cleaves serum C3 into C3a and C3b. C3b can dock covalently on a membrane surface via amide or ester linkages. Downstream of C3 activation, C3 convertase participates in the formation of C5 convertase, a membrane-bound complex that cleaves serum C5 into C5a and C5b. C5b induces sequential recruitment of complement proteins C6, C7, C8, and C9 to form C5b-9, the terminal complement complex, which creates a pore in the membrane (2).