Integrin-dependent interaction of lipid rafts with the actin cytoskeleton in activated human platelets

Abstract

IntroductionThe interactions between the plasma membrane and the cytoskeleton are essential for a wide variety of cellular processes such as endocytosis, cell morphology, cell adhesion, formation of cell-cell contacts and cell motility. These interactions are regulated by signaling complexes formed by the assembly of transmembrane and cytosolic proteins and lipids (Engqvist-Goldstein and Drubin, 2003;Fais and Malorni, 2003;Sechi and Wehland, 2000). These complexes are thought to participate in the control of the elongation of actin filaments leading to the formation of membrane protrusions, or in the contraction of the actomyosin network leading to membrane retraction. Blood platelets represent an attractive model to investigate these mechanisms as the actin cytoskeleton and its interactions with the plasma membrane are critical for their physiological functions (Hartwig et al., 1999). Indeed, platelets must respond rapidly to a vessel injury by a series of coordinated events including adhesion, shape change, spreading, aggregation and clot retraction. αIIbβ3 is the most prominent platelet integrin, and, upon activation by 'insideout' signaling, is capable of binding to several adhesive proteins, including fibrinogen, to support platelet aggregation. During aggregation, αIIbβ3 is also involved in transmitting an 'outside-in' signal leading to the formation of a network of signaling and structural proteins strongly interacting with the newly remodeled actin cytoskeleton (Hartwig et al., 1999). These protein complexes were previously co-isolated with the cytoskeleton as a Triton X-100 insoluble pellet obtained at low speed centrifugation (15,000 g) from aggregated platelets (Fox, 1993). The characterization of this cellular fraction led to the identification of several key signaling molecules recruited to αIIbβ3-based cytoskeletal structures, where they are thought to play an essential role in stabilization of platelet aggregation (Hartwig et al., 1999).At the sites of interaction with the cytoskeleton, the plasma membrane is subjected to important mechanical forces, especially during platelet fibrin clot retraction, a postaggregation event involved in the maintenance of hemostasis. During retraction, integrins take part in the formation of a transmembrane linkage between proteins from the extracellular matrix and from the actomyosin filament network, allowing the transmission of contractile forces. It is currently hypothesized that the areas of membrane interacting with the cytoskeleton have a specific lipid composition adapted to these forces. In this context, cholesterol and sphingolipid enriched membrane microdomains, known as detergent resistant membranes (DRMs) or lipid rafts, are of major interest. Their lipid composition confers a restricted fluidity state called a liquid ordered phase to these membrane microdomains (Brown and London, 2000). Although the existence of rafts has been 759 Dynamic connections between actin filaments and the plasma membrane are crucial for the regulation of blood platelet funct...

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac

“…In agreement with previous reports, 27,28 we found that pretreatment of platelets with a relatively high concentration (5mM) of M␤CD caused mild (Ͻ 20%) inhibition of thrombin induced aggregation, whereas there was no effect at lower M␤CD concentrations ( Figure 2A). In contrast, M␤CD caused a marked, concentration-dependent inhibition of rhodocytin-induced aggregation that reduced the response by greater than 95% at 5mM, with a partial effect observed at lower concentrations (1-2mM).…”

Isoform-specific palmitoylation of JNK regulates axonal development

“…Nevertheless, compared with JNK3 WT, an increase of JNK3 CS and a small decrease of JNK3 WT co-expressed with zD15 were detected at fraction 13, the high-density triton-insoluble fraction enriched with actinassociated cytoskeleton. 29 This implies that palmitoylation may affect JNK3's association with the actin cytoskeleton. Signals for actin cytoskeleton modulation are essential for axonal development.…”

“…Signals for actin cytoskeleton modulation are essential for axonal development. 30 To analyze this quantitatively, 29 we isolated the Triton-insoluble actin cytoskeleton fraction from cortical neurons and HEK293 cells expressing GFP-JNK3, followed by detection of JNK3. Consistently, JNK3 was detected in the Triton-insoluble fraction, which was sensitive to the actin depolymerizers cytochalasin D (Cyt.D) or latrunculin A (Lat.A) (Figure 3b).…”

Reconstituted High-Density Lipoprotein Attenuates Platelet Function in Individuals With Type 2 Diabetes Mellitus by Promoting Cholesterol Efflux

“…29 Lipid rafts have been shown to be integral to platelet signaling pathways, 30 -33 and disruption of rafts is associated with reduced platelet activation. 30 We examined the effect of rHDL incubation on the binding of cholera toxin subunit B (CT-B) to the lipid raft marker ganglioside GM 1 on the surface of platelets. In resting platelets, rHDL was associated with a significant reduction in CT-B binding, suggesting reduced lipid raft assembly (77.3Ϯ1.3%; PϽ0.01).…”