Early Lipid Raft-Related Changes: Interplay between Unilateral Denervation and Hindlimb Suspension

Muscle disuse and denervation leads to muscle atrophy, but underlying mechanisms can be different. Previously, we have found ceramide (Cer) accumulation and lipid raft disruption after acute hindlimb suspension (HS), a model of muscle disuse. Herein, using biochemical and fluorescent approaches the influence of unilateral denervation itself and in combination with short-term HS on membrane-related parameters of rat soleus muscle was studied. Denervation increased immunoexpression of sphingomyelinase and Cer in plasmalemmal regions, but decreased Cer content in the raft fraction and enhanced lipid raft integrity. Preliminary denervation suppressed (1) HS-induced Cer accumulation in plasmalemmal regions, shown for both nonraft and raft-fractions; (2) HS-mediated decrease in lipid raft integrity. Similar to denervation, inhibition of the sciatic nerve afferents with capsaicin itself increased Cer plasmalemmal immunoexpression, but attenuated the membrane-related effects of HS. Finally, both denervation and capsaicin treatment increased immunoexpression of proapoptotic protein Bax and inhibited HS-driven increase in antiapoptotic protein Bcl-2. Thus, denervation can increase lipid raft formation and attenuate HS-induced alterations probably due to decrease of Cer levels in the raft fraction. The effects of denervation could be at least partially caused by the loss of afferentation. The study points to the importance of motor and afferent inputs in control of Cer distribution and thereby stability of lipid rafts in the junctional and extrajunctional membranes of the muscle.

Changes in Membrane Ceramide Pools in Rat Soleus Muscle in Response to Short-Term Disuse

Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNFα receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.

Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virus

Influenza still remains one of the most challenging diseases, posing a significant threat to public health. Host lipid rafts play a critical role in influenza A virus (IAV) assembly and budding, however, their role in polyvalent IAV host binding and endocytosis had remained elusive until now. In the present study, we observed co-localization of IAV with a lipid raft marker ganglioside, GM1, on the host surface. Further, we isolated the lipid raft micro-domains from IAV infected cells and detected IAV protein in the raft fraction. Finally, raft disruption using Methyl-β-Cyclodextrin revealed significant reduction in IAV host binding, suggesting utilization of host rafts for polyvalent binding on the host cell surface. In addition to this, cyclodextrin mediated inhibition of raft-dependent endocytosis showed significantly reduced IAV internalization. Interestingly, exposure of cells to cyclodextrin two hours post-IAV binding showed no such reduction in IAV entry, indicating use of raft-dependent endocytosis for host entry. In summary, this study demonstrates that host lipid rafts are selected by IAV as a host attachment factors for multivalent binding, and IAV utilizes these micro-domains to exploit raft-dependent endocytosis for host internalization, a virus entry route previously unknown for IAV.

Acid-sensing ion channel 3 (ASIC3) cell surface expression is modulated by PSD-95 within lipid rafts

Acid-sensing ion channel 3 (ASIC3) is a H+-gated cation channel primarily found in sensory neurons, where it may function as a pH sensor in response to metabolic disturbances or painful conditions. We previously found that ASIC3 interacts with the postsynaptic density protein PSD-95 through its COOH terminus, which leads to a decrease in ASIC3 cell surface expression and H+-gated current. PSD-95 has been implicated in recruiting proteins to lipid rafts, which are membrane microdomains rich in cholesterol and sphingolipids that organize receptor/signaling complexes. We found ASIC3 and PSD-95 coimmunoprecipitated within detergent-resistant membrane fractions. When cells were exposed to methyl-β-cyclodextrin to deplete membrane cholesterol and disrupt lipid rafts, PSD-95 localization to lipid raft fractions was abolished and no longer inhibited ASIC3 current. Likewise, mutation of two cysteine residues in PSD-95 that undergo palmitoylation (a lipid modification that targets PSD-95 to lipid rafts) prevented its inhibition of ASIC3 current and cell surface expression. In addition, we found that cell surface ASIC3 is enriched in the lipid raft fraction. These data suggest that PSD-95 and ASIC3 interact within lipid rafts and that this raft interaction is required for PSD-95 to modulate ASIC3.

The ADAP/SKAP55 Signaling Module Regulates T-Cell Receptor-Mediated Integrin Activation through Plasma Membrane Targeting of Rap1

ABSTRACT Adhesion of T cells after stimulation of the T-cell receptor (TCR) is mediated via signaling processes that have collectively been termed inside-out signaling. The molecular basis for inside-out signaling is not yet completely understood. Here, we show that a signaling module comprising the cytosolic adapter proteins ADAP and SKAP55 is involved in TCR-mediated inside-out signaling and, moreover, that the interaction between ADAP and SKAP55 is mandatory for integrin activation. Disruption of the ADAP/SKAP55 module leads to displacement of the small GTPase Rap1 from the plasma membrane without influencing its GTPase activity. These findings suggest that the ADAP/SKAP55 complex serves to recruit activated Rap1 to the plasma membrane. In line with this hypothesis is the finding that membrane targeting of the ADAP/SKAP55 module induces T-cell adhesion in the absence of TCR-mediated stimuli. However, it appears as if the ADAP/SKAP55 module can exert its signaling function outside of the classical raft fraction of the cell membrane.

Characterization of Platelet Lipid Rafts Leads to the Identification of RGS-19, Which Forms a Phosphorylation-Dependent Complex with 14-3-3ζ.

Lipid rafts are specialised membrane regions, rich in dynamic multi-protein complexes that are implicated in the regulation of platelet activation processes, particularly those mediated by GPVI and GPIb/IX/V. To elucidate the role of rafts in platelet function, we carried out a comprehensive characterization of the detergent-insoluble proteins associated with rafts from control and VWF-activated platelets using liquid chromatography coupled to tandem mass spectrometry. Over 160 proteins were identified including 72 proteins unique to platelet rafts and several novel platelet signaling and vesicle transport proteins e.g., rab 5, rab 8 and syntaxin 11. Indeed, the high level of signaling and trafficking proteins identified implicates rafts as concentrating platforms for the critical platelet functions of activation and secretion. Moreover, activation through GPIb-IX-V resulted in the translocation of many proteins into or out of rafts. Proteins recruited into rafts upon VWF-activation included GPIbαand regulator of G-protein signalling-19 (RGS-19). We confirmed that the novel platelet protein, RGS-19, upon VWF activation resides exclusively within the raft fraction and using confocal microscopy, RGS-19 co-localised with GPIbα and the raft-marker flottilin, to both the plasma and vesicle membranes of VWF-activated platelets. The phosphorylation-dependent interaction of RGS proteins with 14-3-3 leads to the inhibition of their GTPase-activating protein (GAP) activity. Aligning RGS-19 to other members of the RGS family, we identified that it contained a novel 14-3-3 binding motif. Immunoprecipitation with an antibody to 14-3-3ζ demonstrated that RGS-19 and GPIbα co-immunoprecipitated with 14-3-3ζ in VWF-activated rafts. Furthermore, we demonstrated that acid phosphatase treatment could significantly reduce the co-immunoprecipitation of RGS-19 with 14-3-3ζ from activated rafts. Thus, within 15 sec of VWF activation we are suggesting maximal suppression of the GAP activity of RGS-19 in rafts through a phosphorylation-dependent association with 14-3-3ζ. Our results provide the basis for a new hypothesis concerning how RGS proteins might govern signaling within platelet rafts.

Binding and Internalization of Clostridium perfringens Iota-Toxin in Lipid Rafts

ABSTRACT Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib). The oligomer of Ib formed in membranes induces endocytosis. We examined the binding and internalization of Ib by using Cy3-labeled Ib. Labeled Ib was retained at the membranes of MDCK cells for 60 min of incubation at 37°C, and later it was detected in cytoplasmic vesicles. To determine whether Ib associates with lipid rafts, we incubated MDCK cells with Ib at 4 or 37°C and fractionated the Triton-insoluble membranes. An Ib complex of 500 kDa was localized at 37°C to the insoluble fractions that fulfilled the criteria of lipid rafts, but it did not form at 4°C. The amount of complex in the raft fraction reached a maximum after 60 min of incubation at 37°C. When the cells that were preincubated with Ib at 4°C were incubated at 37°C, the complex was detected in the raft fraction. The treatment of MDCK cells with methyl-β-cyclodextrin reduced the localization of the Ib complex to the rafts and the rounding of the cells induced by Ia plus Ib. When 125I-labeled Ia was incubated with the cells in the presence of Ib at 37°C, it was localized in the raft fraction. Surface plasmon resonance analysis revealed that Ia binds to the oligomer of Ib. We conclude that Ib binds to a receptor in membranes and then moves to rafts and that Ia bound to the oligomer of Ib formed in the rafts is internalized.