Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotes

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not ( P  < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways.     

P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.     

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells

Background

Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear.

Methodology/Principal Finding

In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion.

Conclusion/Significance

Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.     

Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages.

Scavenger receptor (SR)-BI mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. In Chinese hamster ovary (CHO) cells, SR-BI is predominantly associated with caveolae which we have recently demonstrated are the initial loci for membrane transfer of HDL cholesteryl esters. Because cholesterol accumulation in macrophages is a critical event in atherogenesis, we investigated the expression of SR-BI and caveolin-1 in several macrophage cell lines. Human THP-1 monocytes were examined before and after differentiation to macrophages by treatment with 200 nm phorbol ester for 72 h. Undifferentiated THP-1 cells expressed caveolin-1 weakly whereas differentiation up-regulated caveolin-1 expression greater than 50-fold. In contrast, both undifferentiated and differentiated THP-1 cells expressed similar levels of SR-BI. Differentiation of THP-1 cells increased the percent of membrane cholesterol associated with caveolae from 12% +/- 1.9% to 38% +/- 3.1%. The increase in caveolin-1 expression was associated with a 2- to 3-fold increase in selective cholesterol ether uptake from HDL. Two mouse macrophage cell lines, J774 and RAW, expressed levels of SR-BI similar to differentiated THP-1 cells but did not express detectable levels of caveolin-1. In comparison to differentiated THP-1 cells, RAW and J774 cells internalized 9- to 10-fold less cholesteryl ester. We conclude that differentiated THP-1 cells express both caveolin-1 and SR-BI and that their co-expression is associated with enhanced selective cholesteryl ester uptake.     

Stage-specific pathways of Leishmania infantum chagasi entry and phagosome maturation in macrophages

The life stages of Leishmania spp. include the infectious promastigote and the replicative intracellular amastigote. Each stage is phagocytosed by macrophages during the parasite life cycle. We previously showed that caveolae, a subset of cholesterol-rich membrane lipid rafts, facilitate uptake and intracellular survival of virulent promastigotes by macrophages, at least in part, by delaying parasitophorous vacuole (PV)-lysosome fusion. We hypothesized that amastigotes and promastigotes would differ in their route of macrophage entry and mechanism of PV maturation. Indeed, transient disruption of macrophage lipid rafts decreased the entry of promastigotes, but not amastigotes, into macrophages (P<0.001). Promastigote-containing PVs were positive for caveolin-1, and co-localized transiently with EEA-1 and Rab5 at 5 minutes. Amastigote-generated PVs lacked caveolin-1 but retained Rab5 and EEA-1 for at least 30 minutes or 2 hours, respectively. Coinciding with their conversion into amastigotes, the number of promastigote PVs positive for LAMP-1 increased from 20% at 1 hour, to 46% by 24 hours, (P<0.001, Chi square). In contrast, more than 80% of amastigote-initiated PVs were LAMP-1+ at both 1 and 24 hours. Furthermore, lipid raft disruption increased LAMP-1 recruitment to promastigote, but not to amastigote-containing compartments. Overall, our data showed that promastigotes enter macrophages through cholesterol-rich domains like caveolae to delay fusion with lysosomes. In contrast, amastigotes enter through a non-caveolae pathway, and their PVs rapidly fuse with late endosomes but prolong their association with early endosome markers. These results suggest a model in which promastigotes and amastigotes use different mechanisms to enter macrophages, modulate the kinetics of phagosome maturation, and facilitate their intracellular survival.     

心肌肌浆网膜含有膜质微区结构

细胞膜质微区(microdomain)是细胞膜上特殊的结构域,在细胞信号转导和物质运输过程中起着非常重要的作用.绝大多数膜质微区来源于全细胞膜,即包括质膜和细胞器膜.最新研究表明细胞器膜如高尔基体膜也有膜质微区,因此分离了猪心肌浆网膜的膜质微区.首先获得了没有质膜污染的猪心肌浆网,用去污剂TritonX-100处理该肌浆网,获得了去污剂不溶的质膜微区(SR-DRM),该微区富集胆固醇和鞘磷脂.质膜微区的标记脂和蛋白质:神经节苷脂GM1和Caveolin-3也在该区富集.同时还研究了心肌浆网Ca2 -ATPase(SERCA2a)的分布,结果表明,相当数量的SERCA2a分布在膜质微区,并且有正常的生理功能.上述研究结果表明,在心肌浆网膜上有膜质微区的存在,进一步证明膜质微区不仅存在于细胞质膜,也普遍存在于细胞器膜.     

Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [(3)H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MbetaCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MbetaCD, MbetaCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.     

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-beta-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.     

Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process

Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.     

Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.     

Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary interstitial edema.

We evaluated in anesthetized rabbits the compositional changes of plasmalemmal lipid microdomains from lung tissue samples after inducing pulmonary interstitial edema (0.5 ml/kg for 3 h, leading to approximately 5% increase in extravascular water). Lipid microdomains (lipid rafts and caveolae) were present in the detergent-resistant fraction (DRF) obtained after discontinuous sucrose density gradient. DRF was enriched in caveolin-1, flotillin, aquaporin-1, GM1, cholesterol, sphingomyelin, and phosphatidylserine, and their contents significantly increased in interstitial edema. The higher DRF content in caveolin, flotillin, and aquaporin-1 and of the ganglioside GM1 suggests an increase both in caveolar domains and in lipid rafts, respectively. Compositional changes could be ascribed to endothelial and epithelial cells that provide most of plasma membrane surface area in the air-blood barrier. Alterations in lipid components in the plasma membrane may reflect rearrangement of floating lipid platforms within the membrane and/or lipid translocation from intracellular stores. Lipid traffic could be stimulated by the marked increase in hydraulic interstitial pressure after initial water accumulation, from approximately -10 to 5 cmH2O, due to the low compliance of the pulmonary tissue, in particular in the basement membranes and in the interfibrillar substance. Compositional changes in lipid microdomains represent a sign of cellular activation and suggest the potential role of mechanotransduction in response to developing interstitial edema.     

Overexpression of caveolin-1 increases plasma membrane fluidity and reduces P-glycoprotein function in Hs578T/Dox

Cholesterol is a key lipid in mediating the enzyme activity or signaling pathway of many proteins on the plasma membrane in mammalian cells. In this report, we demonstrate for the first time that after overexpressing caveolin-1, the plasma membrane cholesterol level was decreased by about 12% and 30% for doxorubicin-sensitive and doxorubicin-resistant Hs578T breast cancer cells, respectively. However, the total cholesterol level in both cell lines was increased by about 10%. By measuring fluorescence and flow cytometry using the fluorescence dyes 1,6-diphenyl-1,3,5-hexatriene and Merocyanine 540, we found that overexpressing caveolin-1 resulted in a similar increase in membrane fluidity and loosening of lipid packing density as cholesterol depletion by 1 mM methyl-beta-cyclodextrin (MbetaCD) or 2-hydroxypropyl-beta-cyclodextrin (HbetaCD). Moreover, we found that the transport activity of P-gp was significantly inhibited by 1 mM MbetaCD or HbetaCD, which is also similar to the inhibitory effect of caveolin-1 overexpression. Our data demonstrate for the first time that the reduction of the plasma membrane cholesterol level induced by overexpressing caveolin-1 may indirectly inhibit P-gp transport activity by increasing plasma membrane fluidity.     

The sonic hedgehog receptor patched associates with caveolin-1 in cholesterol-rich microdomains of the plasma membrane

The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.     

Effects of cholesterol manipulation on the signaling of the human oxytocin receptor

We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRMs; the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin-2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localization and signaling. Our data indicate that cholesterol depletion in HEK-293 cells does not affect the signaling events mediated by the OTRs located outside DRMs. When treated with 2 mM methyl-beta-cyclodextrin (MbetaCD), the receptors remained outside and continued to inhibit cell growth. On the contrary, the MbetaCD treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that 1) once released from DRMs, the receptors fused to caveolin-2 signal exactly as wild-type OTRs and 2) their DRM location is responsible for the specific OTR signaling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signaling, but, after cell treatment with an MbetaCD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signaling.     

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.     

The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania

Leishmania spp. protozoa are obligate intracellular parasites that replicate in macrophages during mammalian infection. Efficient phagocytosis and survival in macrophages are important determinants of parasite virulence. Macrophage lines differ dramatically in their ability to sustain intracellular Leishmania infantum chagasi (Lic). We report that the U937 monocytic cell line supported the intracellular replication and cell-to-cell spread of Lic during 72?h after parasite addition, whereas primary human monocyte-derived macrophages (MDMs) did not. Electron microscopy and live cell imaging illustrated that Lic promastigotes anchored to MDMs via their anterior ends and were engulfed through symmetrical pseudopods. In contrast, U937 cells bound Lic in diverse orientations, and extended membrane lamellae to reorient and internalize parasites through coiling phagocytosis. Lic associated tightly with the parasitophorous vacuole (PV) membrane in both cell types. PVs fused with LAMP-1-expressing compartments 24?h after phagocytosis by MDMs, whereas U937 cell PVs remained LAMP-1 negative. The expression of one phagocytic receptor (CR3) was higher in MDMs than U937 cells, leading us to speculate that parasite uptake proceeds through dissimilar pathways between these cells. We hypothesize that the mechanism of phagocytosis differs between primary versus immortalized human macrophage cells, with corresponding differences in the subsequent intracellular fate of the parasite.     

Role of cholesterol in human immunodeficiency virus type 1 envelope protein-mediated fusion with host cells

In this study we examined the effects of target membrane cholesterol depletion and cytoskeletal changes on human immunodeficiency virus type 1 (HIV-1) Env-mediated membrane fusion by dye redistribution assays. We found that treatment of peripheral blood lymphocytes (PBL) with methyl-beta-cyclodextrin (MbetaCD) or cytochalasin reduced their susceptibility to membrane fusion with cells expressing HIV-1 Env that utilize CXCR4 or CCR5. However, treatment of human osteosarcoma (HOS) cells expressing high levels of CD4 and coreceptors with these agents did not affect their susceptibility to HIV-1 Env-mediated membrane fusion. Removal of cholesterol inhibited stromal cell-derived factor-1alpha- and macrophage inflammatory protein 1beta-induced chemotaxis of both PBL and HOS cells expressing CD4 and coreceptors. The fusion activity as well as the chemotactic activity of PBL was recovered by adding back cholesterol to these cells. Confocal laser scanning microscopy analysis indicated that treatment of lymphocytes with MbetaCD reduced the colocalization of CD4 or of CXCR4 with actin presumably in microvilli. These findings indicate that, although cholesterol is not required for HIV-1 Env-mediated membrane fusion per se, its depletion from cells with relatively low coreceptor densities reduces the capacity of HIV-1 Env to engage coreceptor clusters required to trigger fusion. Furthermore, our results suggest that coreceptor clustering may occur in microvilli that are supported by actin polymerization.     

Recruitment of protein phosphatase 2A to dorsal ruffles by platelet-derived growth factor in smooth muscle cells: Dephosphorylation of Hsp27

In this study, we investigated the mechanism underlying Hsp27 dephosphorylation in smooth muscle cells. We found that protein phosphatase 2A (PP2A) dephosphorylates Hsp27. In addition, Hsp27 dephosphorylation was regulated by membrane cholesterol content. We showed that PDGF induced a three-fold increase in the proportion of PP2A activity regulated by cholesterol in the Triton-insoluble fraction of cell lysates. Moreover, cholesterol depletion decreased the amount of PP2A recovered in Triton-insoluble fraction. Thus, PDGF might regulate a small pool of PP2A associated with lipid rafts. Isolation of detergent-resistant membrane fragments by Optiprep-gradient density indicated that this pool of PP2A was not associated with caveolae, but was recovered in a higher density fraction (DRM-H) with ganglioside GM1, α-actinin, Hsp27 and p34, a component of Arp2/3 complex. These proteins were also present in dorsal ruffles containing GM1 but not caveolin-1. Phosphorylated Hsp27 levels detected in dorsal ruffles were variable. Cholesterol depletion, which inhibits dorsal ruffle formation, decreased PP2A levels and increased the Hsp27-P to Hsp27 ratio in DRM-H. These findings suggest that Hsp27 is dephosphorylated by PP2A in dorsal ruffles, in non-caveolar lipid raft microdomains. However, similarly to p34, non-phosphorylated Hsp27 is associated to non-raft membrane domains at the leading edge of lamellipodia.     

Participation of caveolae in beta1 integrin-mediated mechanotransduction

We previously reported that caveolin-1 is a key component in a beta1 integrin-dependent mechanotransduction pathway suggesting that caveolae organelles and integrins are functionally linked in their mechanotransduction properties. Here, we exposed BAEC monolayers to shear stress then isolated caveolae vesicles form the plasma membrane. While little beta1 integrin was detected in caveolae derived from cells kept in static culture, shear stress induced beta1 integrin transposition to the caveolae. To evaluate the significance of shear-induced beta1 integrin localization to caveolae, cells were pretreated with cholesterol sequestering compounds or caveolin-1 siRNA to disrupt caveolae structural domains. Cholesterol depletion attenuated integrin-dependent caveolin-1 phosphorylation, Src activation and Csk association with beta1 integrin. Reduction of both caveolin-1 protein and membrane cholesterol inhibited downstream shear-induced, integrin-dependent phosphorylation of myosin light chain. Taken together with our previous findings, the data supports the concept that beta1 integrin-mediated mechanotransduction is mediated by caveolae domains.