RESUMEN
The pathways involved in the intracellular transport and distribution of lipids in general, and sterols in particular, are poorly understood. Cholesterol plays a major role in modulating membrane bilayer structure and important cellular functions, including signal transduction and membrane trafficking. Both the overall cholesterol content of a cell, as well as its distribution in specific organellar membranes are stringently regulated. Several diseases, many of which are incurable at present, have been characterized as results of impaired cholesterol transport and/or storage in the cells. Despite their importance, many fundamental aspects of intracellular sterol transport and distribution are not well understood. For instance, the relative roles of vesicular and non-vesicular transport of cholesterol have not yet been fully determined, nor are the non-vesicular transport mechanisms well characterized. Similarly, whether cholesterol is asymmetrically distributed between the two leaflets of biological membranes, and if so, how this asymmetry is maintained, is poorly understood. In this review, we present a summary of the current understanding of these aspects of intracellular trafficking and distribution of lipids, and more specifically, of sterols.
Asunto(s)
Líquido Intracelular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Esteroles/química , Esteroles/metabolismo , Animales , Transporte Biológico Activo , Humanos , Líquido Intracelular/química , Membrana Dobles de Lípidos/química , Lípidos/químicaRESUMEN
On treatment with chemoattractant, the neutrophil plasma membrane becomes organized into detergent-resistant membrane domains (DRMs), the distribution of which is intimately correlated with cell polarization. Plasma membrane at the front of polarized cells is susceptible to extraction by cold Triton X-100, whereas membrane at the rear is resistant to extraction. After cold Triton X-100 extraction, DRM components, including the transmembrane proteins CD44 and CD43, the GPI-linked CD16, and the lipid analog, DiIC(16), are retained within uropods and cell bodies. Furthermore, CD44 and CD43 interact concomitantly with DRMs and with the F-actin cytoskeleton, suggesting a mechanism for the formation and stabilization of DRMs. By tracking the distribution of DRMs during polarization, we demonstrate that DRMs progress from a uniform distribution in unstimulated cells to small, discrete patches immediately after activation. Within 1 min, DRMs form a large cap comprising the cell body and uropod. This process is dependent on myosin in that an inhibitor of myosin light chain kinase can arrest DRM reorganization and cell polarization. Colabeling DRMs and F-actin revealed a correlation between DRM distribution and F-actin remodeling, suggesting that plasma membrane organization may orient signaling events that control cytoskeletal rearrangements and, consequently, cell polarity.
Asunto(s)
Antígenos CD , Membrana Celular/fisiología , Citoesqueleto/metabolismo , Neutrófilos/fisiología , Actinas/metabolismo , Carbocianinas/metabolismo , Membrana Celular/metabolismo , Polaridad Celular , Humanos , Receptores de Hialuranos/metabolismo , Leucosialina , Miosinas/metabolismo , Neutrófilos/metabolismo , Sialoglicoproteínas/metabolismoRESUMEN
Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine the effect of modulating cellular cholesterol on domain formation. We show that lowering cholesterol levels induces formation of visible micrometer-scale domains in the plasma membrane of several mammalian cell types with complementary distributions of fluorescent lipid analogs with preferences for fluid or ordered domains. A uniform distribution is restored by cholesterol repletion. Unexpectedly, cholesterol depletion does not visibly alter the distribution of a crosslinked or uncrosslinked glycosylphosphatidylinositol-anchored protein (the folate receptor). We also examined the effect of varying cholesterol content on the cold Triton X-100 solubility of several membrane constituents. Although a cholesterol analog, dehydroergosterol, and a glycosylphosphatidylinositol-anchored protein are largely retained after extraction, a lipid analog with saturated 16-carbon acyl chains is largely removed when the cellular cholesterol level is lowered. This result indicates that after cholesterol depletion molecules in the more ordered domains can be extracted differentially by cold nonionic detergents.
Asunto(s)
Colesterol/metabolismo , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Glicosilfosfatidilinositoles/metabolismo , Fluidez de la Membrana , Microscopía Confocal , Microscopía FluorescenteRESUMEN
Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.
Asunto(s)
Vesículas Cubiertas por Clatrina/metabolismo , Clatrina/antagonistas & inhibidores , Clatrina/metabolismo , Endosomas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Células CHO , Cricetinae , Endocitosis , Genes Dominantes/genética , Células HeLa , Humanos , Lipoproteínas LDL/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Transporte de Proteínas , Receptores de Superficie Celular/antagonistas & inhibidores , Transferrina/metabolismo , Proteínas de Transporte VesicularAsunto(s)
Movimiento Celular , Polaridad Celular , Microdominios de Membrana/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Citoesqueleto/fisiología , Detergentes/farmacología , Gangliósido G(M1)/fisiología , Gangliósido G(M3)/fisiología , Lípidos de la Membrana/química , Microdominios de Membrana/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Wild-type Legionella pneumophila grows in human macrophages within a replicative phagosome, avoiding lysosomal fusion, while nonreplicative mutants are killed in lysosomes. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocks phagocytosis of an avirulent mutant, but not of wild-type L. pneumophila, without affecting membrane ruffling and actin polymerization. These results show that wild-type and mutant Legionella strains use different entry pathways. They suggest that PI3Ks are involved in phagocytosis of an avirulent L. pneumophila mutant and regulate the ability of microorganisms to generate a replicative phagosome.
Asunto(s)
Androstadienos/farmacología , Legionella pneumophila/inmunología , Fagocitosis/inmunología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Actinas/metabolismo , Humanos , Fagocitosis/efectos de los fármacos , Células U937 , WortmaninaRESUMEN
A key cellular event in atherogenesis is the interaction of macrophages with lipoproteins in the subendothelium. In vivo, these lipoproteins are bound to matrix and often aggregated, yet most cell-culture experiments explore these events using soluble monomeric lipoproteins. We hypothesized that the internalization and degradation of matrix-retained and aggregated low density lipoprotein (LDL) by macrophages may involve the actin-myosin cytoskeleton in a manner that distinguishes this process from the endocytosis of soluble LDL. To explore these ideas, we plated macrophages on sphingomyelinase-aggregated LDL bound to smooth muscle cell-derived matrix in the presence of lipoprotein lipase. The macrophages internalized and degraded the LDL, which was mediated partially by the LDL receptor-related protein. Cytochalasin D and latrunculin A, which block actin polymerization, markedly inhibited the uptake and degradation of matrix-retained LDL but not soluble LDL. Inhibition of Rho family GTPases by Clostridium difficile toxin B blocked the degradation of matrix-retained and aggregated LDL by >90% without any inhibition of soluble LDL degradation. However, specific inhibition of Rho had no effect, suggesting the importance of Rac1 and Cdc42. Degradation of matrix-retained, but not soluble, LDL was also blocked by inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, and myosin ATPase. These findings define fundamental cytoskeletal pathways that may be involved in macrophage foam cell formation in vivo but have been missed by the use of previous cell culture models.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Miosinas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Células Cultivadas , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismoRESUMEN
RME-1 is an Eps15-homology (EH)-domain protein that was identified in a genetic screen for endocytosis genes in Caenorhabditis elegans. When expressed in a CHO cell line, the worm RME-1 protein and a mouse homologue are both associated with the endocytic recycling compartment. Here we show that expression of a dominant-negative construct with a point mutation near the EH domain results in redistribution of the endocytic recycling compartment and slowing down of transferrin receptor recycling. The delivery of a TGN38 chimaeric protein to the trans-Golgi network is also slowed down. The function of Rme-1 in endocytic recycling is evolutionarily conserved in metazoans as shown by the protein's properties in C. elegans.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Unión al Calcio/fisiología , Compartimento Celular/fisiología , Glicoproteínas , Proteínas de la Membrana , Fosfoproteínas/fisiología , Vesículas Transportadoras/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Células CHO , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Cricetinae , Furina , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/metabolismo , Ratones , Mutación , Fosfoproteínas/química , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Subtilisinas/metabolismo , Transferrina/metabolismoRESUMEN
Cholesterol transport is an essential process in all multicellular organisms. In this study we applied two recently developed approaches to investigate the distribution and molecular mechanisms of cholesterol transport in Caenorhabditis elegans. The distribution of cholesterol in living worms was studied by imaging its fluorescent analog, dehydroergosterol, which we applied to the animals by feeding. Dehydroergosterol accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1-L3 larvae. Later, the bulk of dehydroergosterol accumulates in oocytes and spermatozoa. Males display exceptionally strong labeling of spermatids, which suggests a possible role for cholesterol in sperm development. In a complementary approach, we used a photoactivatable cholesterol analog to identify cholesterol-binding proteins in C. elegans. Three major and several minor proteins were found specifically cross-linked to photocholesterol after UV irradiation. The major proteins were identified as vitellogenins. rme-2 mutants, which lack the vitellogenin receptor, fail to accumulate dehydroergosterol in oocytes and embryos and instead accumulate dehydroergosterol in the body cavity along with vitellogenin. Thus, uptake of cholesterol by C. elegans oocytes occurs via an endocytotic pathway involving yolk proteins. The pathway is a likely evolutionary ancestor of mammalian cholesterol transport.
Asunto(s)
Caenorhabditis elegans/metabolismo , Colesterol/metabolismo , Proteínas del Huevo , Espermatozoides/metabolismo , Animales , Evolución Biológica , Transporte Biológico , Sistema Digestivo/metabolismo , Electroforesis en Gel de Poliacrilamida , Endocitosis , Ergosterol/análogos & derivados , Ergosterol/metabolismo , Ergosterol/farmacocinética , Femenino , Masculino , Microscopía Fluorescente , Modelos Químicos , Mutación , Octoxinol , Faringe/metabolismo , Polietilenglicoles/farmacología , Pruebas de Precipitina , Receptores de Superficie Celular/metabolismo , Espermátides/metabolismo , Espermicidas/farmacología , Esteroles/metabolismo , Sacarosa/metabolismo , Rayos Ultravioleta , Vitelogeninas/metabolismoRESUMEN
To characterize the receptors involved in binding fibrillar amyloid A-beta (fA beta), we compared the uptake of fA beta in microglia from wildtype (MSR-A+/+) and MSR-A knockout (MSR-A-/-) mice. On average, there was a 60% reduction in the uptake of Cy3-fA beta in microglia from the MSR-A-/- mice. Cy3-fA beta uptake in the MSR-A-/- mice was still competable by scavenger receptor ligands, including acetylated low-density lipoprotein (Ac-LDL) and fucoidan. This indicates that uptake by MSR-B and/or other MSRs is also involved in the uptake of fA beta by microglia. However, the significant reduction in the uptake of fA beta in the MSR-A-/- microglia suggests that fA beta gets internalized mostly by MSR-As in microglia. Uptake of modified fA beta (ClqA beta) was similar in the MSR-A-/- microglia as in the wildtype indicating that the uptake of the opsonized fA beta is independent of MSR-A.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Microglía/metabolismo , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Animales , Animales Recién Nacidos , Carbocianinas/metabolismo , Separación Celular , Células Cultivadas , Colorantes Fluorescentes/metabolismo , Inmunoglobulina G/metabolismo , Ratones , Ratones Noqueados , Receptores DepuradoresRESUMEN
We have investigated the transport and canalicular enrichment of fluorescent phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and beta-BODIPY-PC. Fluorescent PC was efficiently transported to the biliary canaliculus (BC) and became enriched on the lumenal side of the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recycling compartment (ARC) in polarized HepG2 cells as shown by colocalization with fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respectively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of PC analogs from the canalicular membrane were found. Evidence for nonvesicular transport included enrichment of the PC-analog beta-BODIPY-PC in the BC (t1/2 = 3.54 min) prior to its accumulation in the SAC/ARC (t1/2 = 18.5 min) at 37 degrees C. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton and at 2 degrees C. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in hepatocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.
Asunto(s)
Polaridad Celular , Hepatocitos/citología , Hepatocitos/metabolismo , Fosfatidilcolinas/metabolismo , Vesículas Transportadoras/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/citología , Canalículos Biliares/metabolismo , Transporte Biológico Activo , Compuestos de Boro/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Endocitosis , Endosomas/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Esfingomielinas/metabolismo , Factores de Tiempo , Transferrina/metabolismo , Células Tumorales CultivadasAsunto(s)
Endocitosis/fisiología , Animales , Transporte Biológico , Endosomas/fisiología , Riñón/metabolismoRESUMEN
Acyl coenzyme A:cholesterol O-acyltransferase (ACAT) is the enzyme responsible for cholesterol esterification in macrophages leading to foam cell formation. The determination of its localization is a critical step in understanding its regulation by cholesterol. Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers of the endoplasmic reticulum, but in addition, ACAT was found in an unidentified paranuclear site. In the present study, we further define the localization of paranuclear ACAT. First, we found that ACAT does not colocalize with sorting endosomes or late endosomes labeled with fluorescent alpha(2)-macroglobulin. The paranuclear ACAT is close to the endocytic recycling compartment labeled with fluorescent transferrin. We also show that the paranuclear structure containing ACAT is very close to TGN38, a membrane protein of the trans-Golgi network (TGN), but farther from Gos28, a marker of cis, medial, and trans Golgi. After treatment with nocodazole, the central localization of ACAT did not colocalize with markers of the TGN. These data indicate that a significant fraction of ACAT resides in membranes that may be a subcompartment of the endoplasmic reticulum in proximity to the TGN and the endocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma membrane, esterification of cholesterol in these membranes may play an important role in macrophage foam cell formation during atherogenesis.
Asunto(s)
Acilcoenzima A/metabolismo , Endosomas/enzimología , Aparato de Golgi/enzimología , Esterol O-Aciltransferasa/metabolismo , Acilcoenzima A/análisis , Animales , Especificidad de Anticuerpos , Arteriosclerosis/metabolismo , Transporte Biológico/fisiología , Células CHO , Línea Celular , Cricetinae , Técnica del Anticuerpo Fluorescente , Cobayas , Macrófagos/enzimología , Esterol O-Aciltransferasa/análisis , Esterol O-Aciltransferasa/inmunologíaRESUMEN
Lipids and other membrane constituents recycle between the plasma membrane and intracellular endocytic compartments. In CHO cells, approximately half of the internalized C(6)-NBD-SM, a fluorescent lipid analogue widely used as a membrane maker, recycles via the endocytic recycling compartment with a t(12) of approximately 12 min (Mayor, S., Presley, J. F., and Maxfield, F. R. (1993) J. Cell Biol. 121, 1257-1269). Surprisingly, the rest returns to the plasma membrane very quickly. A detailed kinetic study presented in this paper indicates that after a brief internalization pulse, 42-62% of the internalized C(6)-NBD-SM returns to the plasma membrane with a t(12) of 1-2 min. Similar results are obtained using HEp2 and nonpolarized Madin-Darby canine kidney cells. Using FM dyes of different hydrophobicity, we show that rapid recycling involves passage through an endocytic organelle that was subsequently identified as the sorting endosome by co-localization with internalized transferrin and low density lipoprotein. These results imply that the membrane internalization rate is much higher than previously estimated, with a t(12) as short as 5-10 min. Rapid internalization and recycling would facilitate processes such as nutrient uptake and cholesterol efflux.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Membrana Celular/metabolismo , Endocitosis/fisiología , Lípidos de la Membrana/metabolismo , Esfingomielinas/farmacocinética , 4-Cloro-7-nitrobenzofurazano/farmacocinética , Animales , Células CHO , Línea Celular , Polaridad Celular , Cricetinae , Perros , Endosomas/fisiología , Colorantes Fluorescentes/farmacocinética , Semivida , Humanos , Cinética , Modelos Biológicos , Células Tumorales CultivadasRESUMEN
Microglia are macrophage-like immune system cells found in the brain. They are associated with Alzheimer's Disease plaques, which contain fibrillar beta-amyloid (fAbeta) and other components such as complement proteins. We have shown previously that murine microglia bind and internalize fAbeta microaggregates via the type A scavenger receptor, but degradation of internalized fAbeta is significantly slower than normal degradation. In this study, we compared internalization by microglia of fAbeta microaggregates to that of anti-Abeta-antibody-coated fAbeta (IgG-fAbeta) microaggregates and found that the uptake of the latter is increased by about 1.5-fold versus unmodified fAbeta. The endocytic trafficking of IgG-fAbeta is similar to that of fAbeta microaggregates, following an endosomal/lysosomal pathway. We also compared the internalization of fAbeta microaggregates to that of complement protein, C1q-coated fAbeta microaggregates, and found that the levels of uptake are also increased by about 1.5-fold. Rates of degradation of both types of modified fAbeta microaggregates are unchanged compared with unmodified fAbeta microaggregates. We demonstrated by blocking studies that internalization of IgG-fAbeta is mediated by Fc receptors. These data suggest that, in vivo, several different microglial receptors may play a part in internalizing fAbeta, but the involvement of other receptors may not increase the degradation of fAbeta.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Complemento C1q/metabolismo , Inmunoglobulina G/metabolismo , Microglía/metabolismo , Animales , Animales Recién Nacidos , Endocitosis , Endosomas/metabolismo , Hidrólisis , Cinética , Lisosomas/metabolismo , Ratones , Receptores Fc/metabolismoRESUMEN
Buffering of intracellular Ca2+ transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]i is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]i buffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]i depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.
Asunto(s)
Calcio/fisiología , Movimiento Celular/fisiología , Miosinas/metabolismo , Neutrófilos/fisiología , Seudópodos/fisiología , Actinas/metabolismo , Azepinas/farmacología , Tampones (Química) , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Miosinas/antagonistas & inhibidores , Miosinas/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Seudópodos/efectos de los fármacos , Talina/metabolismoRESUMEN
Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.
Asunto(s)
Estructuras de la Membrana Celular/fisiología , Membrana Celular/ultraestructura , Endocitosis/fisiología , Lípidos de la Membrana/metabolismo , Animales , Transporte Biológico , Polaridad Celular , Fenómenos Químicos , Química Física , Colesterol/metabolismo , Endosomas/metabolismo , Retroalimentación , Humanos , Enlace de Hidrógeno , Membrana Dobles de Lípidos , Lípidos de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Estructura Molecular , Enfermedades de Niemann-Pick/metabolismo , Fagocitosis/fisiología , Transporte de Proteínas , Transducción de Señal , Enfermedad de Tangier/metabolismoRESUMEN
This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. alpha-Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, alpha-actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion. (Blood. 2000;95:2462-2470)
Asunto(s)
Antígenos CD , Movimiento Celular/fisiología , Polaridad Celular/fisiología , Neutrófilos/patología , Neutrófilos/fisiología , Sialoglicoproteínas/fisiología , Adulto , Secuencia de Aminoácidos , Adhesión Celular/fisiología , Humanos , Leucosialina , Datos de Secuencia Molecular , Proteínas RecombinantesRESUMEN
During cell migration, integrin attachments to the substratum provide the means to generate the traction and force necessary to achieve locomotion. Once the cell has moved over these attachments, however, it is equally important that integrins detach from the substratum. The fate of integrins after detachment may include release from the cell, lateral diffusion across the cell surface, or endocytosis and redelivery to the cell surface. Polymorphonuclear neutrophils (PMNs) become stuck on the extracellular matrix proteins fibronectin and vitronectin when their intracellular free calcium concentration ([Ca(++)]i) is buffered. Taking advantage of this feature of PMN migration, we investigated the fate of integrins to differentiate among various models of migration. We demonstrate that alpha5beta1, one of the fibronectin-binding integrins, is responsible for immobilization of [Ca(++)](i)-buffered PMNs on fibronectin. We find that alpha5 and beta1 are in endocytic vesicles in PMNs and that alpha5 colocalizes with a marker for an endocytic recycling compartment. When [Ca(++)](i) is buffered, alpha5 and beta1 become concentrated in clusters in the rear of the adherent cells, suggesting that [Ca(++)](i) transients are required for alpha5beta1 detachment from the substratum. Inhibition of alpha5beta1 detachment by buffering [Ca(++)](i) results in the depletion of alpha5 from both endocytic vesicles and the recycling compartment, providing compelling evidence that integrins are normally recycled by way of endocytosis and intracellular trafficking during cell migration. This model is further refined by our demonstration that the endocytic recycling compartment reorients to retain its localization just behind the leading lamella as PMNs migrate, indicating that membrane recycling during neutrophil migration has directionality. (Blood. 2000;95:2471-2480)
Asunto(s)
Movimiento Celular/fisiología , Neutrófilos/fisiología , Receptores de Fibronectina/fisiología , Compartimento Celular/fisiología , Polaridad Celular/fisiología , Endocitosis/fisiología , Humanos , Neutrófilos/citologíaRESUMEN
A critical event in atherogenesis is the interaction of arterial wall macrophages with subendothelial lipoproteins. Although most studies have investigated this interaction by incubating cultured macrophages with monomeric lipoproteins dissolved in media, arterial wall macrophages encounter lipoproteins that are mostly bound to subendothelial extracellular matrix, and these lipoproteins are often aggregated or fused. Herein, we utilize a specialized cell-culture system to study the initial interaction of macrophages with aggregated low density lipoprotein (LDL) bound to extracellular matrix. The aggregated LDL remains extracellular for a relatively prolonged period of time and becomes lodged in invaginations in the surface of the macrophages. As expected, the degradation of the protein moiety of the LDL was very slow. Remarkably, however, hydrolysis of the cholesteryl ester (CE) moiety of the LDL was 3-7-fold higher than that of the protein moiety, in stark contrast to the situation with receptor-mediated endocytosis of acetyl-LDL. Similar results were obtained using another experimental system in which the degradation of aggregated LDL protein was delayed by LDL methylation rather than by retention on matrix. Additional experiments indicated the following properties of this interaction: (a) LDL-CE hydrolysis is catalyzed by lysosomal acid lipase; (b) neither scavenger receptors nor the LDL receptor appear necessary for the excess LDL-CE hydrolysis; and (c) LDL-CE hydrolysis in this system is resistant to cellular potassium depletion, which further distinguishes this process from receptor-mediated endocytosis. In summary, experimental systems specifically designed to mimic the in vivo interaction of arterial wall macrophages with subendothelial lipoproteins have demonstrated an initial period of prolonged cell-surface contact in which CE hydrolysis exceeds protein degradation.