RESUMO
Several cell-surface lipid-tethered proteins exhibit a concentration-independent, cholesterol-sensitive organization of nanoscale clusters and monomers. To understand the mechanism of formation of these clusters, we investigate the spatial distribution and steady-state dynamics of fluorescently tagged GPI-anchored protein nanoclusters using high-spatial and temporal resolution FRET microscopy. These studies reveal a nonrandom spatial distribution of nanoclusters, concentrated in optically resolvable domains. Monitoring the dynamics of recovery of fluorescence intensity and anisotropy, we find that nanoclusters are immobile, and the dynamics of interconversion between nanoclusters and monomers, over a range of temperatures, is spatially heterogeneous and non-Arrhenius, with a sharp crossover coinciding with a reduction in the activity of cortical actin. Cholesterol depletion perturbs cortical actin and the spatial scale and interconversion dynamics of nanoclusters. Direct perturbations of cortical actin activity also affect the construction, dynamics, and spatial organization of nanoclusters. These results suggest a unique mechanism of complexation of cell-surface molecules regulated by cortical actin activity.
Assuntos
Actinas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas/metabolismo , Animais , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Microdomínios da Membrana/metabolismo , Microscopia Confocal , Miosinas/metabolismoRESUMO
Detection of microbial constituents by membrane associated and cytoplasmic pattern recognition receptors is the essence of innate immunity, leading to activation of protective host responses. However, it is still unclear how immune cells specifically respond to pathogenic bacteria. Using virulent and nonvirulent strains of Bacillus anthracis, we have shown that secretion of ATP by infected macrophages and the sequential activation of the P2X7 purinergic receptor and nucleotide binding oligomerization domain (NOD)-like receptors are critical for IL-1-dependent host protection from virulent B. anthracis. Importantly, lethal toxin produced by virulent B. anthracis blocked activation of protein kinases, p38 MAPK and AKT, resulting in opening of a connexin ATP release channel and induction of macrophage death. Prevention of cell death or ATP release through constitutive p38 or AKT activation interfered with inflammasome activation and IL-1ß production, thereby compromising antimicrobial immunity.
Assuntos
Antraz/imunologia , Antígenos de Bactérias/metabolismo , Bacillus anthracis/imunologia , Toxinas Bacterianas/metabolismo , Inflamassomos/metabolismo , Macrófagos Peritoneais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antraz/microbiologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Bacillus anthracis/genética , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Células Cultivadas , Conexina 43/metabolismo , Imunidade Inata/genética , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Proteína Oncogênica v-akt/antagonistas & inibidores , Receptores Purinérgicos P2X7/metabolismo , Virulência/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Multiple lipid and protein components of the plasma membrane of a living cell are organized, both compositionally and functionally, at different spatial and temporal scales. For instance, Rab protein domains in membranes the clathrin-coated pit, or the immunological synapse are exquisite examples of functional compartmentalization in cell membranes. These assemblies consist in part of nanoscale complexes of lipids and proteins and are necessary to facilitate some specific sorting and signaling functions. It is evident that cellular functions require a regulated spatiotemporal organization of components at the nanoscale, often comprising of countable number of molecular species. Here, we describe multiple homo-FRET-based imaging methods that provide information about nanoscale interactions between fluorescently tagged molecules in live cells, at optically resolved spatial resolution.