The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution, and tension. ...Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined the fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as the degree of lipid saturation, cholesterol content, double bond position and configuration, and phospholipid headgroup. As a comparison, we investigated the lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g., cholesterol). While the lifetime of Flipper is particularly sensitive to the membrane cholesterol content, the NR12S and NR12A lifetimes are moderately sensitive to both the cholesterol content and lipid acyl chains. Moreover, all of the probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission wavelength dependency results in varying lifetime resolutions at different spectral regions, which are highly relevant for FLIM data acquisition. Our data provide valuable insights on how to perform FLIM with these probes and highlight both their potential and limitations.
Abstract
Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these ...interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
65.
Nanoscale dynamics of cholesterol in the cell membrane Pinkwart, Kerstin; Schneider, Falk; Lukoseviciute, Martyna ...
Journal of biological chemistry/The Journal of biological chemistry,
08/2019, Letnik:
294, Številka:
34
Journal Article
Recenzirano
Odprti dostop
Cholesterol constitutes ∼30–40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping ...molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane.
Lipid transfer from lipoprotein particles to cells is essential for lipid homeostasis. High-density lipoprotein (HDL) particles are mainly captured by cell membrane-associated scavenger receptor ...class B type 1 (SR-B1) from the bloodstream, while low-density and very-low-density lipoprotein (LDL and VLDL, respectively) particles are mostly taken up by receptor-mediated endocytosis. However, the role of the target lipid membrane itself in the transfer process has been largely neglected so far. Here, we study how lipoprotein particles (HDL, LDL, and VLDL) interact with synthetic lipid bilayers and cell-derived membranes and transfer their cargo subsequently. Employing cryo-electron microscopy, spectral imaging, and fluorescence (cross) correlation spectroscopy allowed us to observe integration of all major types of lipoprotein particles into the membrane and delivery of their cargo in a receptor-independent manner. Importantly, the biophysical properties of the target cell membranes change upon delivery of cargo. The concept of receptor-independent interaction of lipoprotein particles with membranes helps us to better understand lipoprotein particle biology and can be exploited for novel treatments of dyslipidemia diseases.
Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons ...(IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG‐I‐like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG‐I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR.
Hertzog et al. report that ZIKV infection generates RIG‐I and MDA5‐stimulatory RNAs and induces type I interferon (IFN). Viral RNA binds directly to RIG‐I while the viral NS5 protein blocks induction of type I IFN. NS5 also blocks type I IFN signaling by antagonising STAT1 phosphorylation and depletion of STAT2.
Lipid membranes in cells fulfill a wide variety of functions essential to life. To dissect the exact roles of membranes and their components, experimental models capable of recapitulating their ...composition and biophysical properties are required. Here, we summarize and discuss different lipid bilayer models used for studying immune cell signaling with a specific focus on the immune synapse.
Understanding the broad variety of functions encoded in cellular membranes requires experimental systems mimicking both their biochemical composition and biophysical properties. Here, we review the interplay between membrane components and the physical properties of the plasma membrane worth considering for biomimetic studies. Later, we discuss the main advantages and caveats of different model membrane systems. We further expand on how the use of model systems has contributed to the understanding of immune cell signaling, with a specific focus on the immunological synapse. We discuss the similarities of immune synapses observed for innate and adaptive immune cells and focus on the physical principles underlying these similarities.
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