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The lipid turnover at RAMPs is slower than that of the PM. A–D) MDCK cells labeled with Di4A and subjected to FRAP for 220 s at the RAMP and at the PM. A) Representative example of recovery of Di4A fluorescence after photobleaching. B) Percentage of the immobile fraction at RAMPs and PM (10 cells were analyzed). C) Representative images of pre‐bleached, bleached, and post‐bleached RAMP. After 3 h, the cells were re‐stained with Di4A to visualize the bleached RAMP lipids. Cells with central RAMPs were used to facilitate the localization of the structure after photobleaching. Only the cells that maintained the patch during the procedure were analyzed (C). D) Relative fluorescent intensity at the indicated steps of the process. Data shown are mean ±SD. Dashed lines indicate the cell contour. Scale bars: 5 μm and 2 μm for insets.
Source publication
Lipid liquid–liquid immiscibility and its consequent lateral heterogeneity have been observed under thermodynamic equilibrium in model and native membranes. However, cholesterol‐rich membrane domains, sometimes referred to as lipid rafts, are difficult to observe spatiotemporally in live cells. Despite their importance in many biological processes,...
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Citations
... Emission signals were collected with two photomultiplier tubes (500-580, 620-750) and the pinhole was set to one Airy unit. Optimized acquisition was performed to retrieve membrane diffusion values as described (33,34). Images of 256 × 256 pixels at 8-bit depth were collected using 80.4 nm pixel size and 4 ms dwell time, for 200 consecutive frames. ...
... We thus used RICS with the Di-4-ANEPPDHQ lipid probe to evaluate membrane fluidity by means of membrane diffusion. This lipophilic dye is also a reporter for lipid lateral packing (33,34). Jurkat cells (Figures 4A, C) and T cell blasts (Figures 4B, D) untreated or treated with bSMase (0.5 U/ml, 3 hours, 37°C, 5% CO 2 ) were labeled with Di-4-ANEPPDHQ and RICS was performed after confocal microscopy. ...
Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-particle tracking analysis of diffusion in the plasma membrane to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with bacterial sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.
... Following abscission of MDCK cells, MBRs move along the outside of the apical membrane toward the centrosome at the cell apex [13]. It was proposed that the MBR delivers a special membrane patch to the centrosome, and once this interaction happens, the cell begins to grow its cilium [86,87]. Physically or genetically removing MBRs from the surface reduced the percentage of the MDCK cells developing a primary cilium [13,79]. ...
Purpose of Review
How stem cells balance proliferation with differentiation, giving rise to specific daughter cells during development to build an embryo or tissue, remains an open question. Here, we discuss recent evidence that cytokinetic abscission regulation in stem cells, particularly neural stem cells (NSCs), is part of the answer. Abscission is a multi-step process mediated by the midbody, a microtubule-based structure formed in the intercellular bridge between daughter cells after mitosis.
Recent Findings
Human mutations and mouse knockouts in abscission genes reveal that subtle disruptions of NSC abscission can cause brain malformations. Experiments in several epithelial systems have shown that midbodies serve as scaffolds for apical junction proteins and are positioned near apical membrane fate determinants. Abscission timing is tightly controlled and developmentally regulated in stem cells, with delayed abscission in early embryos and faster abscission later. Midbody remnants (MBRs) contain over 400 proteins and may influence polarity, fate, and ciliogenesis.
Summary
As NSCs and other stem cells build tissues, they tightly regulate three aspects of abscission: midbody positioning, duration, and MBR handling. Midbody positioning and remnants establish or maintain cell polarity. MBRs are deposited on the apical membranes of epithelia, can be released or internalized by surrounding cells, and may sequester fate determinants or transfer information between cells. Work in cell lines and simpler systems has shown multiple roles for abscission regulation influencing stem cell polarity, potency, and daughter fates during development. Elucidating how the abscission process influences cell fate and tissue growth is important for our continued understanding of brain development and stem cell biology.
... STED-RICS has recently be used to study the biogenesis of ciliary membrane, showing that the midbody supply condensed membrane to the centrosome to generate a ciliary membrane. Thus, STED-RICS enables simultaneous tracking of membrane lateral packing and dynamics at the nanoscale in living cells over time with resolutions down to 100 nm [137]. ...
Cellular membranes are fundamental building blocks regulating an extensive repertoire of biological functions. These structures contain lipids and membrane proteins that are known to laterally self-aggregate in the plane of the membrane, forming defined membrane nanoscale domains essential for protein activity. Membrane rafts are described as heterogeneous, dynamic, and short-lived cholesterol- and sphingolipid-enriched membrane nanodomains (10–200 nm) induced by lipid-protein and lipid-lipid interactions. Those membrane nanodomains have been extensively characterized using model membranes and in silico methods. However, despite the development of advanced fluorescence microscopy techniques, undoubted nanoscale visualization by imaging techniques of membrane rafts in the membrane of unperturbed living cells is still uncompleted, increasing the skepticism about their existence. Here, we broadly review recent biochemical and microscopy techniques used to investigate membrane rafts in living cells and we enumerate persistent open questions to answer before unlocking the mystery of membrane rafts in living cells.
... To simultaneously measure membrane dynamics and packing (57,58), we combined STED-FCS acquisitions with GP analysis. For that, we prepared SLBs containing increasing amounts of cholesterol and measured the intensity fluctuations of NR4A in two different spectral channels (580-625 and 650-700 nm) at different resolutions (i.e., STED laser powers). ...
Understanding the plasma membrane nanoscale organization and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution that permit for long acquisition times and allow for the quantification of membrane biophysical properties, such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time three-dimensional STED recordings of bleb formation and lipid exchange during membrane fusion as well as by STED-fluorescence correlation spectroscopy experiments for the simultaneous quantification of membrane dynamics and lipid packing that correlate in model and live-cell membranes.
... The components of the machinery for ciliary growth are recruited normally to the centrosome zone under those conditions but are unable to elongate the primary cilium correctly and, consequently, the remaining cilia are stunted [46]. This effect of MAL silencing seems to be due to deficient condensation of the centrosome-associated membranes needed to build the ciliary membrane [88,89]. The overexpression of tagged MAL in MDCK cells and in transgenic mice also gives rise to fewer, shortened cilia. ...
The MAL gene encodes a 17-kDa protein containing four putative transmembrane segments whose expression is restricted to human T cells, polarized epithelial cells and myelin-forming cells. The MAL protein has two unusual biochemical features. First, it has lipid-like properties that qualify it as a member of the group of proteolipid proteins. Second, it partitions selectively into detergent-insoluble membranes, which are known to be enriched in condensed cell membranes, consistent with MAL being distributed in highly ordered membranes in the cell. Since its original description more than thirty years ago, a large body of evidence has accumulated supporting a role of MAL in specialized membranes in all the cell types in which it is expressed. Here, we review the structure, expression and biochemical characteristics of MAL, and discuss the association of MAL with raft membranes and the function of MAL in polarized epithelial cells, T lymphocytes, and myelin-forming cells. The evidence that MAL is a putative receptor of the epsilon toxin of Clostridium perfringens, the expression of MAL in lymphomas, the hypermethylation of the MAL gene and subsequent loss of MAL expression in carcinomas are also presented. We propose a model of MAL as the organizer of specialized condensed membranes to make them functional, discuss the role of MAL as a tumor suppressor in carcinomas, consider its potential use as a cancer biomarker, and summarize the directions for future research.
... Our group reported that the midbody remnant (MBR), a structure resulting from the cleavage of the intercellular bridge formed during cytokinesis of MDCK cells moves along the apical membrane to meet the centrosome and enables primary cilium assembly 46 . More recently 39 , we have further investigated the role of the MBR and associated membranes in the assembly of the primary cilium specifically focusing on the role lipid immiscibility and collective dynamic self-assembly may play in ciliary membrane biogenesis. To this end, we developed a method that simultaneously resolves the spatiotemporal distribution of transient nanoscopic membrane subdomains and quantifies its molecular dynamics and lateral packing properties over time. ...
... Interestingly, by monitoring raft-like membrane dynamics in live cells using state-of-the-art super-resolution and fluorescence correlation spectroscopy-based techniques, we found out a membrane refinement process. A remnant-associated membrane patch (RAMP) spatiotemporally remodelled becoming enriched in more condensed membranes to form a centrosome-associated membrane patch (CAMP), which is the precursor of the ciliary membrane 39 . Employing our novel method based on laser interleaved confocal raster image correlation spectroscopy (RICS) and STED-RICS (LICSR) 39 , we unveiled multidimensionally and spatiotemporally lipid lateral packing and dynamics simultaneously. ...
... A remnant-associated membrane patch (RAMP) spatiotemporally remodelled becoming enriched in more condensed membranes to form a centrosome-associated membrane patch (CAMP), which is the precursor of the ciliary membrane 39 . Employing our novel method based on laser interleaved confocal raster image correlation spectroscopy (RICS) and STED-RICS (LICSR) 39 , we unveiled multidimensionally and spatiotemporally lipid lateral packing and dynamics simultaneously. Strikingly, the refinement process before ciliogenesis showed micro-and nano-scale ordered lipid domains collectively organizing seemingly from hot spots of higher lipid condensation; however, we were unable to measure this collective organization in detail. ...
The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.
... Lipid liquid-liquid immiscibility is on the basis of the formation of these specialized lipid environments (Dietrich et al., 2001;Bernardino de la Serna et al., 2004;Veatch and Keller, 2005). Using two different environment-sensitive membrane probes, it was observed that peripheral MBRs are surrounded by a patch of compact membranes, which was named the remnant-associated membrane patch (RAMP) (Bernabé-Rubio et al., 2021). The RAMP is acquired by the MBR soon after completion of cytokinesis, and transits together with the MBR from the periphery to the center of the apical surface (Bernabé-Rubio et al., 2021). ...
... Using two different environment-sensitive membrane probes, it was observed that peripheral MBRs are surrounded by a patch of compact membranes, which was named the remnant-associated membrane patch (RAMP) (Bernabé-Rubio et al., 2021). The RAMP is acquired by the MBR soon after completion of cytokinesis, and transits together with the MBR from the periphery to the center of the apical surface (Bernabé-Rubio et al., 2021). Although the source of the material of the RAMP is not clear, it could be supplied by vesicles present in the MB before abscission (Fielding et al., 2005;Kouranti et al., 2006;Goss and Toomre, 2008;Schiel et al., 2012). ...
... Although the source of the material of the RAMP is not clear, it could be supplied by vesicles present in the MB before abscission (Fielding et al., 2005;Kouranti et al., 2006;Goss and Toomre, 2008;Schiel et al., 2012). It is of particular note that the RAMP has a degree of membrane condensation, lipid diffusion and molecular lateral mobility similar to that of the ciliary membrane, consistent with a precursor-product relationship between the two membranes (Bernabé-Rubio et al., 2021). A significant observation in living cells was that, once the MBR is in the proximity of the centrosome at the center of the apical surface, the RAMP appears to split into two patches, one of which maintains its association with the MBR, and the other, called the centrosome-associated membrane patch (CAMP), localizes to the plasma membrane zone above the centrosome. ...
Primary cilia are solitary, microtubule-based protrusions surrounded by a ciliary membrane equipped with selected receptors that orchestrate important signaling pathways that control cell growth, differentiation, development and homeostasis. Depending on the cell type, primary cilium assembly takes place intracellularly or at the cell surface. The intracellular route has been the focus of research on primary cilium biogenesis, whereas the route that occurs at the cell surface, which we call the “alternative” route, has been much less thoroughly characterized. In this review, based on recent experimental evidence, we present a model of primary ciliogenesis by the alternative route in which the remnant of the midbody generated upon cytokinesis acquires compact membranes, that are involved in compartmentalization of biological membranes. The midbody remnant delivers part of those membranes to the centrosome in order to assemble the ciliary membrane, thereby licensing primary cilium formation. The midbody remnant's involvement in primary cilium formation, the regulation of its inheritance by the ESCRT machinery, and the assembly of the ciliary membrane from the membranes originally associated with the remnant are discussed in the context of the literature concerning the ciliary membrane, the emerging roles of the midbody remnant, the regulation of cytokinesis, and the role of membrane compartmentalization. We also present a model of cilium emergence during evolution, and summarize the directions for future research.
(EUS)
Jendartearen elikadura beharrizanak asetze aldera, eta egungo aldaketa globalaren aurrean, agroekosistemen kalitatea eta emankortasuna bermatu nahi badira, lurzoruko mikroorganismoen biodibertsitatea indartzea funtsezkoa da; tartean, landareekin sinbiosi mutualistak eratzen dituzten onddo mikorriziko arbuskularrena. Nekazaritza ereduak baduenez aurreko guztian eragina, eta agrobiodibertsitatearen kontra, oro har, geroz eta labore-barietate gutxiago erabiltzen denez, nekazaritza konbentzionalak eta ekologikoak Arabako lautadan ekoizten diren lau barietateren mikorrizazio mailan duten eragina aztertu da. Nekazaritza ekologikoak dibertsitatea sustatzen duela ikusi da, eta, ondorioz, mikorrizazioa areagotzeaz gain, barietate batzuen mikorrizek egoera funtzional hobea erakutsi dute.
(EN)
Strengthening the biodiversity of soil microorganisms, including arbuscular mycorrhizal fungi (which form mutualistic symbioses with plants), is essential to meet the food needs of society and to ensure the quality and fertility of agroecosystems given the current global change. Since the agricultural model affects all of the above, and contrary to agrobiodiversity, the use of crop varieties, in general, is decreasing, the influence of conventional and organic farming on the degree of mycorrhization of four varieties produced in the Llanada Alavesa has been analysed. Organic agriculture promoted diversity, which not only increased mycorrhization, but also the mycorrhizae of some varieties showed a better functional state.
Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal‐based cholesterol probe (chol‐N3) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol‐N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live‐cell super‐resolution microscopy, chol‐N3 demonstrates the existence of cholesterol‐rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.
