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Receptor tyrosine kinases of the Trk receptor family regulate various cellular processes, such as proliferation and differentiation, through several cellular signal cascades.

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes

TrkA is specifically activated by NGF reviewed in [ , ]. Downstream signaling by TrkA seems to be dependent on the lipid raft association of TrkA. See Figure 5. A Insulin is the major hormone controlling essential metabolic processes such as glucose and lipid turnover. Insulin binding to the extracellular subunits of the insulin receptor tyrosine kinase IR induces a conformational change and results in the autophosphorylation of several tyrosine residues in the cytoplasmic part of the IR.

In adipocytes, the constitutive complex of the E3 ubiquitin ligase c-Cbl with the c-Cbl associated protein CAP is recruited to the active receptor. B Receptor tyrosine kinases of the Trk receptor family regulate various cellular processes in the mammalian neuronal system, such as proliferation and differentiation by activation through neutrophins. Recently, we have demonstrated that flotillin-1 directly interacts in vitro and in vivo with FRS2, a protein suggested to function in insulin signaling [ ], by means of its N-terminal phosphotyrosine binding PTB domain and competes for the binding with FGFR [ 89 ].

Flotillins are highly conserved proteins which are assumed, mainly on the basis of cell culture studies, to play multifaceted roles in developmental regulation. Hoehne et al.

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According to this study, flotillin proteins are highly expressed in embryonic neuronal tissues, and during later developmental stages flotillins showed an enhanced expression pattern in axon fascicles [ 83 ]. Additionally, flotillin specific antibodies labeled in particular the Drosophila mushroom body, which is a paired structure of insect central brain responsible for higher-order sensory integration and learning. Flotillins showed a categorical neuronal expression, as antibody staining was restricted to optic lobes and central brain but was not observed in glial cells.

A P-element-induced null mutant for flotillin-2 was viable, fertile and with no apparent deficits in behavior or overall activity. In this flotillin-2 null mutant, the expression of flotillin-1 was undetectable even though the mRNA transcript was present [ 83 ]. Additionally, the adult flies showed a variable phenotype affecting bristles, ocelli, eyes, wings, abdominal segments and thoracic structures. A severe wing phenotype, including blistering and melanotic cells, was caused by flotillin overexpression in wing imaginal discs.

Moreover, a later study showed that flotillin-2 deficiency resulted in reduced spreading of the morphogens Wnt and hedgehog, whereas flotillin-2 overexpression resulted in increased morphogen secretion and expanded diffusion. These findings qualify flotillin-2 as a novel component of the Wnt and hedgehog secretion pathway in D rosophila [ 64 ]. Flotillins are also implicated to regulate axon regeneration in a zebrafish model.

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As a consequence of gene duplication in fish, there are two copies of each flotillin gene. To understand the essential role of flotillins in axon regeneration, specific morpholino Mo antisense nucleotides were used to transiently downregulate flotillin-1a, flotillin-1b and flotillin-2a in zebrafish [ 60 ]. Recently, a zebrafish model was employed to study the role of flotillins in cholera intoxication. Consequently, flotillin-1 and flotillin-2 knockdown prevented cholera intoxication in zebrafish embryos. Cell migration is an essential mechanism during the development and maintenance of multicellular organisms.

It requires the constant turnover of cell-matrix adhesion structures, e. Results presented by Ludwig et al. These data suggest that deletion of flotillin-1 leads to the abrogation of flotillin microdomain function. Previous reports have already shown that, upon stimulation with chemoattractants, flotillin microdomains rapidly distribute to the uropod, a contractile structure at the back of leukocytes [ 32 , ]. Ludwig et al. Flotillin overexpression led to an increase of myosin IIa activity, whereas the loss of flotillins caused a reduction as measured by using phosphospecific antibodies [ ].

The regulatory effect of flotillins on myosin IIa activity strengthens the assumption that both proteins exhibit a functional role during cell migration, as myosin IIa was shown to act as a negative regulator of epithelial cell migration, and its ablation results in severe defects in focal adhesion formation and actin stress fiber organization [ , ]. Recent data from several groups have revealed flotillins as important regulators of vital cellular processes such as signaling and endocytosis.

One major challenge in the future will be to dissect how the oligomerization and the covalent modifications of flotillins affect their function during these processes. Endocytosis of flotillins appears to be influenced by upstream signaling [ 30 , 49 , 56 ] and requires clustering of flotillin-1 and flotillin-2 molecules [ 34 , 56 ]. We have shown that EGF stimulation results in uptake of flotillins hetero-oligomers from the plasma membrane, and that hetero-oligomerization appears to be required for the endocytosis [ 30 , 56 ].

Mutation of a single Tyr residue, Tyr, in flotillin-2 is sufficient to prevent the EGF-induced endocytosis of flotillins [ 30 ], and this mutation impairs the hetero-oligomerization of flotillin-2 with flotillin-1 [ 56 ]. Flotillins are phosphorylated by the Src family kinases cSrc and Fyn [ 4 , 49 ], but the direct role of Tyr phosphorylation in the endocytosis is still under debate.

Our data show that inhibition of cSrc kinase, the activity of which is necessary for flotillin-2 phosphorylation [ 30 ], does not impair the endocytosis of flotillin-2 [ 16 ], whereas the mutation of the single Tyr residue Y does [ 30 ]. Thus, it is more likely that this mutation affects the oligomerization of flotillins and thereby also endocytosis. However, phosphorylation of flotillins by the Fyn kinase appears to be sufficient to induce endocytosis of flotillins [ 49 ], suggesting that the Src family kinases might play different roles in terms of flotillin function.

It is also important to keep in mind that PKC has recently been shown to phosphorylate flotillin-1 in Ser, and that this phosphorylation is required for the endocytosis of the dopamin transporter DAT [ 74 ]. Thus, phosphorylation seems to be an important direct or indirect regulator of flotillin trafficking. Not only phosphorylation but also covalent modifications with fatty acids myristate and palmitate take place in the case of flotillins [ 3 , 4 , 53 ]. Mutation of Gly2 prevents the myristoylation and the subsequent palmitoylation of flotillin-2, thus also inhibiting its membrane association and rendering the protein soluble [ 4 ].

Recently, the palmitoyl transferase DHHC5 was shown to palmitoylate flotillin-2 [ 53 ]. Interestingly, growth factor withdrawal, which induces neuronal differentiation, resulted in rapid degradation of DHHC5 [ 53 ], implicating that palmitoylation might be important for the well established function of flotillins in neurite outgrowth in cultured neuronal cells. Since the soluble, non-modified flotillin-2 with Gly2Ala mutation seems to function as a dominant negative protein during cell-matrix adhesion [ 30 ], and successive removal of the three palmitoylation sites affect the membrane association of flotillin-2 [ 4 ], the fatty acid modifications, especially the reversible palmitoylation, are most likely important for the regulation of flotillin function.

Accordingly, we have observed that flotillin-2 expression is necessary for the membrane association of flotillin-1, which is not myristoylated and only palmitoylated in a single residue. Thus, ectopic expression of flotillin-1 in flotillin-2 depleted cells results in a soluble protein that is not capable of exerting its function our unpublished data.

Our recent findings revealed flotillin-1 as a MAPK regulator [ 27 ]. However, no data are so far available on the role of flotillin modifications such as phosphorylation in the regulation of MAPK signaling. Although the most evident cellular localization of prohibitin is the inner mitochondrial membrane, various studies have now shown that prohibitin is a substrate of several cytoplasmic kinases such as Akt, and it becomes Tyr phosphorylated upon insulin stimulation of cells [ , ].

Thus, at least a fraction of prohibitin must be found in another cellular localization such as the plasma membrane. Importantly, prohibitin has been shown to be essential for cell migration that is induced by the Ras-Raf-MAPK pathway [ ], and very recent findings show that phosphorylation of prohibitin, which takes place in a raft domain, is necessary for the Ras-dependent enhancement of cellular metastasis [ ], verifying the results of Rajalingam et al.

Thus, there are several parallels in prohibitin and flotillin function, including MAPK signaling and cell migration, although they appear to be involved in different steps of the signaling. Since some evidence for a hetero-oligomerization of different SPFH-PHB family members, namely prohibitin and stomatin family protein SLP-2 [ ] has been presented, it would be important to study if flotillins and prohibitins are capable of hetero-oligomerizing with each other and how this affects their function, especially with respect to the MAPK signaling.

In the future, it will also be of interest to study if the phosphorylation-incompatible forms of flotillins cause aberrant signaling and if this effect is linked on phosphorylation at specific sites. Our previous data show that the function of flotillin-2 in cell-matrix association is indeed dependent on specific Tyr residues, the mutations of which affect cell spreading [ 30 ]. Early findings already indicated that flotillins may be involved in the regulation of the actin cytoskeleton. Overexpression of flotillin-2 was found to result in induction of filopodia-like protrusions and changes in the cytoskeleton [ 4 , 11 ].

Furthermore, flotillins have been shown to be important for the polarization of various immune cells such as T cells and neutrophils [ 29 , 32 , 33 , 37 , , ], and to be involved in the regulation of the activity of various small GTPases that control e. In fact, flotillin-2 has even been suggested to directly interact with actin [ 36 ], although this has not been verified in further studies. Thus, there are very strong implications that flotillins are required for a proper regulation of the actin cytoskeleton, which is necessary for e.

Consequently, taking also into account the numerous reports on the involvement of flotillins in signaling, it is evident that flotillins are critical factors that regulate the cell physiology and cell growth. Thus, the findings showing that flotillins are involved in some types of cancer are well in accordance with this function.

The strongest evidence for the role of flotillins in cancer to date comes from studies on human malignant melanoma. The direct association of flotillins with human cancers was shown in when the group of M. Duvic demonstrated that flotillin-2 is highly expressed in various melanoma cell lines, and that flotillin-2 expression correlated with the progression of human melanoma [ ]. Furthermore, the thickness of primary melanoma lesions, so-called Breslow depth, which is a prognostic marker for the malignancy of a melanoma lesion, showed a correlation with the increased flotillin-2 expression [ ].

Importantly, benign, non-malignant cells could be converted into tumorigenic, metastatic ones upon overexpression of flotillin-2 [ ]. Furthermore, flotillin-2 was shown to be associated with the thrombin receptor PAR1, the expression of which was dependent on the level of flotillin-2 protein [ ]. Lin et al. Furthermore, they revealed a significant correlation between the expression level of flotillin-1 and poor survival of the patients, suggesting that flotillins might prove useful as novel diagnostic markers for cancer.

Knockdown of flotillin-1 was shown to result in inhibition of Akt activity and increased transcriptional activity of the transcription factor FOXO3a. Knockdown of flotillin-1 also inhibited the proliferation of breast cancer cell lines by inducing a G1-S phase arrest of the cell cycle. In line with this, the expression of the cyclin dependent kinase CDK regulator cyclin D1 was reduced after flotillin-1 silencing, whereas that of the CDK inhibitors p21 Cip1 and p27 Kip1 was considerably increased [ 66 ], suggesting that flotillin-1 is an important regulator of the cell cycle.

This result is strongly supported by our findings showing that in flotillin-1 knockdown cells, cyclin D1 expression after EGF stimulation is severely impaired [ 27 ]. Although flotillins evidently play a role in cancer and their overexpression is associated with malignancy, very little is known about their transcriptional regulation.

Flotillin-2 was shown to be a transcriptional target of the p53 family transcription factors p63 and p73 but not of p53 [ ]. We have recently addressed this question more in detail and shown that flotillins are transcriptionally regulated by the Extracellularly Regulated Kinase signaling and by the Retinoid X Receptor Banning et al. Due to their important role in cancer, a deeper knowledge on the transcriptional regulation of flotillins is essential for understanding the mechanisms of malignancy that results from flotillin upregulation.

APP is subjected to proteolytic processing at several sites by a group of proteases called secretases. It has been strongly implicated that the amyloidogenic processing of APP takes place in lipid rafts, which is supported by the findings showing that APP can be detected in rafts isolated from cortical neurons [ ]. Compelling evidence from various research groups shows that the flotillin family of proteins is partially associated with AD pathology and trafficking [ 47 , , ].

Flotillins are considered as classic raft markers in neuronal tissue since they show an abundant expression in the brain, typically in pyramidal neurons and astrocytes. Kokubo et al. Parallel studies in brain sections from AD, Down syndrome and non-demented subjects with plaques showed enhanced flotillin-1 expression with the progression of AD [ ]. Further insight was provided by Rajendran et al. Furthermore, comparison of flotillin-1 immunoreactivity in the hippocampus, the amygdala and the isocortex of AD and control patients implicated that flotillin-1 principally accumulates in lysosomes of tangle bearing neurons during the advanced stages of AD [ ].

To date, the precise functional significance of APP and flotillin association is enigmatic. Schneider et al. It has previously been shown that the intracellular domain of APP directly interacts with flotillin-1 [ ]. Our unpublished findings suggest that both flotillin-1 and flotillin-2 directly interact with APP, suggesting that both flotillins could facilitate recruitment of APP into microdomains.

Flotillins and other lipid raft-associated proteins might also be involved in other human neuronal diseases. The study by Shin et al. EAE is an experimental animal model, mostly used in rodents, of inflammatory demyelinating diseases of the central nervous system CNS , including multiple sclerosis reviewed in [ ].

Similar results with the caveolin data were obtained for flotillin-1 during the peak stage of EAE. Immunohistochemical analysis showed an enhanced expression of flotillin-1 in the dorsal horn lamina and in some flotillinpositive macrophages and astrocytes, in which it colocalized with the lysosomal marker cathepsin D [ ]. Due to that, they postulated that flotillin-1 inherits an important function in immune and neuronal cells within the CNS during EAE, possibly via the activation of signal transduction and lysosomal activity.

The pathological hallmark of PD is the extensive loss of dopamine secreting cells within the substantia nigra, particularly affecting the ventral component of the pars compacta reviewed in [ ] , which can lead to uncontrolled muscle contraction and movement, dementia, depression and anxiety. During evolution, many pathogens have developed mechanisms to use host-cell lipid rafts as signaling and entry platforms to escape the host immune system reviewed in [ , ]]. Several studies demonstrated that lipid rafts are often used as entry sites for bacteria, e.

In regard to flotillins, Li et al. This bacterium causes diarrheal disease by disrupting the integrity of tight junctions, which are essential structures for physiological homeostasis and defense against pathogen invasion. Furthermore, they detected a shift of flotillin-1 and occludin, a major protein of tight junctions, from Triton X insoluble fractions to Triton X soluble fractions following EPEC infection.

Lipid raft

This led to the conclusion that the loss of tight junction barrier function might be accounted to the redistribution of occludin and flotillin-1 after EPEC infection. Not only bacteria use lipid raft domains as preferred entry sites in host-cells, but also various enveloped and non-enveloped viruses are dependent on an intact lipid raft structure and the presence of cholesterol for successful virus entry reviewed in [ ]]. In the case of the retrovirus human immunodeficiency virus-1 HIV-1 , flotillin-1 was suggested to play a role of in the cellular response to the viral infection since the protein level of flotillin-1 was increased in peripheral blood mononuclear cells after treatment with the HIV-1 component gp [ ].

The primary HIV-1 receptor CD4 and the co-receptor CCR5, both of which are important for HIV-1 host-cell entry, were also shown to associate with flotillin-1 containing lipid rafts in monocytes [ ]. Flotillins seem to be of functional relevance in insulin signaling see above and the metabolic disease diabetes mellitus, as first demonstrated by Baumann et al. A year later, James et al. It is important to note that SHRSP rats are a model of human insulin resistance, defined by the decreased ability of cells or tissues to respond to physiological levels of insulin especially in skeletal muscles and adipose tissue.

Insulin resistance is of major importance in several human diseases, e. The results of both the above studies lead to the assumption that flotillins might play a role in insulin signaling in the skeletal muscle and adipose tissue. Furthermore, during steady state conditions, flotillin-1 was found in perinuclear regions in differentiated skeletal muscle cells, but at the plasma membrane in adipocytes [ 31 ]. This observation suggested that the role of flotillin-1 in targeting GLUT4 to the plasma membrane after insulin stimulation may be different between muscle and adipose tissue. In fact in skeletal muscle cells, the insulin-stimulated signaling cascade leading to the translocation of GLUT4 to the plasma membrane is dependent on flotillin-1 and the muscle-specific protein caveolin-3 [ ].

Lipid rafts also play a role in diabetic xerostomia, or dry mouth. This disease, when associated with diabetes mellitus, is caused by degenerative changes in the salivary glands leading to increased infectious conditions in the oral cavity and in the long-range aggravates the risk of atherosclerosis and cardiovascular diseases reviewed in [ ]. Wang et al. In parotid acinar cells derived from control rats, AQP5 colocalized with flotillin-2 in the cytoplasm and at the apical plasma membrane ten minutes after cemivelime treatment.

In contrast, in the parotid acinar cells derived from diabetic rats, the translocation of these proteins did not occur. In summary, the results showed that AQP5 translocation is a lipid raft-dependent process and inhibition of the muscarinic agonist-induced translocation might be the cause of diabetic xerostomia. In-gel digestion with trypsin was performed as previously described 23 — Peptide samples were resuspended in loading buffer containing 0.

The peptides were ionized with 2.

Membrane lipid rafts

Automatic gating control was set to msec maximum injection time. Quantification of proteins was based on the comparison of extracted ion current intensities for identified peptides as previously described The paired, two-tailed Student's t-test was used to analyze the significance of the difference between groups. Previous studies have demonstrated that ox-LDL induced aggregation of gp 91phox 30 and Fas 31 in lipid rafts.

As shown in Fig.

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These results suggest that treatment with native-LDL led to the formation of lipid rafts in Raw Native-LDL-induced lipid raft clustering in Raw LDL, low-density lipoprotein. Representative confocal microscopy images of lipid rafts and MPO in Raw Yellow spots in the overlaid images were defined as colocalization of both molecules.

The role of lipid rafts in macrophage-mediated LDL oxidation was investigated. Effect of lipid raft disrupters on cell-mediated LDL oxidation. Lipid oxidation was assessed using a thiobarbituric acid assay. Each data point represents the mean of triplicate measurements. Lipid rafts were isolated and purified from non-treated and LDL-stimulated Raw The highest protein concentration levels were located at the bottom of the gradient. Total protein concentration levels in lipid rafts increases following treatment with LDL in Raw Protein concentration in each fraction was measured using a bicinchoninic acid assay.

The experiment was repeated four times. To identify differentially regulated lipid raft-associated proteins following LDL stimulation, label-free quantitative proteomics analysis was performed on lipid raft fractions of macrophages. The distribution of the ratios of protein abundance between the LDL-stimulated macrophages and the resting state is shown in Fig.

Out of the 1, proteins, and proteins were shown to be downregulated and upregulated following LDL stimulation, respectively. Protein groups were then sorted according to biological processes, cellular components and molecular function GO categories Fig. Upregulated proteins comprised GO terms associated with metabolic processes and response to stimuli. Notably, the results also demonstrated enrichment for biological adhesion, localization, and enzyme regulator activity. Furthermore, apoB was identified in lipid rafts when the protein spectra were matched to a human proteome database, which indicated the human origin of LDL.

B Functional classification analysis of upregulated proteins. Numerous interaction groups were apparent, such as Hmox-1 - Bax, Pfn - Cap Notably, ERp29 was associated with calreticulin. To further validate the proteomic identification results, the expression levels of one of the upregulated proteins, ERp29, were analyzed by western blotting.

The floated low density fractions 6, 7 and 8 lipid rafts observed one-third of the way down the gradient represented the lipid raft fractions following gradient ultra-centrifugation of the cell lysates. It was demonstrated that flotillin-1 localized not only in lipid raft fractions, but also in non-raft fractions high density fractions.

It should be noted that this was not due to unsuccessful isolation of the lipid raft, but a phenomenon observed in previous studies 22 , Notably, the transferrin receptor, a marker for the non-raft plasma membrane, was distributed at the bottom of the gradient Fig. Bar graphs show the band density ratio of Flot-1 Fig.

Western blot analysis of the distribution of flotillin-1 and ERp29 in lipid raft fractions isolated from Raw The expression of A flotillin-1, the protein marker for lipid rafts; B ERp29; and C transferrin receptor, a non-raft plasma membrane protein. Quantitative analysis of the ratio of the relative fold increase over the control for D flotillin-1 and E ERp The results represent the mean value of three independent experiments.

It has been demonstrated that ox-LDL-induces lipid raft-redox signaling in the coronary arterial endothelium The present study demonstrated that LDL increases the formation of lipid rafts. Numerous changes occurred in lipid rafts following treatment with LDL in macrophages. Oxidation of LDL has an important role in the pathogenesis of atherosclerosis and vascular diseases.

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However, the precise mechanisms underlying the role of LDL remain to be elucidated, and the enzymes responsible for these mechanisms also have yet to be identified 2. Certain studies have suggested that LDL oxidation does not take place in the blood circulation, and must occur in the arterial wall due to the fact that blood contains numerous antioxidant molecules 33 , However, a previous study recently reported results that supported the possibility of LDL oxidation in the circulation There may be two major ways in which cell-mediated LDL oxidation may occur in cells: i Cell oxidative stress activated by microbial infection causes normal levels of LDL oxidative damage, termed passive oxidation; and ii cell oxidative stress activated by high-level LDL, termed active LDL oxidation The results of the present study demonstrated that LDL induced lipid raft clustering in macrophages.

These data suggested that LDL signaling may be associated with lipid rafts. MPO is an important enzyme in innate immunity and defense against pathogens The first study to suggest that MPO is implicated in atherogenesis was conducted in The present study demonstrated that MPO was aggregated in lipid rafts following LDL cultivation with macrophages for 9 h.

Thus, lipid rafts are likely to have coalesced, at the resolution limit of two-photon microscopy, leading to the formation of a continuous, percolating raft-like phase. Two-photon microscopic measurement of the generalized polarization of Laurdan. Control and sphingolipid-enriched Gaucher-type macrophages were labeled by Laurdan and the generalized polarization GP of the indicator, characterizing membrane hydration, was measured by two-photon microscopy.

Images were recorded in a plane corresponding to the flat plasma membrane adjacent to the coverslip. Hydrated and less hydrated membrane domains are shown in green and red, respectively. The generalized polarization values shown in these images are not comparable to the ones in Fig. The corresponding transmission images showing the morphology of cells are displayed in the middle.

The images on the left were segmented into two masks corresponding to high and low values of generalized polarization shown in red and green, respectively. The same threshold was applied for both images, and it was chosen by visual inspection. The percentages displayed on the masks are the fractional areas of the high-GP mask. Atomic force microscopy AFM is a versatile technique suitable for determining topographical and nanomechanical properties of the membrane 28 , 30 , When the AFM cantilever is retracted from the cell, membrane tethers, corresponding to nanotubes, are formed between the membrane and the AFM tip.

These tethers are believed to characterize the stiffness of the membrane and its interaction with the underlying cytoskeleton 32 , Stepwise changes in the retraction force spectrum correspond to extension and rupture of individual tethers allowing us to enumerate them and to measure the single-tether force Fig. The median force was 30 pN and 27 pN in control and Gaucher-type macrophages, respectively Fig.

Thus, the Gaucher-type membrane tethers require less force to be extended, hence their surface tension or bending rigidity is smaller The Gaucher phenotype led to a doubling in the number of tethers, with the mean tether number increasing from 4. We concluded that the cell membrane of Gaucher-type macrophages was more prone to the formation of membrane nanotubes than that of control cells. Measurement of membrane tether formation and elastic properties using atomic force microscopy. A , B Membrane tether formation was analyzed on the force vs.

Lipid rafts

Tether force was determined as the abrupt change in force upon detachment of a tether from the cantilever and the distribution of tether forces in control and Gaucher-type cells was calculated A. Such step-like changes in the force curve were enumerated and the distributions of their numbers for control and Gaucher-type cells are displayed B. C , D The elastic modulus was determined in the extension phase of the force vs. Although the histogram of the elastic modulus showed a hardly detectable shift to lower values in Gaucher-type cells, two measures of central tendency clearly implied that the cell became significantly more compliant upon accumulation of glycosphingolipids Fig.

The data convincingly shows that Gaucher-type cells have a significantly lower elastic modulus regardless of cellular height Fig. Since membrane stiffness and viscosity have been linked to endocytosis 28 , the results described in the previous sections led us to assume that the rate of endocytosis of certain ligands may be affected by the Gaucher phenotype. Therefore, we carried out quantitative analysis of endocytosis of transferrin receptor and subunit B of cholera toxin, typical examples for non-raft and raft-dependent internalization pathways, respectively 35 , In conclusion, both the FRAP and the endocytosis experiments imply that glycosphingolipid accumulation present in Gaucher macrophages mainly affects the non-raft components of the cell membrane as far as their properties related to lateral diffusion and membrane dynamics are concerned.

Quantitative evaluation of the endocytosis of transferrin and subunit B of cholera toxin. The endocytosed-fraction of the ligands was determined as the relative fraction of fluorescence intensity in the cytoplasmic mask compared to the total cellular fluorescence on a cell-by-cell basis. Since the Gaucher phenotype seems to influence the biophysical and cell biological properties of the cell membrane, we aimed at characterizing the difference between control and Gaucher cells regarding signal transduction. Cells were also labeled with fluorescent transferrin and DAPI to segment the confocal images to the cytoplasm and the nucleus Fig.

We have independently confirmed the results of these microscopic measurements using flow cytometry. These results imply that the gross cellular alterations induced by accumulation of glycosphingolipids in the plasma membrane and other membranes lead to inhibition of STAT1 signaling. Cells were analyzed by microscopy A—C or flow cytometry after trypsinization D. The level of tyrosine phosphorylated STAT1 was calculated in the whole cell A , in the nucleus B or in the cytoplasm C using quantitative image analysis, or it was determined from the mean of flow cytometric histograms D.

The fluorescence intensity values were normalized to the unstimulated control. The error bars represent the standard error of the mean of four independent experiments in the case of microscopy and three independent experiments in the case of flow cytometric results. The biological properties of the plasma membrane depend on its biophysical characteristics which are, in turn, influenced by the individual and ensemble properties of its lipid and protein constituents The aim of the current manuscript was to elucidate the effects of glycosphingolipid accumulation on the physical and cell biological properties of the cell membrane in an in vitro model of Gaucher disease and to reveal novel molecular mechanisms behind the emergence of the disease symptoms.

Generalized polarization and fluorescence anisotropy measurements revealed that the plasma membrane became less hydrated and more restrictive to rotational diffusion as a result of glycosphingolipid accumulation. The reported 2—fold increase in the glycosphingolipid content in in vitro and in vivo models of Gaucher disease are substantial enough to give rise to such alterations in the biophysical properties of the cell membrane 7 , 8 , 9.

We next performed FRAP experiments to measure the lateral mobility of membrane constituents. FRAP is a robust and model-independent approach, and although the absolute diffusion coefficients provided by this method are sensitive to bleaching correction artifacts, the relative changes are as reliable as those reported by fluorescence correlation spectroscopy FCS.

According to FRAP, the lateral mobility of non-raft protein and lipid constituents was significantly restricted in Gaucher-type cells, while raft-resident proteins and lipids were either unaffected or less significantly inhibited in their lateral mobility. Two-photon microscopy of Laurdan-labeled cell membranes revealed that the fractional area of membrane exhibiting low hydration, corresponding to raft-like domains, increased as a result of glycosphingolipid accumulation. The raft-like phase became so abundant that it formed a continuous, percolating phase isolating and restricting the movement of constituents residing in the non-raft phase explaining why non-raft components were preferentially affected by glycosphingolipid accumulation When interpreting the results of FRAP experiments it must be kept in mind that diffusion of membrane proteins and lipids in restricted displaying fast local motion within a confined zone and slow hopping between adjacent compartments according to single particle tracking and super-resolved FCS measurements Diffusion parameters revealed by FRAP are determined by both of the above diffusion processes.

Although FRAP is unable to resolve the diffusion coefficient characterizing local motion within a confinement zone, our conclusions are valid since they are based on the assumption that enlargement of glycosphingolipid-enriched domains leads to a lower rate of long-range diffusion due to enhanced confinement of non-raft membrane components. It is noteworthy that highly dissimilar membrane constituents lipids, GPI-anchored and transmembrane proteins have been shown to display comparable diffusion characteristics as far as long-range diffusion is concerned suggesting that the barriers restricting diffusion affect all of them in a similar way AFM is another sensitive tool for interrogating the biophysical properties of the membrane 30 , Step-like decreases in the force measured by the device in the retraction phase correspond to detachment of single membrane tethers from the AFM cantilever.

These membrane tethers correspond to nanotubes which, when formed as a result of physiological processes, are responsible for intercellular communication In-plane membrane tension, bending stiffness and membrane-to-cytoskeleton attachment contribute to the tether force The fact that the number of membrane tethers formed was more than two-times higher in Gaucher-type cells than in control ones suggests that the resistance of the membrane to bending was reduced. The formation of membrane tethers or nanotubes involves the generation of negatively and positively curved membrane at the base and around the circumference, respectively.

The accumulation of inverted cone-shaped glycosphingolipids in the outer leaflet may promote the formation of highly positively curved membranes and thereby can explain the more frequent formation of membrane tethers. Lipid rafts and glycosphingolipids have already been linked to the formation of membrane nanotubes 45 , Since the formation of membrane tethers upon retracting the AFM cantilever does not involve biological processes, our results imply that the altered biophysical properties of the membrane upon glycosphingolipid accumulation per se lead to easier formation of membrane tethers.

As membrane nanotubes are involved in immunoregulation, their altered number in Gaucher-type cells may contribute to immune system irregularities in Gaucher disease 47 , It is unknown whether the dynamics of the cytoskeleton in general, and actin filaments in particular, is directly modified in Gaucher disease.

However, it has been shown that alterations in the lipid composition of the cell membrane lead to changes in the elastic properties of the plasma membrane and the cell, and that these changes depend on the actin meshwork 33 , It has been hypothesized that anchoring of the actin cytoskeleton to the membrane is behind this phenomenon.

Indeed, it has been shown that lipid raft domains are involved in connecting actin to the cell membrane 50 , We assume that the increased area of lipid rafts in glycosphingolipid-enriched membranes of Gaucher cells leads to rearrangement or dilution of actin-to-membrane linkages with consequent changes in the dynamics of the actin cytoskeleton.

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The aforementioned changes in the biophysical properties of the plasma membrane led to significant alterations in cell biological processes connected to the cell membrane:. Although the raft-dependent endocytosis of cholera toxin was not different in control and Gaucher-type cells, the raft-independent, clathrin-dependent endocytosis of transferrin was significantly inhibited upon glycosphingolipid accumulation 35 , Generation of the negatively curved membrane of endocytic vesicles becomes more difficult if inverted cone-shaped glycosphingolipids, favoring the formation of positively curved membranes, accumulate in the extracellular leaflet.

Although glycosphingolipids are preferentially present in rafts, their concentration also increases in non-raft domains in Gaucher cells, which may hinder the formation of endocytic vesicles outside rafts 7. Alternatively, the significantly inhibited lateral diffusion of transferrin receptor, revealed by FRAP measurements, may also lead to the decreased rate of endocytosis of transferrin.

In contrast, the raft-dependent endocytosis of cholera toxin did not change upon glycosphingolipid accumulation in the cell membrane, because the biophysical properties of rafts did not change substantially in Gaucher cells according to our FRAP measurements. This diminished STAT signaling may seem to be somewhat surprising at first sight given that lipid rafts are believed to be crucial in the signal transduction of STAT However, lipid rafts often play a bipartite role by promoting the activation of membrane receptors and keeping their activation under tight control at the same time While mild cholesterol extraction induces T cell activation, strong cholesterol depletion inhibits the same process 54 , Similarly, although lipid rafts inhibit ligand binding by the epidermal growth factor receptor, they potentiate EGFR-dependent signaling 56 , In summary, the accumulation of glycosphingolipids in the cell membrane led to the increase in the size of raft-like domains which became a percolating, continuous phase Fig.

According to percolation theory the plasma membrane is an interrupted system in which obstacles form islands in a continuous conducting phase at low obstacle concentrations. However, the obstacles form an obstructed phase isolating islands of the conducting phase from each other at high obstacle concentrations above the percolation transition.

The aforementioned model has been put forward several times 58 , 59 , and the fence-and-picket structure of the plasma membrane also involves a high density of obstacles formed by cytoskeleton-anchored transmembrane proteins In the context of our experimental system raft-like microdomains correspond to obstacles.

An increase in the size of raft-like microdomains and the consequent formation of a continuous raft phase have already been reported upon glucosylceramide loading or changes in temperature 40 , While the diffusion of non-raft lipids and proteins was found to be significantly inhibited due to the corralling effect of the percolating raft phase, the movement of raft components was not significantly affected. A similar bipartite effect of glycosphingolipid accumulation was observed for endocytosis. The results not only reveal new potential cellular pathways behind the symptoms of Gaucher disease, but also characterize the profound changes associated with glycosphingolipid accumulation in the plasma membrane.

Model for sphingolipid enrichment-induced changes in the plasma membrane. The membrane is assumed to be composed of liquid-disordered L d and liquid-ordered L o domains with the latter corresponding to lipid rafts enriched in sphingolipids. Membrane components mainly diffuse within their own domains. Upon sphingolipid enrichment, the physical landscape of the membrane changes due to enlargement of L o domains causing their coalescence and consequent confinement of non-raft components.

For serum-starvation, the cells were kept in the above medium containing 0. A background region of interest ROI was drawn outside cells and the membrane of the bleached cell was determined by performing manually-seeded watershed segmentation in every image so that the membrane mask moved with the changing shape of the membrane Fig. S3 Raw intensity values were double-normalized to the pre-bleach intensity and to bleaching taking place during recovery:. Double-normalized FRAP curves were averaged, and the following empirical equation was fitted to the mean recovery curves, R t :.

Generalized polarization GP of Laurdan fluorescence was calculated according to the following formula 27 :. Data were collected with a custom-built microscope built around an Olympus upright frame with a Femto2D scanner Femtonics Ltd, Budapest, Hungary.

The red edge emission of Laurdan I red was detected using a LP filter, while its fluorescence in the blue range I blue was recorded between and nm After incubation, cells were put on ice to prevent further endocytosis followed by fixation with 3. In order to locate and identify cells we adapted a method based on semi-automatic detection of nuclei and the cell membrane Image analysis was carried out with DipImage and Matlab. The membrane was thickened inwardly and this shape was used as the membrane mask.

The area inside the membrane mask was defined as the intracellular mask. Image segmentation is demonstrated in Fig. Images were background-corrected before quantitative evaluation. The cantilever was calibrated using the thermal method yielding a typical spring constant of 0. At least 10 cells were manipulated from each cell type. Nuclear and membrane segmentation was carried out using the DAPI-stained nuclei and transferrin-stained membranes as described for endocytosis measurements.

STAT and p-STAT intensities were evaluated in the cell mask corresponding to the area inside the membrane mask including the membrane itself. The nuclear fraction of STAT was calculated by dividing the fluorescence intensity of STAT measured in the nuclear mask by the total cellular intensity. Nuclear staining with DAPI and membrane staining with AlexaFluortransferrin were not carried out with these samples. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Escriba, P. Membranes: a meeting point for lipids, proteins and therapies. Membrane lipid therapy: Modulation of the cell membrane composition and structure as a molecular base for drug discovery and new disease treatment. Butters, T. Gaucher disease. Grabowski, G.