Caveolae and Lipid Rafts in Membrane Traffic
Membrane traffic mediates the exchange of components between the different cellular organelles. Membrane proteins and lipids are synthesized in the endoplasmic reticulum and from there are transported to their subcellular sites of action 109, 110 . While peripheral membrane proteins as well as single lipids bound to lipid transfer proteins can shuttle between different membranes via the cytoplasm or through contacts between membranes 4 , most membrane turnover is mediated by vesicular traffic....
The Role of Caveolin Phosphorylation in Cells
Our current hypothesis is that caveolin organizes signaling complexes containing Fyn, Csk, and Abl that regulate cortical actin remodeling. Caveolin phosphorylation is required for the recruitment of both Csk and Abl into the complexes, which leads to negative regulation of the Src-family kinases that are resident in the caveolae. When the phosphorylation of caveolin is catalyzed by the Src-kinases themselves, this is a mechanism for the transient activation rapid Csk-induced attenuation of...
Mechanism of Raft Action in Prion Conversion
It has been shown that direct cell-to-cell contact between infected cells and unin-fected cells i.e., with PrPc and PrPSc being on opposite cell surfaces 71 , can efficiently induce the passage of prion infection between cells. The importance of this membrane environment in the conversion reaction has been underscored by Fig. 10.1 Different models for the role of rafts in prion formation, based on the apparent implication of rafts in many features of prion protein trafficking. A Rafts may...
References Bro
1 Razani, B., Woodman, S.E., Lisanti, M.P. 2002 Pharmacol. Rev. 54, 431-467. 2 Parton, R.G. 2003 Nat. Rev. Mol. Cell Biol. 4, 162-167. 3 Li, W.P., Liu, P., Pilcher, B.K., Anderson, R.G. 2001 J. Cell Sci. 114, 1397-1408. 4 Liu, P., Rudick, M., Anderson, R.G. 2002 J. Biol. Chem. 277, 41295-41298. 5 Razani, B., Schlegel, A., Liu, J., Lisanti, M. P. 2001 Biochem. Soc. Trans. 29, 494-499. 6 Liscovitch, M., Burgermeister, E., Jain, N., Ravid, D., Shatz, M., Tencer, L. 2004 In Mattson, M.P. Ed....
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy FCS provides information on the diffusion of fluorescently tagged molecules by observing the correlation of fluorophore fluctuation through a small optically delimited detection volume 60-65 . The resultant fluorescence fluctuations provide an autocorrelation curve, which could be used to calculate diffusion constant of the molecules. Whilst FCS retains the statistical advantages of FRAP, it can be performed with dilute fluorophores and with relatively less...
Organization and Function of Receptor Tyrosine Kinases is Linked to Lipid
Receptor tyrosine kinases are prominent examples of the proteins involved in cell signaling that form molecular associations and superstructures in the cell membrane. Cooperative interactions between or among receptor tyrosine kinases play a pivotal role in signal transduction. This includes homo- and hetero-dimerizations as well as potentially higher-order associations, which may be either dependent or independent of ligands. In addition to protein-protein interactions, lipid microdomains also...
Scanning NearField Optical Microscopy SNOM
This technique combines the enhanced lateral and vertical resolution characteristic of AFM with simultaneous measurements of topographic and optical signals. SNOM can achieve a spatial resolution beyond the diffraction limit by scanning a sub-wavelength-sized aperture, confining the excitation beam to the close proximity of the sample. The resolution depends on the size of the aperture and the distance between the probe and the sample, rather than on the wavelength. The most commonly used SNOM...
Chaperone Complexes Involved in Cholesterol Transport in Specialized Tissues
The intestine has a major role in the overall regulation of systemic cholesterol levels. We have identified a third chaperone complex that is involved in the net uptake and transport of cholesterol in the intestine 80 . Annexin II and caveolin-1 form a chaperone complex with cyp40 and cypA that binds cholesterol in the intestine. Ablation of annexin II or caveolin-1 in the zebrafish disrupted cholesterol uptake. Further, the drug ezetimibe decreased cholesterol levels in the circulation by...
An Internalization Wave of Caveolae can be Stimulated by Virus
Studies on the entry of simian 40 SV40 virus by Pelkmans and coworkers 52-54 have documented that caveolae can actually play a role in nonconstitutive endocytosis. Thus, after binding of SV40 virus to the cell surface via the MHC class I molecule, the virus particles move laterally in the plasma membrane to end up in caveolae. Although these caveolae are initially immobile, the virus initiates a complex signaling cascade leading to a profound disorganization of the cortical actin cytoskeleton...
Index
AA see amino acid Ab see amyloid b-peptide ABCA1 see ATP-binding cassette - caveolin phosphorylation 121 acceptor, fluorescence correlation spectroscopy 59 acceptor channels, FRET 146 ACTH see adrenocorticotropic actin plasma membrane attachment sites, phosphorylation 128 actin polymerization 131 actin remodeling 127 - insulin-induced 131 activation, Csk 125-127 activation-induced cell death AICD , lipid rafts 157 acylation, signal transduction 92 AD see Alzheimer's disease ADAM10,...
Specific Heat Of Lipids
1 Danielli, J.F. and H. Davson, A contribution to the theory of permeability of thin films. J. Cell. Comp. Physiol. 1935 5 Nat. Rev. Mol. Cell. Biol. 2005 6 3 209-220. 5 Allan, D., Mapping the lipid distribution in the membranes of BHK cells mini-review . Mol. Membr. Biol. 1996 13 2 81-84. 2 Singer, S.J. and G.L. Nicolson, The fluid mosaic model of the structure of cell membranes. Science 1972 175 23 720-731. 6 Tran, D., et al., Ligands internalized through coated or noncoated invaginations...
Caveolin A Scaffold for eNOS
Since NO is a very labile and highly reactive messenger molecule with autocrine and paracrine functions, the site of NO production should logically have a major influence on its biological activity. The discovery in 1996 of the location of eNOS in caveolae 6,7 was therefore viewed as the proof of concept that compartmentation of the enzyme is critical to fine-tune NO synthesis. This specific locale of eNOS had been suspected based on the double acylation process that characterizes eNOS...
Introduction Vax
Caveolae are free cholesterol sphingolipid FC SPH -rich microdomains of the cell surface that are assembly sites for many transmembrane signaling complexes. Other proteins associated with caveolae include the transporters of some small ligands glucose, inorganic ions and catalysts of cell FC homeostasis. The highest levels of caveolae are found in differentiated primary cells, including pulmonary type-1 cells, adipocytes, endothelial cells, smooth muscle cells, and fibroblasts. In blood...
The Immunological Synapse
One of the best-characterized examples of specialized signaling domains where the dynamic assembly of both small- and large-scale protein associations have a crucial role, is the contact region formed between a T cell and a target or an antigen-presenting cell APC , termed the immunological synapse IS 63,64 . Formation of the IS is initiated by recognition of the peptide antigen presented in complex with MHC glycoproteins on the APC or target cell by the T cell receptor complex TCR on the...
Molecular Mechanisms Implicated in the ProSurvival Action of Caveolin1
The phosphoinositide 3-kinase PI3K Akt cell survival pathway has recently emerged as a major target for regulation by caveolin-1 in a variety of cancer cells 12,13,86 . A possible role of caveolin-1 in PI3K Akt pathway activation was shown in multiple myeloma cells which, unlike most other hematopoietic-derived cells, express high levels of caveolin-1 86 . Caveolin-1 was found to co-localize in lipid rafts fractions, and to co-exist in an immunoprecipitable complex, with receptors for...
Determination of Domain Size and Overlap between Fluorescence Distributions
The lateral distribution of cell-surface proteins and their co-localization in membrane microdomains can be studied by digital imaging microscopy with a resolution of 200-300 nm, as set by Abbe's law 35 . The application of confocal laser scanning microscopy or multiphoton excitation 36 greatly improves image sharpness and contrast by excluding photons arising from out-of-focus planes. This resolution is not sufficient to directly indicate molecular associations however, it allows the...
Caveolin and its Role in Intracellular Chaperone Complexes 175
William V. Everson and Eric J. Smart 8.1 Caveolae and Caveolin-1 175 8.2 Caveolin Protein Structure, Domains, and Membrane Interactions 177 8.3 Caveolin Expression and Localization in the Cell 178 8.4 Caveolin Expression and Localization Varies Depending on the Physiological State of Cells in Culture 180 8.5 Caveolin-1 Expression Confers a New Level of Regulation 182 8.6 Caveolae Cholesterol and Caveolin Localization to Caveolae 182 8.7 Caveolin and Cholesterol Cross Membranes During...
Caveolin and Focal Adhesion Complex
When cells come into contact with the extracellular matrix ECM , they usually extend filopodia. Then, integrins located at the tip of filopodia bind to the ECM and initiate the formation of focal adhesions. Actin-rich lamellipodia are then generated as the cell spreads on the ECM 16 . As the integrins bind to the ECM they become clustered in the plane of the cell membrane and associate with the cytoske-letal and signaling complex that promote the assembly of actin filaments. The reorganization...
Caveolin and Rhofamily GTPases
Rho GTPases participate in the regulation of polarity, microtubule dynamics and cell shape, the latter property being regulated by the Rho GTPases adjusting the organization of the actin cytoskeleton. For example, Cdc42 induces filopodia, Rac induces lamellipodia, and Rho induces focal adhesion and associated stress fibers 13 Fig. 9.2 . Rho GTPases are a group of molecular switches which control complex cellular processes. They cycle between two conformational states one state is bound to GTP...
Introduction Eip
Caveolae are specialized invaginated domains of the plasma membrane that act as organizing centers for signaling molecules. Caveolae are formed in membranes by the caveolins, a family of three related gene products caveolins-1, -2, and -3 1-4 . Caveolins form homo- and hetero-oligomers that make up the characteristic striated coat of caveolae 5 . Expression of caveolin is necessary and sufficient to induce cell-surface caveolae 6-8 . In addition to their role as coat proteins that drive the...
Caveolin and Cholesterol Cross Membranes During Trafficking
During the process of vesicle trafficking, membrane topology is normally conserved - that is, material found in the extracellular exofacial leaflet of the plasma membrane is topologically conserved. For example, it remains associated with the exofacial leaflet during endocytosis and after fusion with organelles. The exofacial leaflet is thus the inner leaflet of vesicles and the interior leaflet of organelles such as the ER. Proteins and lipids associated with the cytofacial leaflet cytoplasmic...
Phosphocaveolin is Enriched at Sites of Attachment of the Actin Cytoskeleton to
Significant data links caveolae and caveolins to the actin cytoskeleton 102,103 . In confluent, quiescent cells and in tissues, caveolin-1 is primary localized to the plasma membrane. The caveolin-1 at cell surface is tethered to the actin cytoskeleton 104,105 . Caveolin binds to actin through filamin, a protein that regulates cortical actin assembly 106 . Agents that disrupt the actin cytoskeleton cause rapid internalization of caveolin 105 . Caveolin-1 also redistributes in response to shear...
Role of Caveolae in Endocytosis of Cholera Toxin
In addition to the recent studies on internalization of virus mentioned above, studies on cholera toxin CT uptake have also been important for the ongoing discussion of the role of caveolae in endocytosis. CT belongs to a large group of protein toxins with enzymatically active A-moieties, and B-moieties that act as ligands and bind to cell- surface components. These toxins must be endocytosed before they exert their toxic effect s . In the case of CT, the B-moieties bind to the ganglioside GM1...
Proximity Measurement Using HeteroFRET
Another FRET technique which has been used extensively to detect proximities between fluorophores is that of hetero-FRET 80 . Several hetero-FRET methods exist see Fig. 3.5 , and most have been applied to study GPI-anchored proteins for a review, see 75 . In the early studies, Kenworthy and Edidin 80 , when examining FRET between donor and acceptor labeled antibodies against 5'-nucleotidase 5'-NT a GPI-AP , reported that most proteins on the surface of fixed cells were monomers. These authors...
Caveolae Linked to Nongenomic Actions and Uptake of Estrogen
The initial paradigm of sex hormone action in the cell begins with the delivery of sex hormones to the cell surface, passage across the plasma membrane, interaction with a receptor localized in the cytosol, and transport of the hormone-receptor complex to the nucleus, where the sex hormone-bound receptor acts to alter specific gene transcription. During the early 1990s, a few studies showed that the initial model proposing that the steroid receptor was the cytosolic chaperone for these...
Fluorescence Resonance Energy Transfer FRET
Techniques utilizing Forster-type FRET offer convenient tools for mapping the spatial distribution and molecular vicinity relations of membrane proteins on live cells in situ, without any major interference with the physiological condition of the cells. Several techniques have been developed to measure FRET on cell surfaces 5,41-43 . FRET is a process wherein energy is transferred non-radiatively from an excited donor fluorophor to a nearby acceptor via dipole-dipole coupling 44 . In order for...
The Origin of the Raft Hypothesis
Membrane heterogeneities resurfaced primarily as a necessity to explain a specific biological observation of preferential sorting of glycosphingolipids to apical membranes in polarized epithelial cells 4 . This idea was finally developed into a notion of lipid rafts by Simons and coworkers, and was centered on cholesterol-containing domains as the cornerstone of the raft model of life in a living cell membrane 5 Fig. 3.2A . Lipid rafts were hypothesized as specialized regions of cell membrane...
Caveosomes Intracellular CaveolinAssociated Structures
In addition to reaching the TGN, internalized CT is also found in caveosomes 90 , the organelle reached by internalized SV40 virus 52 . The caveosome is a non-acidic, caveolin-associated compartment that does not stain for EEA1, a marker of early endosomes reached by the clathrin-dependent endocytic pathway. Moreover, caveosomes do not accumulate Lysotracker, a lysosomal marker, and they do not stain for TGN46 and mannosidase II, both of which are Golgi markers 52 . Evidence that the...
Proximity Measurement Using HomoFRET
Homo-FRET measures the loss of fluorescence anisotropy, resulting from the FRET process. It is a relatively less well-known approach to FRET, and is also referred to as depolarization-FRET 74,75 . In this type of FRET, fluorescence emission excited by polarized light is measured for the extent of loss of fluorescence anisotropy using appropriately placed excitation and emission polarizers Fig. 3.6 . Due to a finite spread of allowed angular dependence between the donor and acceptor...
The Caveolin Scaffold Hypothesis
The scaffold hypothesis is a widely cited early interpretation of the link between caveolin and caveola-associated proteins 84 . This hypothesis proposes that proteins associated in vivo with caveolin can be identified by a scaffold recognition sequence that is responsible for their co-assembly, and identifies the protein-protein contact domain. Based on the selectivity of a peptide library, the scaffold sequence motif was identified as -FxxFxxxxF-, FxxxxF xxF- or -fxfxxxxf- where F is an...
References Jyp
1 Hooper NM. Detergent-insoluble glycos-phingolipid cholesterol-rich membrane do mains, lipid rafts and caveolae review . Mol. Membr. Biol. 16 145-156, 1999. 2 Iwabuchi K, Handa K, Hakomori S. Separation of glycosphingolipid-enriched microdomains from caveolar membrane characterized by presence of caveolin. Methods Enzymol. 312 488-494, 2000. 3 Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, Lisanti MP. Caveolins, liquid-ordered domains, and signal transduction....
Fluorescence Recovery After Photobleaching
Fluorescence recovery after photobleaching FRAP is a well-established technique which is used to study the lateral mobility and fluorescence dynamics of proteins in membranes 47,48 . After labeling of the surface molecules with a fluorescent tag, a defined micrometer-sized area on the cell membrane is photobleached irreversible photo-destruction within a very short time, using a focused laser beam. This is followed by an observation of the recovery of fluorescence in the bleached area by...
The Site of PrPc Conversion The Role of Rafts in the Different Intracellular
A major unresolved question is where PrPc-PrPSc transconformation occurs. Both PrPc and PrPSc have been localized at the plasma membrane and have been shown to undergo endocytosis 46,47 , so it is likely that the plasma membrane and or the endolysosomal compartment participate in PrPSc formation. In addition, in the case of inherited diseases, pulse-chase experiments indicate that the pathological conversion of mutant PrP to the PrPSc-like conformer proceeds in a stepwise manner, via a series...
Caveolae May Show Local ShortRange Motility A Role in Transendothelial Transport
In the above-mentioned bleaching studies a special FRAP protocol was included in which the entire interior of the cells was bleached, leaving only a narrow, fluorescent rim of GFP-tagged caveolin at the cell periphery 31 . These FRAP experiments highlighted two important points The total fluorescence intensity of the peripheral rim including caveolae at the plasma membrane decreased only very slowly, further supporting the notion that caveolae are not becoming efficiently internalized see also...
PrPc and Prion Diseases
Transmissible spongiform encephalopathies also known as prion diseases have become an interesting example of how lipid rafts are involved in regulating protein trafficking and processing for a review, see 26 . Prion diseases include a large group of neurodegenerative disorders including scrapie in sheep and goats, bovine spongiform encephalopathies, chronic wasting disease in deer and elk, and Creutzfeldt-Jakob disease CJD in humans 27-29 . These pathologies are caused by an agent consisting of...
Role of Rafts in Proteolytic Attack on PrPc
PrPc is a complex multi-domain protein that contains potential toxic sequences PrPc 106-126 78 . It has been shown that the PrPc 106-126 peptide could trigger neurotoxicity and lead to an apoptotic response by insertion into membranes 79 . Like APP, PrPc undergoes protein kinase C PKC -regulated proteolysis by identical proteases of the disintegrin family, which also inactivate the peptide. Indeed, the normal cleavage of PrPc, directed by ADAM10 a disintegrin and metalloprotease occurs inside...
SingleMolecule Studies
Single-particle tracking studies, which involves observing the motion of a single molecule tagged with nanometer-sized colloidal gold or fluorescent dye 35 , has been used to elucidate the fine structure of biological membranes Fig. 3.4 . Sheets and coworkers used video-enhanced bright field microscopy on 40-nm colloidal gold labeled to antibodies against Thy-1 or cholera toxin B CtxB subunit to study the movement of Thy-1 or GM1 respectively, in C3H 10T1 2 fibroblasts 36 . They divided the...
Caveolae and Membrane Tension MechanoSensitivity and MechanoRegulation
So far, we have two possible mechanisms by which invaginated membrane domains may form at the plasma membrane. The raft model of Section 2.3 tells us that a phase separation in the membrane promotes membrane curvature because of chemical immiscibility, the driving force is the domain line tension. The curvature instability model of Section 2.5, takes the point of view that membrane curvature promotes the phase separation because of the aggregation of proteins into patches of preferred...