Visualization of Detergent Solubilization of Membranes: Implications for the Isolation of Rafts

Garner, Ashley E.; Smith, D. A.; Hooper, Nigel M.

doi:10.1529/biophysj.107.114108

Cited by 80 publications

(57 citation statements)

References 45 publications

(80 reference statements)

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“…We therefore next tested the non-micelle forming bile salt taurodehydrocholate [22] as negative control and the bile salt derived detergent, CHAPS (CMC 6mM, [23]) as positive control. Octylglucoside as a microdomain destabilizing detergent was included as a third control (CMC 25mM, [23]). …”

Section: Resultsmentioning

confidence: 99%

Interaction of bile salts with rat canalicular membrane vesicles: Evidence for bile salt resistant microdomains

Guyot

Stieger

2011

Journal of Hepatology

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Section: Resultsmentioning

confidence: 99%

Interaction of bile salts with rat canalicular membrane vesicles: Evidence for bile salt resistant microdomains

Guyot

Stieger

2011

Journal of Hepatology

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“…On the other hand, they stand in contrast to the findings of another study (17) showing that the amount of lipid raft-associated CSK in thymus cells is not affected by the absence of PAG. However, we would like to point out that different detergents and cell/detergent ratios were used in these various studies and that the association of proteins with lipid rafts is sensitive to these parameters (75,76).…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

Dráberová

Potůčková

Hálová

et al. 2014

Molecular and Cellular Biology

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The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN-and/or a FYNdependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved. Mast cells are widely distributed in the body, where they play important roles in innate as well as adaptive immune responses (1). To fulfill their role in adaptive immune responses, the cells express the high-affinity IgE receptor FcεRI on their plasma membranes. Aggregation of this tetrameric immunoreceptor, ␣␤␥2, induces cell signaling events leading to the release of preformed inflammatory mediators and the de novo synthesis and release of leukotrienes, cytokines, and chemokines. The first welldefined biochemical step after FcεRI triggering is tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs in the cytoplasmic tail of the FcεRI ␤ and ␥ subunits by the SRC family protein tyrosine kinase (PTK) LYN (2, 3). The phosphorylated ␤ and ␥ subunits then serve as binding and activation sites for LYN kinase and spleen tyrosine kinase (SYK), respectively. These two enzymes, together with FYN and other kinases, then phosphorylate various adaptor proteins and enzymes with a variety of functions in signal transduction pathways. The exact molecular events preceding LYN-mediated tyrosine phosphorylation of the FcεRI ␤ subunit are not clear, and several models have been proposed, including the transphosphorylation model (4), the lipid raft model (5), and the PTK-protein tyrosine phosphatase (PTP) interplay model (6).Our previous study with murine bone marrow-derived mast cells (BMMCs) showed that FcεRI triggering induced transient hyperphosphorylation of LYN kinase on its C-terminal regulatory tyrosine (Tyr 487), leading to the formation of a closed inactive conformation where the SRC homology 2 (...

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“…Therefore, oligomerization and avidity (multiple binding) of receptors and other signaling molecules is likely to be catalyzed by lipid rafts, which, in principle, is based on a process of lipid aggregation and coalescence forming specific lipid microdomains in the cell membrane. Rafts are defined as lipid microdomains with ordered (Lo) structure in the liquid phase of the membrane [108][109][110][111][112][113][114][115][116][117][118][119][120]. The terms lipid microdomains, rafts and detergent-insoluble membranes are often used interchangeably.…”

Section: The Rising Of the Raft: Interfacing Membrane Biophysics And Cementioning

confidence: 99%

Ceramide signaling in cancer and stem cells

Bieberich

2008

Future Lipidology

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Most of the previous work on the sphingolipid ceramide has been devoted to its function as an apoptosis inducer. Recent studies, however, have shown that in stem cells, ceramide has additional nonapoptotic functions. In this article, ceramide signaling will be reviewed in light of 'systems interface biology': as an interconnection of sphingolipid metabolism, membrane biophysics and cell signaling. The focus will be on the metabolic interconversion of ceramide and sphingomyelin or sphingosine-1-phosphate. Lipid rafts and sphingolipid-induced protein scaffolds will be discussed as a membrane interface for lipid-controlled cell signaling. Ceramide/sphingomyelin and ceramide/ sphingosine-1-phosphate-interdependent cell-signaling pathways are significant for the regulation of cell polarity, apoptosis and/or proliferation, and as novel pharmacologic targets in cancer and stem cells. Keywordsapoptosis; cancer; cell polarity; ceramide; ES cells; lipid rafts; sphingomyelin; sphingosine-1-phosphate Ceramide & its derivatives: from metabolism to cell signalingThe structural core of ceramide is the sphingoid base sphingosine (Figure 1), a lipid that is synthesized from the amino acid serine and the activated fatty acid palmitoyl-CoA (Figure 2). Sphingosine (or sphingenine) was first isolated and characterized by the German pathologist and 'father of neurochemistry ' Johann Ludwig Wilhelm Thudichum (1829-1901 [1,2]. He coined the term sphingolipids, which is derived from 'sphingos', the genitive of Sphinx. In Greek, 'sphingo' means 'to strangle'. According to the legend, a fate of death was meant for everyone who could not solve the riddles posed by the Sphinx. Quite ironically, this is how many sphingolipid researchers may feel when faced with analyzing the function of ceramide. The riddle of ceramide, its function and how it acts as signaling lipid, is far from being solved. To ease our fears when confronted with the Sphinx in sphingolipid biochemistry, a related Greek word, 'sphingein', meaning 'to bind tight', alludes to the possibility of solving the enigma of ceramide by identifying its binding proteins.In addition to ceramide, more than a hundred different sphingolipids are known. The most comprehensive collection of sphingolipids, with respect to their characteristics and structures, can be found at [401], a Webpage supported by the National Institutes of General Medical Sciences [3,4]. Sphingolipids are essential components of cellular membranes and have been (Figure 3) . Among these, their roles as pro-or anti-apoptotic and pro-or anti-proliferative signaling lipids are the most important. Most recently, our group has found that establishment of cell polarity during embryonic development is another biological process regulated by sphingolipids [28]. To define a profile of functions for individual sphingolipids is difficult because of their rapid metabolic interconversion. To add to this predicament, several derivatives of ceramide have functions similar to ceramide itself. Hence, novel techniques are need...

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Visualization of Detergent Solubilization of Membranes: Implications for the Isolation of Rafts

Cited by 80 publications

References 45 publications

Interaction of bile salts with rat canalicular membrane vesicles: Evidence for bile salt resistant microdomains

Interaction of bile salts with rat canalicular membrane vesicles: Evidence for bile salt resistant microdomains

Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

Ceramide signaling in cancer and stem cells

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