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K Simons

Publications and source records attributed to K Simons.

At least 19 recordsLinked to original sources
PubMed2001

Cholesterol depletion reduces apical transport capacity in epithelial Madin-Darby canine kidney cells.

Reduction of the cholesterol level in membranes of epithelial Madin-Darby canine kidney (MDCK) cells reverses the apical-to-basolateral transport ratio of the apical membrane marker protein influenza virus haemagglutinin and the secreted glycoprotein gp80. At the same time, basolateral transport of the vesicular stomatitis virus G protein is unaffected [Keller and Simons (1998) J. Cell Biol. 140, 1357-1367]. To investigate whether cholesterol depletion influences apical sorting mechanisms specifically, or apical transport capacity more generally, we studied the effect of cholesterol depletion on the secretion of three different classes of molecules from the apical and basolateral surfaces of MDCK cell layers: glycoprotein gp80, sulphated proteoglycans and proteins, and non-glycosylated rat growth hormone. In each case, cholesterol depletion reduced the fraction secreted to the apical medium and increased the fraction secreted basolaterally. The fact that this was observed for all sulphated proteins and proteoglycans and for the non-glycosylated rat growth hormone, which is randomly secreted in untreated cells, indicates that cholesterol depletion reduces the apical transport capacity, rather than interfering with specific recognition and sorting processes.

Animals↗
PubMed2001

Raft-partitioning of the ubiquitin ligases Cbl and Nedd4 upon IgE-triggered cell signaling.

The high affinity receptor for IgE, FcepsilonRI on mast cells and basophils plays an essential role in immunological defense. Upon multivalent antigen binding, FcepsilonRI becomes phoshorylated by the protein-tyrosine kinase Lyn, as a result of receptor clustering in lipid rafts. FcepsilonRI has been shown to be ubiquitinated. Ubiquitination can lead to degradation by proteasomes, but it can also act as a sorting signal to internalize proteins destined to the endosomal/lysosomal pathway. We have analyzed whether FcepsilonRI ubiquitination takes place within rafts. We report biochemical and imaging evidence in rat basoleukemia cells for the presence of ubiquitinated FcepsilonRI in clustered rafts upon receptor activation. Moreover, we demonstrated that the ubiquitin ligases Cbl and Nedd4 colocalize with FcepsilonRI patches and showed that both ligases become associated with lipid rafts after activation of IgE signaling. Because Cbl is known to interact with the FcepsilonRI signaling complex, ubiquitination is likely to be an important parameter regulating IgE-triggered signaling occurring in rafts.

Animals↗
PubMed2001

Multicolour imaging of post-Golgi sorting and trafficking in live cells.

The biogenesis and maintenance of asymmetry is crucial to many cellular functions including absorption and secretion, signalling, development and morphogenesis. Here we have directly visualized the segregation and trafficking of apical (glycosyl phosphatidyl inositol-anchored) and basolateral (vesicular stomatitis virus glycoprotein) cargo in living cells using multicolour imaging of green fluorescent protein variants. Apical and basolateral cargo segregate progressively into large domains in Golgi/trans-Golgi network structures, exclude resident proteins, and exit in separate transport containers. These remain distinct and do not merge with endocytic structures suggesting that lateral segregation in the trans-Golgi network is the primary sorting event. Fusion with the plasma membrane was detected by total internal reflection microscopy and reveals differences between apical and basolateral carriers as well as new 'hot spots' for exocytosis.

Adenoviridae↗
PubMed2001

Lessons in science politics.

Today, biology has become a big science, which devours huge amounts of money. In the United States, a phenomenal growth in biomedical science funding has paralleled the increase in research costs. But this did not occur spontaneously, and the American research community has had to learn how to influence budget policy. In Europe, we are now also faced with a similar problem and need to learn how to campaign for better research funding.

Academies and Institutes↗
PubMed2001

Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast.

Correct sorting of proteins is essential to generate and maintain the identity and function of the different cellular compartments. In this study we demonstrate the role of lipid rafts in biosynthetic delivery of Pma1p, the major plasma membrane proton ATPase, to the cell surface. Disruption of rafts led to mistargeting of Pma1p to the vacuole. Conversely, Pma1-7, an ATPase mutant that is mistargeted to the vacuole, was shown to exhibit impaired raft association. One of the previously identified suppressors, multicopy AST1, not only restored surface delivery but also raft association of Pma1-7. Ast1p, which is a peripheral membrane protein, was found to directly interact with Pma1p inducing its clustering into a SDS/Triton X100-resistant oligomer. We suggest that clustering facilitates partition of Pma1p into rafts and transport to the cell surface.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Depletion of rafts in late endocytic membranes is controlled by NPC1-dependent recycling of cholesterol to the plasma membrane.

In mammalian cells, cholesterol is thought to associate with sphingolipids to form lateral membrane domains termed rafts. Increasing evidence suggests that rafts regulate protein interactions, for example, during signalling, intracellular transport and host-pathogen interactions. Rafts are present in cholesterol-sphingolipid-enriched membranes, including early and recycling endosomes, but whether rafts are found in late endocytic organelles has not been analyzed. In this study, we analyzed the association of cholesterol and late endosomal proteins with low-density detergent-resistant membranes (DRMs) in normal cells and in cells with lysosomal cholesterol-sphingolipid accumulation. In normal cells, the majority of [(3)H]cholesterol released from [(3)H]cholesterol ester-LDL associated with detergent-soluble membranes, was rapidly transported to the plasma membrane and became increasingly insoluble with time. In Niemann-Pick C1 (NPC1) protein-deficient lipidosis cells, the association of LDL-cholesterol with DRMs was enhanced and its transport to the plasma membrane was inhibited. In addition, the NPC1 protein was normally recovered in detergent-soluble membranes and its association with DRMs was enhanced by lysosomal cholesterol loading. Moreover, lysosomal cholesterol deposition was kinetically paralleled by the sequestration of sphingolipids and formation of multilamellar bodies in late endocytic organelles. These results suggest that late endocytic organelles are normally raft-poor and that endocytosed LDL-cholesterol is efficiently recycled to the plasma membrane in an NPC1-dependent process. The cholesterol-sphingolipid accumulation characteristic to NPC disease, and potentially to other sphingolipidoses, causes an overcrowding of rafts forming lamellar bodies in the degradative compartments.

Androstenes↗
PubMed2000

How cells handle cholesterol.

Cholesterol plays an indispensable role in regulating the properties of cell membranes in mammalian cells. Recent advances suggest that cholesterol exerts many of its actions mainly by maintaining sphingolipid rafts in a functional state. How rafts contribute to cholesterol metabolism and transport in the cell is still an open issue. It has long been known that cellular cholesterol levels are precisely controlled by biosynthesis, efflux from cells, and influx of lipoprotein cholesterol into cells. The regulation of cholesterol homeostasis is now receiving a new focus, and this changed perspective may throw light on diseases caused by cholesterol excess, the prime example being atherosclerosis.

Animals↗
PubMed2000

Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb.

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca(2+)-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca(2+), a approximately 35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca(2+)-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.

Amino Acid Sequence↗
PubMed2000

Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy.

Monitoring the fusion of constitutive traffic with the plasma membrane has remained largely elusive. Ideally, fusion would be monitored with high spatial and temporal resolution. Recently, total internal reflection (TIR) microscopy was used to study regulated exocytosis of fluorescently labeled chromaffin granules. In this technique, only the bottom cellular surface is illuminated by an exponentially decaying evanescent wave of light. We have used a prism type TIR setup with a penetration depth of approximately 50 nm to monitor constitutive fusion of vesicular stomatitis virus glycoprotein tagged with the yellow fluorescent protein. Fusion of single transport containers (TCs) was clearly observed and gave a distinct analytical signature. TCs approached the membrane, appeared to dock, and later rapidly fuse, releasing a bright fluorescent cloud into the membrane. Observation and analysis provided insight about their dynamics, kinetics, and position before and during fusion. Combining TIR and wide-field microscopy allowed us to follow constitutive cargo from the Golgi complex to the cell surface. Our observations include the following: (1) local restrained movement of TCs near the membrane before fusion; (2) apparent anchoring near the cell surface; (3) heterogeneously sized TCs fused either completely; or (4) occasionally larger tubular-vesicular TCs partially fused at their tips.

Bacterial Proteins↗
PubMed2000

Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast.

Lipid rafts, formed by lateral association of sphingolipids and cholesterol, have been implicated in membrane traffic and cell signaling in mammalian cells. Sphingolipids also have been shown to play a role in protein sorting in yeast. Therefore, we wanted to investigate whether lipid rafts exist in yeast and whether these membrane microdomains have an analogous function to their mammalian counterparts. We first developed a protocol for isolating detergent-insoluble glycolipid-enriched complexes (DIGs) from yeast cells. Sequencing of the major protein components of the isolated DIGs by mass spectrometry allowed us to identify, among others, Gas1p, Pma1p, and Nce2p. Using lipid biosynthetic mutants we could demonstrate that conditions that impair the synthesis of sphingolipids and ergosterol also disrupt raft association of Gas1p and Pma1p but not the secretion of acid phosphatase. That endoplasmic reticulum (ER)-to-Golgi transport of Gas1p is blocked in the sphingolipid mutant lcb1-100 raised the question of whether proteins associate with lipid rafts in the ER or later as shown in mammalian cells. Using the sec18-1 mutant we found that DIGs are present already in the ER. Taken together, our results suggest that lipid rafts are involved in the biosynthetic delivery of proteins to the yeast plasma membrane.

Biological Transport↗
PubMed2000

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells.

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.

Alkaline Phosphatase↗
PubMed2000

Induction of caveolae in the apical plasma membrane of Madin-Darby canine kidney cells.

In this paper, we have analyzed the behavior of antibody cross-linked raft-associated proteins on the surface of MDCK cells. We observed that cross-linking of membrane proteins gave different results depending on whether cross-linking occurred on the apical or basolateral plasma membrane. Whereas antibody cross-linking induced the formation of large clusters on the basolateral membrane, resembling those observed on the surface of fibroblasts (Harder, T., P. Scheiffele, P. Verkade, and K. Simons. 1998. J. Cell Biol. 929-942), only small ( approximately 100 nm) clusters formed on the apical plasma membrane. Cross-linked apical raft proteins e.g., GPI-anchored placental alkaline phosphatase (PLAP), influenza hemagglutinin, and gp114 coclustered and were internalized slowly ( approximately 10% after 60 min). Endocytosis occurred through surface invaginations that corresponded in size to caveolae and were labeled with caveolin-1 antibodies. Upon cholesterol depletion the internalization of PLAP was completely inhibited. In contrast, when a non-raft protein, the mutant LDL receptor LDLR-CT22, was cross-linked, it was excluded from the clusters of raft proteins and was rapidly internalized via clathrin-coated pits. Since caveolae are normally present on the basolateral membrane but lacking from the apical side, our data demonstrate that antibody cross-linking induced the formation of caveolae, which slowly internalized cross-linked clusters of raft-associated proteins.

Alkaline Phosphatase↗
PubMed2000

Protein expression in Drosophila Schneider cells.

We expressed recombinant secreted, membrane, and cytosolic proteins in stably transfected Drosophila Schneider (SL-3) cells. To allow easy cloning of N- and C-terminal fusion proteins containing epitope- and His-tags for the detection of recombinant proteins and purification by affinity chromatography we constructed new expression vectors. To exemplify the general applicability of protein expression in Schneider cells we characterized the expression system with respect to inducibility, localization of the recombinant proteins, yields of purified proteins, and presence of posttranslational and cotranslational modifications. Secreted proteins became quantitatively N-glycosylated in SL-3 cells and the N-glycan of a Golgi-resident membrane protein was found to be Endo-H-resistant. Myristoylation of AnxXIIIb, a member of the annexin family, could be demonstrated and glycosylphosphatidylinositol-anchored proteins containing their lipid anchor were expressed efficiently in SL-3 cells. Since generation of stable cell lines and mass culture of SL-3 cells is cheap and easy, they provide an attractive eukaryotic expression system.

Alkaline Phosphatase↗
PubMed2000

Photoscreening.

Explore the source record for details and available documents.

Amblyopia↗
PubMed2000

Jamming the endosomal system: lipid rafts and lysosomal storage diseases.

Some lysosomal storage diseases result from the accumulation of lipids in degradative compartments of the endocytic pathway. Particularly striking is the example of the Niemann-Pick (NP) syndrome. NP syndromes types A and B are characterized by the accumulation of sphingomyelin, whereas cholesterol typically accumulates in NP type C. These two different lipids, sphingomyelin and cholesterol, are normal constituents of specific lipid microdomains called rafts. Because accumulation of raft lipids is observed not only in NP diseases but also in many other lipidoses, we forward the hypothesis that lysosomal storage diseases can be caused by the accumulation of lipid rafts in late endosomes/lysosomes.

Endocytosis↗