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K Simons

Publications and source records attributed to K Simons.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons.

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called beta-amyloid (Abeta) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Abeta that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-beta-cyclodextrin, we show that the formation of Abeta is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Abeta formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Abeta formation to occur and imply a link between cholesterol, Abeta, and Alzheimer's disease.

Alzheimer Disease↗
PubMed1998

Lipid domain structure of the plasma membrane revealed by patching of membrane components.

Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.

Alkaline Phosphatase↗
PubMed1998

Neuronal polarity: essential role of protein-lipid complexes in axonal sorting.

The viral glycoprotein hemagglutinin (HA) and the endogenous glycosylphosphatidylinositol-anchored protein Thy-1 are efficiently targeted to the axonal surface of fully polarized hippocampal neurons in culture. Here we have shown that in these cells HA and Thy-1 interact with sphingolipid-cholesterol rafts and are included in detergent-insoluble glycolipid-enriched complexes. Axonal HA and Thy-1, but not two dendritic membrane proteins, resisted extraction to detergents at 4 degrees C. Both HA and Thy-1 became detergent-soluble in neurons with reduced levels of cholesterol or sphingolipids. Missorting of the axonal Thy-1 but not of a dendritic membrane protein occurred in sphingolipid-deprived cells. These results indicate that neurons sort a subset of axolemmal proteins by a mechanism that requires the formation of protein-lipid rafts. The involvement of rafts in axonal membrane sorting may explain the neurological deficits observed in patients with certain types of Niemann-Pick disease.

Animals↗
PubMed1998

Cholesterol is required for surface transport of influenza virus hemagglutinin.

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid-cholesterol rafts. By reducing the cholesterol level of living cells by 60-70% with lovastatin and methyl-beta-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-beta-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.

Animals↗
PubMed1998

Caveolin-1 and -2 in the exocytic pathway of MDCK cells.

We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.

Amino Acid Sequence↗
PubMed1998

Protein and lipid sorting from the trans-Golgi network to the plasma membrane in polarized cells.

The targeting of proteins and lipids to the cell surface domains of polarized cells is not a simple bulk flow process but requires sorting into distinct apical and basolateral pathways from the trans-Golgi network. Here, we describe the sorting determinants in the cargo molecules, the cellular sorting machineries responsible for the hierarchical read-out of the signals, and the mechanisms of cargo delivery. Furthermore, we discuss the implications of these findings for protein-lipid interactions in other cellular machineries.

Animals↗
PubMed1997

Interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain.

Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes.

Amino Acid Sequence↗
PubMed1997

Functional rafts in cell membranes.

A new aspect of cell membrane structure is presented, based on the dynamic clustering of sphingolipids and cholesterol to form rafts that move within the fluid bilayer. It is proposed that these rafts function as platforms for the attachment of proteins when membranes are moved around inside the cell and during signal transduction.

Animals↗
PubMed1997

Local interactions and the optimization of protein folding.

The role of local interactions in protein folding has recently been the subject of some controversy. Here we investigate an extension of Zwanzig's simple and general model of folding in which local and nonlocal interactions are represented by functions of single and multiple conformational degrees of freedom, respectively. The kinetics and thermodynamics of folding are studied for a series of energy functions in which the energy of the native structure is fixed, but the relative contributions of local and nonlocal interactions to this energy are varied over a broad range. For funnel shaped energy landscapes, we find that 1) the rate of folding increases, but the stability of the folded state decreases, as the contribution of local interactions to the energy of the native structure increases, and 2) the amount of native structure in the unfolded state and the transition state vary considerably with the local interaction strength. Simple exponential kinetics and a well-defined free energy barrier separating folded and unfolded states are observed when nonlocal interactions make an appreciable contribution to the energy of the native structure; in such cases a transition state theory type approximation yields reasonably accurate estimates of the folding rate. Bumps in the folding funnel near the native state, which could result from desolvation effects, side chain freezing, or the breaking of nonnative contacts, significantly alter the dependence of the folding rate on the local interaction strength: the rate of folding decreases when the local interaction strength is increased beyond a certain point. A survey of the distribution of strong contacts in the protein structure database suggests that evolutionary optimization has involved both kinetics and thermodynamics: strong contacts are enriched at both very short and very long sequence separations.

Kinetics↗
PubMed1997

Identification of components of trans-Golgi network-derived transport vesicles and detergent-insoluble complexes by nanoelectrospray tandem mass spectrometry.

Epithelial cells have to deliver newly synthesized proteins to the apical and the basolateral plasma membrane domains of the polarized cell surface. Sorting takes place in the trans-Golgi network and at least two vesicular carriers exist for apical and basolateral delivery. After immuno-isolation, the composition of these vesicle preparations was analyzed by two-dimensional (2-D) gel electrophoresis and detergent extraction. In this paper we compare the constituents of detergent-insoluble complexes in different cell lines of polarized or nonpolarized origin and present the identification of five previously uncharacterized proteins. We show that our protein identification strategy can be successfully applied to the problem of small hydrophobic proteins from organisms that have not been substantially sequenced. The high sensitivity of nanoelectrospray tandem mass spectrometry allowed us to identify two proteins that belong to the p23/p24 family of putative cargo receptors for vesicular trafficking. Furthermore we have mapped CD9 and CD81, two members of a large family of proteins consisting of highly hydrophobic four transmembrane proteins. In addition we have identified caveolin-2 as a constituent of basolateral transport vesicles. We have also extended our analysis of immuno-isolated vesicles to a more basic pI range and show that this region on 2-D gels is devoid of proteins. With these approaches and with the previously published data we have now identified most of the major low molecular weight proteins recovered in detergent-insoluble glycolipid-enriched complexes.

Amino Acid Sequence↗
PubMed1997

Mapping the protein composition of trans-Golgi network (TGN)-derived carrier vesicles from polarized MDCK cells.

In polarized MDCK cells, proteins and lipids are sorted in the trans-Golgi network /TGN) and packaged into different vesicular carriers that are delivered to the apical or basolateral cell surface. To gain insight into the sorting and trafficking machinery, we have previously isolated TGN-derived carrier vesicles from perforated MDCK cells. The composition of immuno-isolated apical and basolateral carriers was mapped by two-dimensional (2-D) gel electrophoresis. Here we describe the identification of several components of the vesicle fraction by using three different methods. 2-D gel comigration was performed with carrier vesicles isolated from metabolically labeled MDCK cells and human epidermal keratinocyte lysates. This allowed us to assign eleven known components by a comparison with the comprehensive keratinocyte 2-D gel database. These comprised two members of the 14-3-3 family of proteins that have been implicated in vesicular trafficking. Five proteins were purified from preparative 2-D gels and identified by peptide microsequencing, including the beta1 and beta2 subunit of trimeric G proteins and an annexin II variant. A member of the SNARE family of proteins was identified by immunoblotting. The combination of 2-D gel electrophoresis and 2-D gel databases allows the rapid assessment of the purity of subcellular fractions and to characterize components involved in vesicular transport.

Amino Acid Sequence↗
PubMed1997

Robert Feulgen Lecture 1997. Lipid microdomains and membrane trafficking in mammalian cells.

This overview summarizes the data for how epithelial cells sort and deliver proteins and lipids to the apical and basolateral cell surface domains. The basolateral pathway uses a Rab-SNARE mechanism for docking and fusion, while the apical route employs a different machinery. This latter mechanism is based on lipid microdomains, composed of clusters of sphingolipids and cholesterol, which function as rafts for apical delivery. The sphingolipid-cholesterol raft mechanism seems to be employed generally by mammalian cells to transport raft-associated proteins to their post-Golgi destinations.

Animals↗
PubMed1997

Penalization versus part-time occlusion and binocular outcome in treatment of strabismic amblyopia.

OBJECTIVE: The purpose of the study is to compare the visual outcome of occlusion versus penalization treatment of strabismic amblyopia, with particular attention to binocularity outcome. DESIGN: The study design was a retrospective study. PARTICIPANTS: Patients with strabismic amblyopia, 75 receiving penalization alone, 87 with a history of occlusion treatment who were later treated by penalization, and 30 treated by means of part-time occlusion (2 to 6 hours/day) participated in this study. MAIN OUTCOME MEASURES: Logarithm of the minimum angle of resolution (logMAR) visual acuity and binocularity index were measured. RESULTS: No statistically significant difference was found between outcomes for the penalization groups with and without a history of occlusion, either by univariate analysis or by multivariate analysis controlling for initial-visit age, acuity, and binocularity status. One marginally significant outcome difference was found between the pure penalization and part-time occlusion groups by univariate analysis, but no significant difference was found in the multivariate analyses controlling for the same three variables at the initial visit. All visual outcome differences between the pure penalization and part-time occlusion groups were less than 1 logMAR line visual acuity or less than a half-unit on the binocularity index. CONCLUSIONS: The study provided no evidence of a difference in visual function outcome between penalization and occlusion, in terms of either statistical or clinical significance, although limitations of the patient samples used preclude these data from showing conclusively that there was no such difference. The lack of any other study adequately comparing these two treatment methods, in combination with the current study's demonstration of the difficulty of making adequate retrospective-based comparison despite a large patient base (n = 1413), suggests that a large prospective, randomized comparative treatment trial is needed. If atropine penalization, with its high acceptability to patients and parents, is found to produce results comparable with those of occlusion in cases of mild-to-moderate amblyopia, as the current and previous smaller studies suggest, then reconsideration of the standard of care for such amblyopia cases is indicated.

Accommodation, Ocular↗
PubMed1997

Full-time atropine, intermittent atropine, and optical penalization and binocular outcome in treatment of strabismic amblyopia.

OBJECTIVE: The purpose of the study is to evaluate the monocular and binocular outcome of three types of "penalization" (blurring of the sound eye) treatment of amblyopia: traditional full-time atropine or optical penalization and a new intermittent atropine regimen involving atropine instillation 1 to 3 days a week. DESIGN: The study design was a retrospective study. PARTICIPANTS: A total of 163 patients with strabismic amblyopia treated by full-time atropine (n = 38), intermittent atropine (n = 73), or optical (n = 52) penalization participated. MAIN OUTCOME MEASURES: Logarithm of the minimum angle of resolution (logMAR) visual acuity, and binocularity index were determined. RESULTS: All three forms of penalization produced statistically significant mean reduction in amblyopia (1.7-2.7 logMAR lines) and mean improvement in binocularity by the end-of-treatment or long-term follow-up visit or both, with minimal mean loss after discontinuation or slight mean improvement on these measures at long-term mean follow-up of 1.9 to 4 years across groups. Few patients achieved high-grade stereoacuity. Compliance was high. Comparable efficacy was found for all three treatment groups after controlling for age, depth of amblyopia, and binocularity at the initial visit. Initial-visit amblyopia depth was strongly and significantly associated with amblyopia depth at both post-treatment visits. Pretreatment and post-treatment binocularity showed a similar strong relationship. Surprisingly, however, there was no consistent or significant association found between depth of amblyopia and binocularity in any visit combination. Post-treatment measures of these two variables also were not associated with initial-visit age or refractive error at any clinically significant level. Mean treatment duration was 1.1 to 2.9 years and was not found to be associated with visual outcome. Amblyopia reversal was found in one (full-time atropine) case at a clinically important level. CONCLUSIONS: The authors confirmed previous reports of penalization's efficacy as a primary treatment of moderate amblyopia (20/100 or better acuity) and, in some cases, relatively severe amblyopia (>20/100) and also confirmed its ability to significantly improve mean binocularity. Amblyopia and binocularity appear to respond to treatment independently and, within the postinfancy age range of the sample studied, the responses appear to be independent of initial-visit age. The high acceptability to patients and parents of atropine penalization, and particularly of the intermittent regimen introduced here, suggests the need for prospective-study-based re-evaluation of the relative merits of penalization and occlusion as the standard of care for mild-to-moderate amblyopia.

Accommodation, Ocular↗
PubMed1997

Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains.

There is accumulating evidence that lateral assemblies (rafts) of sphingolipids and cholesterol form platforms that serve to support numerous cellular events in membrane traffic and signal transduction. Raft membrane microdomains are thought to function by preferentially associating with specific proteins while excluding others. The basic forces driving raft formation are lipid interactions which are, per se, weak and transient. Sphingolipid rafts should therefore be considered to be dynamic structures in which cholesterol plays an important role as a linker. Caveolins influence these dynamics by forming stabilized raft domains in intracellular membranes as well as at the plasma membrane. Recent data suggest that clustering of raft components could regulate raft dynamics and therefore represents an important feature in the function of these membrane microdomains.

Animals↗
PubMed1997

Myosin II is associated with Golgi membranes: identification of p200 as nonmuscle myosin II on Golgi-derived vesicles.

A variety of peripheral membrane proteins associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells. A monoclonal antibody (AD7) raised against Golgi membranes recognizes a peripheral membrane protein, p200, which associates with vesicles budding off the trans-Golgi network (TGN). Based on preliminary findings, a potential association between p200 and myosin on Golgi membranes was investigated. Immunofluorescence staining of cultured cells under a variety of fixation conditions was carried out using an antibody raised against chick brush border nonmuscle myosin II. We show that, in addition to being found in the cytoplasm or associated with stress fibres, nonmuscle myosin II is also specifically localized on Golgi membranes. Myosin II was also detected on Golgi membranes by immunoblotting and by immunogold labeling at the electron microscopy level where it was found to be concentrated on Golgi-derived vesicles. The association of myosin II with Golgi membranes is dynamic and was found to be enhanced following activation of G proteins. Myosin II staining of Golgi membranes was also disrupted by brefeldin A (BFA). Colocalization of the AD7 and myosin II antibodies at the light and electron microscopy levels led us to investigate the nature of the 200 kDa protein recognized by both antibodies. The 200 kDa protein immunoprecipiated by the AD7 antibody was isolated from MDCK cells and used for microsequencing. Amino acid sequence data enabled us to identify p200 as the heavy chain of nonmuscle myosin IIA. In addition, an extra protein (240 kDa) recognized by the AD7 antibody specifically in extracts of HeLa cells, was sequenced and identified as another actin-binding protein, filamin. These results show that nonmuscle myosin II is associated with Golgi membranes and that the vesicle-associated protein p200, is itself a heavy chain of myosin II.

Actins↗
PubMed1997

Post-Golgi biosynthetic trafficking.

Eukaryotic cells have developed complex machineries to distribute proteins and lipids from the Golgi complex. Contrary to what has originally been postulated, delivery of proteins to the cell surface is not a simple bulk flow process but involves sorting into distinct pathways from the trans-Golgi network. Here we describe the various routes emerging from the trans-Golgi network in different cell types, and we discuss the mechanisms that mediate sorting into these pathways. While much remains to be learned about these sorting mechanisms, it is apparent that a number of pathways previously believed to be restricted to certain cell types might be used more commonly.

Animals↗