Dataset Information

Isolation of detergent resistant microdomains from cultured neurons: detergent dependent alterations in protein composition.

ABSTRACT:

Background

Membrane rafts are small highly dynamic sterol- and sphingolipid-enriched membrane domains that have received considerable attention due to their role in diverse cellular functions. More recently the involvement of membrane rafts in neuronal processes has been highlighted since these specialized membrane domains have been shown to be involved in synapse formation, neuronal polarity and neurodegeneration. Detergent resistance followed by gradient centrifugation is often used as first step in screening putative membrane raft components. Traditional methods of raft isolation employed the nonionic detergent Triton X100. However successful separation of raft from non-raft domains in cells is dependent on matching the detergent used for raft isolation to the specific tissue under investigation.

Results

We report here the isolation of membrane rafts from primary neuronal culture using a panel of different detergents that gave rise to membrane fractions that differed in respect to cholesterol and protein content. In addition, proteomic profiling of neuronal membrane rafts isolated with different detergents, Triton X100 and CHAPSO, revealed heterogeneity in their protein content.

Conclusions

These data demonstrate that appropriate selection of detergent for raft isolation is an important consideration for investigating raft protein composition of cultured neurons.

SUBMITTER: Williamson R

PROVIDER: S-EPMC2955047 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Publications

Isolation of detergent resistant microdomains from cultured neurons: detergent dependent alterations in protein composition.

Williamson Ritchie R Thompson Andrew J AJ Abu Mika M Hye Abdul A Usardi Alessia A Lynham Steven S Anderton Brian H BH Hanger Diane P DP

BMC neuroscience 20100922

<h4>Background</h4>Membrane rafts are small highly dynamic sterol- and sphingolipid-enriched membrane domains that have received considerable attention due to their role in diverse cellular functions. More recently the involvement of membrane rafts in neuronal processes has been highlighted since these specialized membrane domains have been shown to be involved in synapse formation, neuronal polarity and neurodegeneration. Detergent resistance followed by gradient centrifugation is often used as ...[more]

PMID: 20858284

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Project description:Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum.

Dataset Information

Isolation of detergent resistant microdomains from cultured neurons: detergent dependent alterations in protein composition.

Background

Results

Conclusions

Publications

Isolation of detergent resistant microdomains from cultured neurons: detergent dependent alterations in protein composition.

Similar Datasets

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