A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains.

The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20-50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor.

Two motifs within the tau microtubule-binding domain mediate its association with the hsc70 molecular chaperone.

Tau, a microtubule-associated protein with multiple phosphorylation sites, forms aggregates that correlate with neurodegeneration in Alzheimer's disease and several other neurodegenerative diseases, termed tauopathies. Hsc70 is a highly expressed constitutive chaperone that can drive conformational change in proteins, prevent the aggregation of its substrates, recognize misfolded substrates, and facilitate their degradation. Here, we show that hsc70 binds to the microtubule-binding domain of tau in vitro and in vivo, without an absolute requirement for tau phosphorylation. Binding requires a carboxy-terminal region of hsc70 comprising its peptide-binding and variable domains. We have identified two hsc70 binding sites on tau and hydrophobic amino acids crucial for hsc70 binding. Interestingly, these hsc70 binding sites correspond to the beta-structure elements that have been previously reported to facilitate tau aggregation. Thus, it is possible that hsc70 binding might directly inhibit tau-tau interactions that precede tau oligomerization and aggregation. Our results provide an important stimulus for research into how the hsc70-tau interaction might affect tau fate in normal cells and in disease.

Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

Polymorphism analysis of HOPA: a candidate gene for schizophrenia.

HOPA is a 25 kb Xq13 gene that codes for a member of the thyroid receptor co-activator protein (TRAP) family of nuclear receptor co-activators. In our prior research, polymorphisms in the opposite paired (Opa) domain of HOPA have been associated with a syndrome of aberrant behavior, most prominently psychosis, and hypothyroidism. These Opa domain polymorphisms are intriguing because subsequent research has demonstrated that changes in the Opa domain of the C. elegans orthologue of HOPA results in altered neurogenesis and release of transcriptional suppression. In an effort to determine whether other allelic polymorphisms in this gene exist and may potentially contribute to increased susceptibility to neuropsychiatric illness, we have performed single stranded conformational polymorphism (SSCP) analysis of all 45 exons and each of the two potential promoter regions of HOPA using DNA from a panel of patients with psychosis. We found a rare promoter polymorphism in an individual with schizoaffective disorder and extremely low thyroid stimulating hormone (TSH). The most common exonic polymorphism in HOPA is the previously demonstrated HOPA(12 bp) polymorphism. Transmission disequilibrium analysis of the HOPA(12 bp) polymorphism showed segregation with affected status in six of eight instances. We suggest that this evidence supports previous associations of HOPA(12 bp) with a broad range of neuropsychiatric illness and conclude that further studies of this uncommon polymorphism are merited.