No abstract

Lipid rafts are membrane platforms that spatially organize molecules for specific signaling pathways that regulate various cellular functions. Cholesterol is critical for liquidordered raft formation by serving as a spacer between the hydrocarbon chains of sphingolipids, and alterations in the cholesterol contents of the plasma membrane causes disruption of rafts. The role that S receptors play in cancer is not clear, although it is frequently up-regulated in human cancer cells and tissues and S receptors inhibit proliferation in carcinoma and melanoma cell lines, induce apoptosis in colon and mammary carcinoma cell lines, and reduce cellular adhesion in mammary carcinoma cell lines. In this study, we provide molecular and functional evidence for the involvement of the enigmatic S1 receptors in lipid raft modeling by S1 receptor-mediated cholesterol alteration of lipid rafts in breast cancer cell lines. Cholesterol binds to cholesterol recognition domains in the COOH terminus of the S1 receptor. This binding is blocked by S receptor drugs because the cholesterol-binding domains form part of the S receptor drugbinding site, mutations of which abolish cholesterol binding. Furthermore, we outline a hypothetical functional model to explain the myriad of biological processes, including cancer, in which these mysterious receptors are involved. The findings of this study provide a biological basis for the potential therapeutic applications of lipid raft cholesterol regulation in cancer therapy using S receptor drugs. [Cancer Res 2007; 67(23):11166-75]

To understand the function of cellular protein networks, spatial and temporal context is essential. Fluorescence correlation spectroscopy (FCS) is a single-molecule method to study the abundance, mobility and interactions of fluorescence-labeled biomolecules in living cells. However, manual acquisition and analysis procedures have restricted live-cell FCS to short-term experiments of a few proteins. Here, we present high-throughput (HT)-FCS, which automates screening and time-lapse acquisition of FCS data at specific subcellular locations and subsequent data analysis. We demonstrate its utility by studying the dynamics of 53 nuclear proteins. We made 60,000 measurements in 10,000 living human cells, to obtain biophysical parameters that allowed us to classify proteins according to their chromatin binding and complex formation. We also analyzed the cell-cycle-dependent dynamics of the mitotic kinase complex Aurora B/INCENP and showed how a rise in Aurora concentration triggers two-step complex formation. We expect that throughput and robustness will make HT-FCS a broadly applicable technology for characterizing protein network dynamics in cells.

HsSAS-6 homodimers are present in the cytoplasm and assemble into ninefold symmetrical arrays at centrosomes, thus initiating procentriole formation.