Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts

Cell Communication and Signaling, Dec 2011

Background Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. Results Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. Conclusions Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

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Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts

Cell Communication and Signaling Analysis of detergent-free lipid rafts isolated from + CD4 T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts Colleen Kennedy 0 2 Matthew D Nelson 0 1 Anil K Bamezai 0 0 Department of Biology, Villanova University , 800 Lancaster Avenue, Villanova, PA 19085 , USA 1 Current Address: Department of Neurology, University of Pennsylvania , 415 Curie Blvd., Philadelphia, PA 19104 , USA 2 Current Address: Physician Assistant Program, Cornell Medical College , 1300 York Avenue, New York, NY 10065 , USA Background: Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. Results: Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. Conclusions: Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen. raft coalescence; CD4+ T cells; antigen presenting cells; electron microscopy; raft-ELISA - Background Signals emanating from the plasma membrane have spatial and temporal components [1-5]. Spatial distribution and accessibility of signaling proteins on the plasma membrane can potentially have profound effects on the outcome of signaling. While knowledge of temporal signaling events has rapidly advanced, the spatial distribution of signaling proteins remains unclear. More so, how the spatial distribution of signaling molecules relates to temporal signaling is unknown. However, recently, re-organization on the plasma membrane of quiescent cells was recognized after triggering signaling from the membrane [6-11]. Lipid raft membrane domains are rich in cholesterol and sphingolipids and known to compartmentalize signaling proteins [12-17]. Heterogeneity of lipid rafts, with respect to protein composition, on the plasma membrane may provide an additional level of spatial segregation [18-26]. Ligand and receptor induced molecular interactions on the plasma membrane trigger a signaling cascade that culminates into specific gene expression. Compositional heterogeneity of lipid rafts on the surface of quiescent cells and their subsequent coalescence, when the receptors engage their ligands, might promote interactions between appropriate signaling proteins [14,27]. However, this is only one of several proposed models to explain signal transduction from the plasma membrane to the interior of the cell [28-35]. Lipid rafts assemble to form an immunological synapse, a central structure at the contact site of CD4+ T cells and antigen presenting cells involved in regulating cell signaling [36-45]. These early signaling events are crucial in generating a response by T cells, especially since CD4+ T cells are capable of generating specific cellular responses after the engagement of the same antigen receptor, ranging from differentiation to Th1 or Th2 or Th17 (T helper cell subsets). In light of the observation that lipid rafts are compositionally heterogeneous, it remains unclear whether distinct sub-populations of rafts assemble at or around the synapse and thus, contribute to signal transduction and distinct cellular responses. Methods allowing enumeration of lipid rafts as on a single raft and sub-population basis in quiescent, activated, and differentiating cells will aid in addressing the role of lipid rafts in signaling. To enumerate lipid rafts in T cells, we have used a published detergent-free isolation procedure [46]. Lipid rafts isolated from a T cell line in the presence and absence of a specific antigen were visualized by transmission electron microscopy. It was surprising to find that lipid rafts isolated from co-cultures of CD4+ T cell and antigen presenting cells in the absence of antigen show raft coalescence/clustering. Materials and methods Cell Culture Mouse CD4+ T-T hybrid of Th1 phenotype YH16.33 [47] and A20 [48] cell lines (generous gifts from Dr. Ken Rock, University of Massachusetts Medical Ctr, MA) were grown in Dulbeccos modified eagle medium (DMEM) with 4.5 g/ml of glucose (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum, L-glutamine (Atlanta Biologicals, Atlanta, GA), sodium pyruvate, penicillin/streptomycin, and (...truncated)


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Colleen Kennedy, Matthew D Nelson, Anil K Bamezai. Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts, Cell Communication and Signaling, 2011, pp. 31, 9, DOI: 10.1186/1478-811X-9-31