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
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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)