ASACUSA Status Report

ASACUSA progress during 2009 and plans for 201

Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved

Role of cytomegalovirus in driving cytotoxic CD28null T cells

Accumulation of CD28null T cells has been traditionally considered a sign of aging, as the percentage of these cells is increased in the elderly. However, the permanent loss of CD28 on T cells is caused by chronic antigenic stimulation. In that sense, CMV infection seems to be an important factor, particularly in the CD4 T-cell compartment, where significant expansions of these cells have been observed in CMV-seropositive individuals only and independently of age. In contrast to this, the CD28null CD8 T-cell subset is more heterogeneous, consisting of different type of cells with diverse origins, phenotype, and functions. Indeed, contrarily to their CD4 counterparts, CD28null CD8 T cells can be found expanded in the absence of CMV. CD28null CD4 T cells are cytotoxic and produce high amounts of pro-inflammatory cytokines. Expansions of these cells has been shown to be associated with many diseases and seems to have a relevant role in CVD (cardiovascular diseases). We, therefore, propose that CMV-related CVD risk may be mediated in part by CD28null CD4 T-cells, capable of damaging the vasculature