Sunimar>'. A mathematical model is presenied which may be applied to describe and analyse daia from microscopic phagocyiosis assays. The meihod has been used to investigate the phagocytosis of opsonized yeast by peripheral blood neutrophils Ireatcd with purified recombinam human granulocyte-macrophage colony-stimulating factor {rH GM-CSF) m vitro. Under limiting conditions of scrum opsonization, rH GM-CSF decreased the proportion of non-phagocyiic cells and increased the mean number of ingested yeast per cell. Slimulation of phagocytosis was dose-dependent and occurred with concentrations of rH GM-CSF in the range 10-320 uniis/ml. The effect was dependent on a heat-labile component in serum and was not attributable to endotoxin contamination of the preparation.

Phosphorylation of the negative regulatory element of the tyrosine kinase Lck by Csk downmodulates T-cell receptor induced signalling. Being constitutively active, Csk spatial organization is responsible for regulating this signalling interaction. Here, we used stoichiometrically accurate, multiplexed, single-molecule super-resolution microscopy (DNA-qPAINT) to image the nanoscale spatial architecture of Csk and two binding partners implicated in its membrane association -PAG and TRAF3. Combined with a newly developed co-clustering analysis framework, we provide a powerful resource for dissecting signalling pathways regulated by spatio-temporal organisation. We found that Csk forms nanoscale clusters proximal to the plasma membrane that are lost post-stimulation and rerecruited at later time points. Unexpectedly, these clusters do not directly co-localise with PAG at the membrane, but instead provide a ready pool of monomers to down-regulate signalling. By generating CRISPR/Cas9 knock-out T-cells, our data also identify that protein tyrosine phosphatase non-receptor type 22 (PTPN22) is essential for Csk nanocluster rerecruitment and for maintenance of the synaptic PAG population.