Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein–Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa–1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity

Visualizing Single-Cell Secretion Dynamics with Single-Protein Sensitivity

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein–Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa–1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity

Null Mutation in PGAP1 Impairing Gpi-Anchor Maturation in Patients with Intellectual Disability and Encephalopathy

<div><p>Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene <i>PGAP1</i>. <i>PGAP1</i> was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type <i>PGAP1</i> cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene <i>PGAP1</i>. Our results add <i>PGAP1</i> to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development.</p></div

Molecular modeling of PGAP1.

<p>(A) Model of PGAP1 highlighting the position of Leu197. The two views differ by a rotation of 90° around the horizontal axis. (B) Interactions of Leu197 (green) with residues Leu184 and Ile194 of the hydrophobic core. (C) Interactions of Ile198 (green) in the Leu197del mutant. Clashes with the adjacent amino acids Leu184 and Ile194 are indicated by cyan arrows. Residues 203–316 are not shown in (B) and (C) for reasons of clarity.</p

FACS analysis of GPI-APs on LCLs and their PI-PLC sensitivity.

<p>(A, B) Cells from one of the affected siblings (III-3) and the parents were transfected with empty pMEoriP vector (A) and pMEoriP-FLAG-humanPGAP1 (B). Cells from the healthy sister were used without transfection. Four days after transfection, cells were treated with (solid lines) or without (dotted lines) 10 unit/ml of PI-PLC for 1.5 h at 37°C, and the surface expression of CD59, DAF and CD48 were assessed by flow cytometry.</p

Pedigree of family MR079 and a PGAP1 mutation.

<p>Pedigree of family MR079 and a PGAP1 mutation.</p