Real-time single-cell imaging of protein secretion

Abstract

Protein secretion, a key intercellular event for transducing cellular signals, is thought to be strictly regulated. However, secretion dynamics at the single-cell level have not yet been clarified because intercellular heterogeneity results in an averaging response from the bulk cell population. To address this issue, we developed a novel assay platform for real-time imaging of protein secretion at single-cell resolution by a sandwich immunoassay monitored by total internal reflection microscopy in sub-nanolitre-sized microwell arrays. Real-time secretion imaging on the platform at 1-min time intervals allowed successful detection of the heterogeneous onset time of nonclassical IL-1β secretion from monocytes after external stimulation. The platform also helped in elucidating the chronological relationship between loss of membrane integrity and IL-1β secretion. The study results indicate that this unique monitoring platform will serve as a new and powerful tool for analysing protein secretion dynamics with simultaneous monitoring of intracellular events by live-cell imaging.

Mechanisms of unconventional secretion of IL-1 family cytokines

“…The mechanisms underlying pyroptosis are unknown, but appear to initially involve the appearance of caspase-1-dependent plasma membrane pores of 1.1-2.4 nm diameter [27], followed by dissipation of ionic gradients, cell swelling, and osmotic lysis. A number of studies have highlighted the temporal association of IL-1b secretion and cell lysis [15,28,29], promoting a model in which IL-1b is passively released during cell lysis. This was most recently proposed from studies in which cytokine secretion from single cells was monitored in real-time by total internal reflection fluorescence microscopy [28,29].…”

“…A number of studies have highlighted the temporal association of IL-1b secretion and cell lysis [15,28,29], promoting a model in which IL-1b is passively released during cell lysis. This was most recently proposed from studies in which cytokine secretion from single cells was monitored in real-time by total internal reflection fluorescence microscopy [28,29]. The authors highlighted the tight temporal coupling of IL-1b release and loss of plasma membrane integrity, and showed that cell lysis is the ultimate fate of IL-1b-producing monocyte/macrophages.…”

“…An alternative explanation is that cell lysis and IL-1b secretion are temporally coupled because they both rely on activated caspase-1. Further, the single-cell studies [28,29] did not appear to discriminate between pro-IL-1b (which is passively released upon cell lysis [30][31][32]) and mature IL-1b. Two lines of evidence argue against a model of IL-1b passive release by cell lysis as the major secretory mechanism: (1) we showed that inflammasome-activated neutrophils produce caspase-1-dependent IL-1b but do not undergo caspase-1-dependent cell lysis or passive protein release [33]; and (2) the osmoprotectant glycine blocks osmotic lysis and resultant passive protein release in macrophages without affecting the kinetics of mature IL-1b secretion [27,[30][31][32].…”

Biosensors for Cell Analysis

“…To create a cell-friendly microenvironment, microwell arrays are typically composed of biocompatible materials such as PDMS (49)(50)(51), PEG (52,53), collagen (54), agarose (55), or silicon (56). PDMS, collagen, and agarose-based microarrays can be fabricated via soft lithography approaches.…”

“…Recently, a microfabricated platform was developed for real-time detection of cytokine secretion using total internal reflection fluorescence microscopy (50). IL-1β secreted from stimulated human peripheral blood monocytes was monitored at 1 minute intervals, demonstrating cell-tocell heterogeneity in the onset of cytokine secretion.…”