SCIMP is a spatiotemporal transmembrane scaffold for Erk1/2 to direct pro-inflammatory signaling in TLR-activated macrophages

Immune cells are armed with Toll-like receptors (TLRs) for sensing and responding to pathogens and other danger cues. The role of extracellular-signal-regulated kinases 1/2 (Erk1/2) in TLR signaling remains enigmatic, with both pro- and anti-inflammatory functions described. We reveal here that the immune-specific transmembrane adaptor SCIMP is a direct scaffold for Erk1/2 in TLR pathways, with high-resolution, live-cell imaging revealing that SCIMP guides the spatial and temporal recruitment of Erk2 to membrane ruffles and macropinosomes for pro-inflammatory TLR4 signaling. SCIMP-deficient mice display defects in Erk1/2 recruitment to TLR4, c-Fos activation, and pro-inflammatory cytokine production, with these effects being phenocopied by Erk1/2 signaling inhibition. Our findings thus delineate a selective role for SCIMP as a key scaffold for the membrane recruitment of Erk1/2 kinase to initiate TLR-mediated pro-inflammatory responses in macrophages

Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion.

IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion

Fast automated cell phenotype image classification

BACKGROUND: The genomic revolution has led to rapid growth in sequencing of genes and proteins, and attention is now turning to the function of the encoded proteins. In this respect, microscope imaging of a protein's sub-cellular localisation is proving invaluable, and recent advances in automated fluorescent microscopy allow protein localisations to be imaged in high throughput. Hence there is a need for large scale automated computational techniques to efficiently quantify, distinguish and classify sub-cellular images. While image statistics have proved highly successful in distinguishing localisation, commonly used measures suffer from being relatively slow to compute, and often require cells to be individually selected from experimental images, thus limiting both throughput and the range of potential applications. Here we introduce threshold adjacency statistics, the essence which is to threshold the image and to count the number of above threshold pixels with a given number of above threshold pixels adjacent. These novel measures are shown to distinguish and classify images of distinct sub-cellular localization with high speed and accuracy without image cropping. RESULTS: Threshold adjacency statistics are applied to classification of protein sub-cellular localization images. They are tested on two image sets (available for download), one for which fluorescently tagged proteins are endogenously expressed in 10 sub-cellular locations, and another for which proteins are transfected into 11 locations. For each image set, a support vector machine was trained and tested. Classification accuracies of 94.4% and 86.6% are obtained on the endogenous and transfected sets, respectively. Threshold adjacency statistics are found to provide comparable or higher accuracy than other commonly used statistics while being an order of magnitude faster to calculate. Further, threshold adjacency statistics in combination with Haralick measures give accuracies of 98.2% and 93.2% on the endogenous and transfected sets, respectively. CONCLUSION: Threshold adjacency statistics have the potential to greatly extend the scale and range of applications of image statistics in computational image analysis. They remove the need for cropping of individual cells from images, and are an order of magnitude faster to calculate than other commonly used statistics while providing comparable or better classification accuracy, both essential requirements for application to large-scale approaches

Visualizing and clustering high throughput sub-cellular localization imaging

The expansion of automatic imaging technologies has created a need to be able to efficiently compare and review large sets of image data. To enable comparisons of image data between samples we need to define the normal variation within distinct images of the same sample