A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways

Abstract

Eukaryotic cells have evolved to segregate cellular functions into specialized intracellular membrane compartments. Information regarding the precise intracellular localization of a poorly characterized protein is thus important when evaluating its function. However, localization per se, without additional information, is not sufficient to indicate the potential function of cytoplasmic, or cytoplasmic and nuclear proteins that account for almost half of the proteome diversity, when compared with proteins located in dedicated organelles, such as the Golgi apparatus or lysosomes. To tackle this need for additional information, we have implemented a live-imaging screening strategy that is based on changes in localization of fluorescently labeled cytoplasmic proteins studied upon the activation of cells. Indeed, cytoplasmic and nuclear proteins often respond to the activation of a given signaling cascade by translocating to another cell compartment, for instance by moving from the cytoplasm to the plasma membrane, or to the nucleus. Classical signaling proteins that translocate following receptor activation include AKT/PKB (1), raf1 (2), or NF-KB (3). Therefore, finding that a given cytoplasmic protein responds to the activation of a given signaling cascade provides further insight into its function.

Chloride intracellular channel protein 2 in cancer and non-cancer human tissues: relationship with tight junctions

“…CLIC2 is the least investigated subtype among the CLIC family members. Its known physiological function may be the modulation of ryanodine receptor activities to regulate Ca 2+ -mediated intracellular signaling in skeletal and cardiac muscles 18,19 CLIC2 may form chloride ion channels in vitro when it is inserted into an artificial lipid bilayer 7 Compared with the localization of CD31 or PECAM, a transmembrane protein belonging to the immunoglobulin superfamily, 20 CLIC2 was distributed in the cytoplasm and not as a transmembrane protein as described previously, 21 suggesting that CLIC2 does not function as a chloride channel in the plasma membrane. Tight junctions and CLIC2; relationship with the hematogenous spread of cancer Endothelia in the blood vessels of non-cancer tissues express tight junction proteins claudins 1 and 5, ZO-1 and occludin in addition to CLIC2 as revealed by immunohistochemical staining.…”

Identification of a Munc13-sensitive step in chromaffin cell large dense-core vesicle exocytosis

“…We detected three endogenously expressed isoforms in murine chromaffin cells, Munc13-1, ubMunc13-2, and Baiap3 ( Figure 1 ). Baiap3, somewhat surprisingly given its prominent expression and ability to translocate to membranes in a Ca 2+ -dependent manner ( Lecat et al, 2015 ), does not appear to be involved in LDCV priming in this cell type. However, Munc13-4, which regulates SNARE- mediated vesicle exocytosis in the hematopoietic system ( Boswell et al, 2012 ; Feldmann et al, 2003 ; Shirakawa et al, 2004 ), and is the closest relative of Baiap3 ( Koch et al, 2000 ), can promote LDCV priming, albeit less efficiently than Munc13-1 and ubMunc13-2.…”

Identification of EGFLAM, SPATC1L and RNASE13 as novel susceptibility loci for aortic aneurysm in Japanese individuals by exome-wide association studies

“…The SPATC1L is located at chromosomal region 21q22.3 (NCBI Gene) and is expressed in various tissues and organs including vascular smooth muscle (The Human Protein Atlas). SPATC1L is distributed in the cytoplasm, nucleus, and perinuclear region of cells, and it translocates to the sites of cell-cell junctions in response to stimulation of cells with the neuropeptide neurokinin A ( 24 ). Expression of SPATC1L was also found to modulate the response of cells to N -methyl- N ′-nitro- N -nitrosoguanidine and may thereby protect cells from cell death induced by this DNA-damaging agent ( 25 ).…”