Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics

Abstract

Signaling complexes are often organized in a spatiotemporal manner and on a minute timescale. Proximity labeling based on engineered ascorbate peroxidase APEX2 pioneered in situ capture of spatiotemporal membrane protein complexes in living cells, but its application to cytosolic proteins remains limited due to the high labeling background. Here, we develop proximity labeling probes with increased labeling selectivity. These probes, in combination with label-free quantitative proteomics, allow exploring cytosolic protein assemblies such as phosphotyrosine-mediated protein complexes formed in response to minute-scale EGF stimulation. As proof-of-concept, we systematically profile the spatiotemporal interactome of the EGFR signaling component STS1. For STS1 core complexes, our proximity proteomics approach shows comparable performance to affinity purification-mass spectrometry-based temporal interactome profiling, while also capturing additional—especially endosomally-located—protein complexes. In summary, we provide a generic approach for exploring the interactome of mobile cytosolic proteins in living cells at a temporal resolution of minutes.

Regulation of ERK2 activity by dynamic S-acylation

“…This plasma-membrane localized PAT is unlikely a writer of ERK1/2 S-acylation, given its inability to increase fatty acid incorporation (Figure 5A) (Ohno et al, 2006). However, proximity proteomics experiments indicate that it is associated with Grb2, an adaptor protein that links the MAPK pathway to activated RTKs (Ke et al, 2021). These associations suggest it may have a role as a scaffolding protein in the EGFR signaling complex.…”

Bioengineering Approaches for the Advanced Organoid Research

“…Forebrain subdivisions that contain positional axes [22] Dual patterning from bipotent progenitors Self-organizing neuromuscular organoids [197] Stepwise modulation of signaling cues Cerebrospinal fluid production of choroid plexus-forming brain organoids [198] Stepwise modulation of signaling cues Hair-bearing skin organoids [199] Interorganoid communication Assembly of region-specific models Mixed dorsal and ventral forebrain organoids [54] Co-culture with connective tissue Promoted formation of alveolar organoid by addition of mesenchymal stem cells [213] Co-culture with connective tissue/organ-on-a-chip Structural arrangement in mesenchymal bodies [214] Organ-on-a-chip Recapitulating the connections between GI microbiome and CNS [207] Organ-on-a-chip/bioprinting Multi-organ interactions upon drug administration [208] Bioprinting Self-patterned 3D tissue models [209] Topographical patterning/profiling Light-induced small molecule release Spatiotemporally controlled neural stem cell fate [200] Light-induced patterning Axon guidance with NGF-patterned matrix [201] AI-based optimization Predicted experimental parameters for PSC self-organization [202] Microrheological characterization Mechanical properties of collagen gels and cell ECM interactions [203] Super-resolution imaging Cellular composition of organoids with high resolution 3D imaging [216] Spatial transcriptomics Visualization of the distribution of mRNAs [215] Spatial proteomics Spatiotemporal profiling of signaling interactomes [217] understood how to suppress the chaotic differentiation of mesenchymal stem cells and implement connective tissues with diverse composition and characteristics in the body. Advancements in analytical methods, for instance, single-cell sequencing, enable researchers to decipher the transcriptional profile of cell populations that compose organoids or the connective tissues at the single-cell level.…”

“…[215] In addition to the spatial structure of RNA expression, recent innovations in optical imaging technologies, such as super-resolution confocal microscopy, multiphoton laser scanning microscopy, and lightsheet fluorescence microscopy, have enabled the high-resolution 3D visualization of an entire immunolabeled organoid at the subcellular level. [216] Furthermore, recently developed proximity proteomics enabled spatiotemporal profiling of signaling interactomes, [217] which would potentially provide the analytic approach for the proteome in organoids. Collectively, deep sequencing and deep imaging systems can facilitate our understanding of the spatial distribution and dynamic interactions between multiple types of cells within organoids.…”

Recent progress in mass spectrometry-based strategies for elucidating protein–protein interactions

“…Recently, Ke et al designed and evaluated 12 different biotin-phenol analogues as proximity labelling probes for APEX2 [31]. Among these probes, the BP5 and BN2 were found to generate free radicals and conjugates to tyrosine residues with high efficiency and selectivity.…”