1.
Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.
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Mumford, TR
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Rae, D
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Brackhahn, E
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Idris, A
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Gonzalez-Martinez, D
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Pal, AA
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Chung, MC
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Guan, J
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Rhoades, E
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Bugaj, LJ
Abstract:
Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here, we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity called CluMPS (clusters magnified by phase separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context. A record of this paper's transparent peer review process is included in the supplemental information.
2.
Oncogenic protein condensates modulate cell signal perception and drug tolerance.
Abstract:
Drug resistance remains a central challenge towards durable cancer therapy, including for cancers driven by the EML4-ALK oncogene. EML4-ALK and related fusion oncogenes form cytoplasmic protein condensates that transmit oncogenic signals through the Ras/Erk pathway. However, whether such condensates play a role in drug response or resistance development is unclear. Here, we applied optogenetic functional profiling to examine how EML4-ALK condensates impact signal transmission through transmembrane receptor tyrosine kinases (RTKs), a common route of resistance signaling. We found that condensates dramatically suppress signaling through activated RTKs including EGFR. Conversely, ALK inhibition restored and hypersensitized RTK signals. Modulation of RTK sensitivity occurred because EML4-ALK condensates sequestered downstream adapters that are required to transduce signals from both EML4-ALK and ligand-stimulated RTKs. Strikingly, EGFR hypersensitization resulted in rapid and pulsatile Erk signal reactivation within 10s of minutes of drug addition. EGFR reactivation originated from paracrine signals from neighboring apoptotic cells, and reactivation could be blocked by inhibition of either EGFR or matrix metalloproteases. Paracrine signals promoted survival during ALK inhibition, and blockade of paracrine signals accelerated cell killing and suppressed drug tolerance. Our results uncover a regulatory role for protein condensates in cancer signaling and drug response and demonstrate the potential of optogenetic profiling for drug discovery based on functional biomarkers in cancer cells.
