Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter
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 (<jats:underline>Clu</jats:underline>sters<jats:underline>M</jats:underline>agnified by<jats:underline>P</jats:underline>hase<jats:underline>S</jats:underline>eparation). 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..
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Preprint| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
bioRxiv.org - (2024) vom: 16. Jan. Zur Gesamtaufnahme - year:2024 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Mumford, Thomas R. [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2022.07.13.499962 |
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| PPN (Katalog-ID): |
XBI036541257 |
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| 245 | 1 | 0 | |a Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter |
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| 520 | |a 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 (<jats:underline>Clu</jats:underline>sters<jats:underline>M</jats:underline>agnified by<jats:underline>P</jats:underline>hase<jats:underline>S</jats:underline>eparation). 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. | ||
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| 700 | 1 | |a Rae, Diarmid |4 aut | |
| 700 | 1 | |a Brackhahn, Emily |4 aut | |
| 700 | 1 | |a Idris, Abbas |4 aut | |
| 700 | 1 | |a Gonzalez-Martinez, David |4 aut | |
| 700 | 1 | |a Pal, Ayush Aditya |4 aut | |
| 700 | 1 | |a Chung, Michael C. |4 aut | |
| 700 | 1 | |a Guan, Juan |4 aut | |
| 700 | 1 | |a Rhoades, Elizabeth |4 aut | |
| 700 | 1 | |a Bugaj, Lukasz J. |4 aut | |
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