Biology subjects

Gonzalez-Martinez, D.

Publications and source records attributed to Gonzalez-Martinez, D..

4 recordsLinked to original sources

bioRxiv2025

Low-order assemblies drive oncogenic RTK fusion signaling without condensation

Receptor tyrosine kinase (RTK) fusions are a large class of oncoproteins found in [~]5% of cancers. Key questions remain, however, about how RTK fusions transmit oncogenic signals, including how these largely cytoplasmic proteins activate downstream pathways that originate at the plasma membrane. Fusions are multimeric and can form mesoscale condensates in cancer cells, and condensation has been implicated as an essential mechanism to enable signal transmission from the cytoplasm. However, whether condensates play a causal role, or whether smaller diffuse assemblies are sufficient to transduce signals, has been challenging to establish. Here we apply advanced microscopy, single-cell analysis, and synthetic fusions to determine the principles by which multimerization and condensation drive signaling from cytoplasmic RTK fusions. For EML4-ALK, a prominent fusion that forms condensates, we found poor correlation between condensation and signaling. By contrast, EML4-ALK activity was abundant in the diffuse phase, and the kinetics of diffuse-phase activity aligned more closely with downstream Erk signaling than did kinetics of signaling within condensates. Synthetic RTK fusions showed that cytoplasmic ALK or RET fusion dimers--and even constitutively active monomers--were sufficient to induce strong Ras-Erk signaling despite the absence of condensates, and diffuse fusions were sufficient to transform cells in vitro and in subcutaneous tumor models. A panel of various other cancer-driving RTK fusions showed that low-order multimerization was universal across fusions, whereas mesoscale condensation was rare and did not correlate with signaling. Our results suggest that low-order fusion multimerization is sufficient to drive its phosphorylation, which is necessary and sufficient to trigger downstream oncogenic signaling.

cancer biology↗

bioRxiv2022

Visual detection of submicroscopic protein clusters with a phase-separation-based fluorescent reporter.

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.

bioengineering↗

bioRxiv2022

High-throughput feedback-enabled optogenetic stimulation and spectroscopy in microwell plates

The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.

synthetic biology↗

bioRxiv2022

Oncogenic protein condensates modulate cell signal perception and drug tolerance

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 is unclear. Here, we used optogenetics to find that condensates suppress signaling through endogenous RTKs including EGFR. Notably, ALK inhibition hypersensitized RTK signals, which are known to drive resistance. Suppression of RTKs occurred because condensates sequestered downstream adapter proteins that are required for RTK signal transmission. Strikingly, EGFR hypersensitization resulted in rapid and pulsatile Erk signal reactivation, which originated from neighboring apoptotic cells. Paracrine signals promoted survival during ALK inhibition, and blockade of paracrine signals suppressed drug tolerance. Our results uncover a regulatory role for RTK fusion condensates in cancer drug response and demonstrate the potential of optogenetics for uncovering functional biomarkers of cancer cells.

cancer biology↗