Biology subjects

Mumford, T. R.

Publications and source records attributed to Mumford, T. R..

5 recordsLinked to original sources

bioRxiv2026

Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

The mammalian kidney relies on a branched network of collecting ducts for fluid transport and homeostasis. Replicating this network in vitro would parallelize function in synthetic replacement kidneys, yet current organoids have limited branching capacity. Here, we establish a developmentally-informed strategy to control organoid budding through optogenetic control of a receptor tyrosine kinase, RET. We first show pharmacological manipulation of RET signaling controls the extent of branching in mouse embryonic kidneys and human stem cell-derived kidney organoids. Next, we develop an optogenetic RET receptor (optoRET) that signals in a ligand-independent manner via blue light-mediated clustering. Epithelial cells expressing optoRET reproduce stereotyped RET signaling, scattering, and symmetry breaking in response to blue light. Human kidney organoids undergo budding with controllable orientation in response to spatially patterned optoRET stimulation. Our results establish ligand-free optogenetic control of branching and inspire new synthetic biology strategies for epithelial organoid design. HighlightsGDNF-RET controls branching and tip cell state in mouse and human kidney tissues. OptoRET reproduces endogenous RET signaling and morphogenesis in cell lines. OptoRET enables ligand-free budding in human renal epithelial organoids. Spatially patterned optoRET stimulation controls budding orientation.

bioengineering↗

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↗

bioRxiv2025

Nuclear-import receptors remodel the dilute phase to suppress phase transitions of RNA-binding proteins with prion-like domains.

AbstractRNA-binding proteins (RBPs) with prion-like domains, including FUS, hnRNPA1, and hnRNPA2, assemble into functional, metastable condensates that organize ribostasis, but can also transition into self-templating fibrils implicated in neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS). How nuclear-import receptors (NIRs) antagonize this pathological transition has remained unresolved. Here, we establish that NIRs regulate the phase behavior of prion-like cargos by remodeling the dilute phase. Quantitative analyses across length scales reveal that Karyopherin-{beta}2 (Kap{beta}2) preferentially binds cargo in the dilute phase to lower the effective concentration of free RBPs thereby elevating the saturation concentration for phase separation and suppressing mesoscale clustering. ALS-linked FUSP525L, which binds Kap{beta}2 weakly, evades this regulation to form pathogenic assemblies. Thus, NIRs harness polyphasic linkage, the thermodynamic relationship between ligand binding and phase equilibria, to reshape the landscape of prion-like RBP assembly states, establishing a paradigm for how ATP-independent chaperones regulate phase behavior to prevent disease-linked aggregation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/688546v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1dd32cforg.highwire.dtl.DTLVardef@132ba1eorg.highwire.dtl.DTLVardef@1593c80org.highwire.dtl.DTLVardef@19311d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor

Optogenetic clustering is a versatile method to control protein activity in living cells, tissues, and organisms. Here we show that the BcLOV4 photoreceptor both clusters and translocates to the plasma membrane in response to blue light, representing a new class of light-dependent behavior. We demonstrate that dual translocation and clustering can be harnessed for novel single-component optogenetic tools, including for activation of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are causally linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~3-4-fold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

bioengineering↗

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↗