Biology subjects

Mumford, T.

Publications and source records attributed to Mumford, T..

3 recordsLinked to original sources

bioRxiv2024

Multiplexed dynamic control of temperature to probe and observe mammalian cells.

Temperature is aa critical parameter for biological function, yet there is a lack of approaches to modulate the temperature of biological specimens in a dynamic and high-throughput manner. We present the thermoPlate, a device for programmable control of temperature in each well of a 96-well plate, in a manner compatible with mammalian cell culture and live cell imaging. The thermoPlate maintains precise feedback control of temperature patterns independently in each well, with minutes-scale heating and cooling through {Delta}T [~]15-20{degrees}C. A computational model that predicts thermal diffusion guides optimal design of heating protocols. The thermoPlate allowed systematic characterization of both synthetic and natural thermo-responsive systems. We first used the thermoPlate in conjunction with live-cell microscopy to characterize the rapid temperature-dependent phase separation of a synthetic elastin-like polypeptide (ELP53). We then measured stress granule (SG) formation in response to heat stress, observing differences in SG dynamics with each increasing degree of stress. We observed adaptive formation of SGs, whereby SGs formed but then dissolved in response to persistent heat stress (> 42{degrees}C). SG adaptation revealed a biochemical memory of stress that depended on both the time and temperature of heat shock. Stress memories continued to form even after the removal of heat and persisted for 6-9 hours before dissipating. The capabilities and open-source nature of the thermoPlate will empower the study and engineering of a wide range of thermoresponsive phenomena.

bioengineering↗

bioRxiv2024

A temperature-inducible protein module for control of mammalian cell fate

Inducible protein switches allow on-demand control of proteins in response to inputs including chemicals or light. However, these inputs either cannot be controlled with precision in space and time or cannot be applied in optically dense settings, limiting their application in tissues and organisms. Here we introduce a protein module whose active state can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization through temperature), exists as a monomer in the cytoplasm at elevated temperatures but both oligomerizes and translocates to the plasma membrane when temperature is lowered. The original Melt variant switched states between 28-32{degrees}C, and state changes could be observed within minutes of temperature change. Melt was highly modular, permitting thermal control over diverse processes including signaling, proteolysis, nuclear shuttling, cytoskeletal rearrangements, and cell death, all through straightforward end-to-end fusions. Melt was also highly tunable, giving rise to a library of variants with switch point temperatures ranging from 30-40{degrees}C. The variants with higher switch points allowed control of molecular circuits between 37{degrees}C-41{degrees}C, a well-tolerated range for mammalian cells. Finally, Melt permitted thermal control of cell death in a mouse model of human cancer, demonstrating its potential for use in animals. Thus Melt represents a versatile thermogenetic module for straightforward, non-invasive, spatiotemporally-defined control of mammalian cells with broad potential for biotechnology and biomedicine.

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↗