Biology subjects

Bugaj, L.

Publications and source records attributed to Bugaj, L..

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↗

bioRxiv2023

Enhancing single-cell western blotting sensitivity using diffusive analyte blotting and antibody conjugate amplification

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein contents often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity western blots on single cells (scWesterns) are attractive since they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 103 molecules, a 520-fold improvement. This enables us to detect 85% and 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies respectively, compared to 47% of cells using in-gel detection. These results suggest compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents -- not previously accessible for in-gel use -- for further signal amplification and detection of low abundance targets.

bioengineering↗