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

Temperature is a 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 ΔT ~15-20°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°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.

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

Inducible protein switches are used throughout the biosciences to allow on-demand control of proteins in response to chemical or optical inputs. However, these inducers 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. Using custom devices for rapid and high-throughput temperature control during live-cell microscopy, we find that the original Melt variant fully switches states between 28-32°C, and state changes can be observed within minutes of temperature changes. Melt was highly modular, permitting thermal control over diverse intracellular processes including signaling, proteolysis, and nuclear shuttling through straightforward end-to-end fusions with no further engineering. Melt was also highly tunable, giving rise to a library of Melt variants with switch point temperatures ranging from 30-40°C. The variants with higher switch points allowed control of molecular circuits between 37°C-41°C, a well-tolerated range for mammalian cells. Finally, Melt could thermally regulate important cell decisions over this range, including cytoskeletal rearrangement and apoptosis. Thus Melt represents a versatile thermogenetic module that provides straightforward, temperature-based, real-time control of mammalian cells with broad potential for biotechnology and biomedicine.

Simple visualization 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. A record of this paper's transparent peer review process is included in the supplemental information.

Complete chloroplast genome of the marine red alga Rhodochorton tenue (Rhodochortonaceae, Rhodophyta) from San Juan Island, Washington.

We present the complete chloroplast genome sequence of Rhodochorton tenue from San Juan Island, Washington. The chloroplast genome of R. tenue is 192,037 bp in length, contains 244 genes, and is similar in content to Acrochaetium secundatum. Rhodochorton tenue is genetically distinct from Rhodochorton purpureum from the North Atlantic Ocean.

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor.

Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, 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.

Long-term changes in kelp forests in an inner basin of the Salish Sea.

Kelp forests form an important biogenic habitat that responds to natural and human drivers. Global concerns exist about threats to kelp forests, yet long-term information is limited and research suggests that trends are geographically distinct. We examined distribution of the bull kelp Nereocystis luetkeana over 145 years in South Puget Sound (SPS), a semi-protected inner basin in a fjord estuary complex in the northeast Pacific Ocean. We synthesized 48 historical and modern Nereocystis surveys and examined presence/absence within 1-km segments along 452 km of shoreline. Compared to the earliest baseline in 1878, Nereocystis extent in 2017 decreased 63%, with individual sub-basins showing up to 96% loss. Losses have persisted for decades, across a range of climate conditions. In recent decades, Nereocystis predominantly occurred along shorelines with intense currents and mixing, where temperature and nutrient concentrations did not reach thresholds for impacts to Nereocystis performance, and high current speeds likely excluded grazers. Losses predominated in areas with elevated temperature, lower nutrient concentrations, and relatively low current velocities. The pattern of long-term losses in SPS contrasts with stability in floating kelp abundance during the last century in an area of the Salish Sea with greater wave exposure and proximity to oceanic conditions. These findings support the hypothesis that kelp beds along wave-sheltered shorelines exhibit greater sensitivity to environmental stressors. Additionally, shorelines with strong currents and deep-water mixing may provide refugia within sheltered systems.

Reverse and Forward Engineering Multicellular Structures with Optogenetics.

Understanding how cells self-organize into functional higher-order structures is of great interest, both towards deciphering animal development, as well as for our ability to predictably build custom tissues to meet research and therapeutic needs. The proper organization of cells across length-scales results from interconnected and dynamic networks of molecules and cells. Optogenetic probes provide dynamic and tunable control over molecular events within cells, and thus represent a powerful approach to both dissect and control collective cell behaviors. Here we emphasize the breadth of the optogenetic toolkit and discuss how these methods have already been used to reverse-engineer the design rules of developing organisms. We also offer our perspective on the rich potential for optogenetics to power forward-engineering of tissue assembly towards the generation of bespoke tissues with user-defined properties.

Mitochondrial and plastid genome analysis of the heteromorphic red alga Mastocarpus papillatus (C. Agardh) Kützing (Phyllophoraceae, Rhodophyta) reveals two characteristic florideophyte organellar genomes.

Analysis of the marine red algal species Mastocarpus papillatus (C. Agardh) Kützing using paired-end 36 bp Illumina sequences resulted in the assembly of its complete mitochondrial and plastid genomes. The mitogenome is 25,906 bp in length and contains 50 genes, and the plastid genome is 184,382 bp with 234 genes. The mitochondrial and plastid genomes show high gene synteny with published Florideophyceae.