Protocol. 

Membraneless organelles (MLOs) formed via protein phase separation have garnered significant attention recently due to their relevance to cellular physiology and pathology. However, there is a lack of tools available to study their behavior and control their bioactivity in complex biological systems. This chapter describes a new optogenetic tool based on water-soluble chlorophyll protein (WSCP), a red light-induced singlet oxygen-generating protein, to control synthetic MLOs. Upon exposure to red light, WSCP generates singlet oxygen, which triggers the crosslinking of the proteins in the MLOs, resulting in their liquid-to-solid phase transition. The effective delivery of chlorophylls enables the successful reconstitution of WSCP in living cells, thus offering a potential approach to biological regulation at the subcellular level.

Light-sheet fluorescence microscopy has revolutionized biology by visualizing dynamic cellular processes in three dimensions. However, light scattering in thick tissue and photobleaching of fluorescent reporters limit this method to studying thin or translucent specimens. In this study, we applied nondiffractive ultrasound beams in conjunction with a cross-amplitude modulation sequence and nonlinear acoustic reporters to enable fast and volumetric imaging of targeted biological functions. We reported volumetric imaging of tumor gene expression at the cubic centimeter scale using genetically encoded gas vesicles and localization microscopy of cerebral capillary networks using intravascular microbubble contrast agents. Nonlinear sound-sheet microscopy provides a ~64× acceleration in imaging speed, ~35× increase in imaged volume, and ~4× increase in classical imaging resolution compared with the state of the art in biomolecular ultrasound.

Spider silk is recognized for its exceptional mechanical properties and biocompatibility, making it a versatile platform for developing functional materials. In this study, a modular functionalization strategy for recombinant spider silk is presented using SpyTag/SpyCatcher chemistry, a prototype of genetically encoded click chemistry. The approach involves AlphaFold2-aided design of SpyTagged spider silk coupled with bacterial expression and biomimetic spinning, enabling the decoration of silk with various SpyCatcher-fusion motifs, such as fluorescent proteins, enzymes, and cell-binding ligands. The silk threads can be coated with a silica layer using silicatein, an enzyme for silicification, resulting in a hybrid inorganic–organic 1D material. The threads installed with RGD or laminin cell-binding ligands lead to enhanced endothelial cell attachment and proliferation. These findings demonstrate a straightforward yet powerful approach to 1D protein materials.