Biology subjects

Lu, G. J.

Publications and source records attributed to Lu, G. J..

7 recordsLinked to original sources

bioRxiv2026

Transcriptomic and proteomic responses to gas vesicle collapse in native and engineered bacterial systems

Gas vesicles (GVs) are air-filled protein nanostructures produced by microbes to regulate buoyancy and have emerged as powerful tools in biomedical imaging, particularly as acoustic reporter genes. Their mechanically robust shells enclose a stable air compartment, which collapses irreversibly when subjected to sufficient hydrostatic or acoustic pressure, leaving behind large protein sheets. This collapse phenomenon underlies key applications such as differential imaging and controlled cavitation and is also believed to occur naturally during buoyancy regulation, yet its physiological consequences remain poorly understood. Here, we used transcriptomic and proteomic approaches to investigate cellular responses to GV collapse. In the native GV-producing cyanobacterium Dolichospermum flos-aquae, RNA sequencing revealed a distinct transcriptional response characterized by the upregulation of heat shock proteins, indicative of a stress reaction to intracellular protein aggregation. In contrast, bioluminescence reporter assays in E. coli heterologously expressing GVs showed no comparable activation of heat shock promoters. To test the hypothesis that collapsed GVs can be recognized by specific proteins inside cells, we conducted LC-MS/MS-based pull-down assays in both species but did not identify strong candidate binders. While no definitive recognition mechanism was uncovered, our omics-based study provides a rich dataset for cellular responses to GV collapse in both the native and heterologous systems. These findings suggest that cellular responses to collapsed GVs may be more complex than previously recognized, and that both improved assay sensitivity and additional focused experiments will be needed to elucidate how cells detect and manage large intracellular protein aggregates such as collapsed gas vesicles. Proteomics data are available via ProteomeXchange (PXD060779), and RNA sequencing data via NCBI GEO (GSE289028).

synthetic biology↗

bioRxiv2025

Temporal gene regulation enables controlled expression of gas vesicles and preserves bacterial viability

Gas vesicles (GVs) are genetically encodable, air-filled protein nanostructures that have rapidly emerged as a versatile platform for biomedical imaging, cell tracking, and therapeutic delivery. However, their heterologous expression in non-native hosts such as Escherichia coli can be challenging due to the complex assembly process, which often involves around ten different proteins and can lead to proteotoxic stress and impair cell growth. Here, we report the observation of a drop in cell density occurring 8 to 16 hours after GV induction in E. coli. To address these, we developed a dual-inducer transcriptional regulation system that enables orthogonal control of GV assembly factor proteins and the shell protein over a range of stoichiometries. Sequential induction in time, in which assembly factor expression is initiated before shell protein expression, restored normal bacterial growth and prevented lysis without compromising GV production. Further analysis revealed that varying the interval between the two induction steps affected both GV yields and cellular stress. By preserving cell integrity with GV expression, our approach enhances the utility of GV-expressing bacteria in applications that demand population-wide cellular stability and facilitates their broader application in biomedical engineering and synthetic biology.

synthetic biology↗

bioRxiv2023

Elucidating the assembly of gas vesicles by systematic protein-protein interaction analysis

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process to assemble this shell remains elusive: while 6-8 assembly factor proteins were identified as essential, none of them have a defined function. As one of the first steps to elucidate the assembly mechanism, we devise a high-throughput in vivo assay to determine the interactions of all 11 proteins in a GV operon. Complete or partial deletions of the operon establish the interdependence relationship of the interaction on the background GV proteins with additional information on assembly tolerance and cellular burden. Clusters of GV protein interactions are revealed, which establishes the plausible protein complexes important for the assembly process of these protein organelles. We anticipate our findings will set the stage for solving the molecular mechanism of GV assembly and designing GVs that efficiently assemble in heterologous hosts during biomedical applications.

synthetic biology↗

bioRxiv2023

50-nm gas-filled protein nanostructures to enable the access of lymphatic cells by ultrasound technologies

Ultrasound imaging and ultrasound-mediated gene and drug delivery are rapidly advancing diagnostic and therapeutic methods; however, their use is often limited by the need of microbubbles, which cannot transverse many biological barriers due to their large size. Here we introduce 50-nm gas-filled protein nanostructures derived from genetically engineered gas vesicles that we referred to as 50nmGVs. These diamond-shaped nanostructures have hydrodynamic diameters smaller than commercially available 50-nm gold nanoparticles and are, to our knowledge, the smallest stable, free-floating bubbles made to date. 50nmGVs can be produced in bacteria, purified through centrifugation, and remain stable for months. Interstitially injected 50nmGVs can extravasate into lymphatic tissues and gain access to critical immune cell populations, and electron microscopy images of lymph node tissues reveal their subcellular location in antigen-presenting cells adjacent to lymphocytes. We anticipate that 50nmGVs can substantially broaden the range of cells accessible to current ultrasound technologies and may generate applications beyond biomedicine as ultrasmall stable gas-filled nanomaterials.

synthetic biology↗

bioRxiv2023

Spatial Organization of Gas Vesicles is Governed by Phase-separable GvpU

Gas vesicles (GVs) are microbial protein organelles that support cellular buoyancy, and the recent engineering of GVs has led to multiple applications including reporter gene imaging, acoustic control, and payload delivery. GVs often cluster into a honeycomb pattern to minimize their occupancy of cytosolic space; however, the molecular mechanism behind this process and its influence on cellular physiology remain unknown. Here, we identified GvpU as the protein governing this process. GvpU-mediated clustering is selective to the genotype of GVs, allowing the design of GV variants with genetically encodable clustering states. Furthermore, we uncovered that the clustering is modulated by phase transition behaviors encoded in the intrinsically disordered region of GvpU through a balanced contribution of acidic and aromatic residues, and such phase transition can directly modulate cellular fitness. Collectively, our findings elucidate the protein player, molecular mechanism, and functional roles of GV clustering, and its programmability for biomedical applications.

synthetic biology↗

bioRxiv2021

Measuring gas vesicle dimensions by electron microscopy

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that during air drying on the EM grid, GVs flatten, leading to a ~1.5-fold increase in their apparent diameter. We demonstrate that GVs diameter can be accurately determined by direct measurements from cryo-EM samples or alternatively indirectly derived from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported data and provide accurate methods to measure GV dimensions.

biochemistry↗

bioRxiv2019

Biomolecular Contrast Agents for Optical Coherence Tomography

Optical coherence tomography (OCT) has gained wide adoption in biological and medical imaging due to its exceptional tissue penetration, 3D imaging speed and rich contrast. However, OCT plays a relatively small role in molecular and cellular imaging due to the lack of suitable biomolecular contrast agents. In particular, while the green fluorescent protein has provided revolutionary capabilities to fluorescence microscopy by connecting it to cellular functions such as gene expression, no equivalent reporter gene is currently available for OCT. Here we introduce gas vesicles, a unique class of naturally evolved gas-filled protein nanostructures, as the first genetically encodable OCT contrast agents. The differential refractive index of their gas compartments relative to surrounding aqueous tissue and their nanoscale motion enables gas vesicles to be detected by static and dynamic OCT at picomolar concentrations. Furthermore, the OCT contrast of gas vesicles can be selectively erased in situ with ultrasound, allowing unambiguous assignment of their location. In addition, gas vesicle clustering modulates their temporal signal, enabling the design of dynamic biosensors. We demonstrate the use of gas vesicles as reporter genes in bacterial colonies and as purified contrast agents in vivo in the mouse retina. Our results expand the utility of OCT as a unique photonic modality to image a wider variety of cellular and molecular processes.

bioengineering↗