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SUMMARYGas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically-encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryo-electron tomography to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder—a site that may act as an elongation center. High-resolution subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a β-sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes.

The ability to physically manipulate specific cells is critical for the fields of biomedicine, synthetic biology, and living materials. Ultrasound has the ability to manipulate cells with high spatiotemporal precision via acoustic radiation force (ARF). However, because most cells have similar acoustic properties, this capability is disconnected from cellular genetic programs. Here, we show that gas vesicles (GVs)—a unique class of gas-filled protein nanostructures—can serve as genetically encodable actuators for selective acoustic manipulation. Because of their lower density and higher compressibility relative to water, GVs experience strong ARF with opposite polarity to most other materials. When expressed inside cells, GVs invert the cells’ acoustic contrast and amplify the magnitude of their ARF, allowing the cells to be selectively manipulated with sound waves based on their genotype. GVs provide a direct link between gene expression and acoustomechanical actuation, opening a paradigm for selective cellular control in a broad range of contexts.

SUMMARYAcoustic reporter genes based on gas vesicles (GVs) have enabled the use of ultrasound to noninvasively visualize cellular function in vivo. The specific detection of GV signals relative to background acoustic scattering in tissues is facilitated by nonlinear ultrasound imaging techniques taking advantage of the sonomechanical buckling of GVs. However, the effect of geometry on the buckling behavior of GVs under exposure to ultrasound has not been studied. To understand such geometric effects, we developed computational models of GVs of various lengths and diameters and used finite element simulations to predict their threshold buckling pressures and post-buckling deformations. We demonstrated that the GV diameter has an inverse cubic relation to the threshold buckling pressure, whereas length has no substantial effect. To complement these simulations, we experimentally probed the effect of geometry on the mechanical properties of GVs and the corresponding nonlinear ultrasound signals. The results of these experiments corroborate our computational predictions. This study provides fundamental insights into how geometry affects the sonomechanical properties of GVs, which, in turn, can inform further engineering of these nanostructures for high-contrast, nonlinear ultrasound imaging.STATEMENT OF SIGNIFICANCEGas vesicles (GVs) are an emerging class of genetically encodable and engineerable imaging agents for ultrasound whose sonomechanical buckling generates nonlinear contrast to enable sensitive and specific imaging in highly scattering biological systems. Though the effect of protein composition on GV buckling has been studied, the effect of geometry has not previously been addressed. This study reveals that geometry, especially GV diameter, significantly alters the threshold acoustic pressures required to induce GV buckling. Our computational predictions and experimental results provide fundamental understanding of the relationship between GV geometry and buckling properties and underscore the utility of GVs for nonlinear ultrasound imaging. Additionally, our results provide suggestions to further engineer GVs to enable in vivo ultrasound imaging with greater sensitivity and higher contrast.

Phagocytic clearance and lysosomal processing of pathogens and debris are essential functions of the innate immune system. However, the assessment of these functions in vivo is challenging because most nanoscale contrast agents compatible with non-invasive imaging techniques are made from non-biodegradable synthetic materials that do not undergo regular lysosomal degradation. To overcome this challenge, we describe the use of an all-protein contrast agent to directly visualize and quantify phagocytic and lysosomal activities in vivo by ultrasound imaging. This contrast agent is based on gas vesicles (GVs), a class of air-filled protein nanostructures naturally expressed by buoyant microbes. Using a combination of ultrasound imaging, pharmacology, immunohistology and live-cell optical microscopy, we show that after intravenous injection, GVs are cleared from circulation by liver-resident macrophages. Once internalized, the GVs undergo lysosomal degradation, resulting in the elimination of their ultrasound contrast. By non-invasively monitoring the temporal dynamics of GV-generated ultrasound signal in circulation and in the liver and fitting them with a pharmacokinetic model, we can quantify the rates of phagocytosis and lysosomal degradation in living animals. We demonstrate the utility of this method by showing how these rates are perturbed in two models of liver dysfunction: phagocyte deficiency and non-alcoholic fatty liver disease. The combination of proteolytically-degradable nanoscale contrast agents and quantitative ultrasound imaging thus enables non-invasive functional imaging of cellular degradative processes.

Optical coherence tomography (OCT) has gained wide adoption in biological research 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 class of naturally evolved gas-filled protein nanostructures, as 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. 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 to image a wider variety of cellular and molecular processes.

The structure-driven assembly of multimeric protein complexes and the formation of intracellular phase-like protein condensates have been the subject of intense research. However, the assembly of larger superstructures comprising cellular components, such as protein nanoparticles driven by general physical rather than specific biochemical interactions, remains relatively uncharacterized. Here, we use gas vesicles (GVs)—genetically encoded protein nanoparticles that form ordered intracellular clusters—as a model system to study the forces driving multiparticle assembly under cytoplasm-like conditions. Our calculations and experimental results show that the ordered assembly of GVs can be achieved by screening their mutual electrostatic repulsion with electrolytes and creating a crowding force with dissolved macromolecules. The precise balance of these forces results in different packing configurations. Biomacromolecules such as polylysine and DNA are capable of driving GV clustering. These results provide basic insights into how physically driven interactions affect the formation of protein superstructures, offer guidance for manipulating nanoparticle assembly in cellular environments through synthetic biology methods, and inform research on the biotechnology applications of GVs.

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 GVs dimensions.

Ultrasound is playing an emerging role in molecular and cellular imaging thanks to new micro- and nanoscale contrast agents and reporter genes. Acoustic methods for the selective in vivo detection of these imaging agents are needed to maximize their impact in biology and medicine. Existing ultrasound pulse sequences use the nonlinearity in contrast agents’ response to acoustic pressure to distinguish them from mostly linear tissue scattering. However, such pulse sequences typically scan the sample using focused transmissions, resulting in a limited frame rate and restricted field of view. Meanwhile, existing wide-field scanning techniques based on plane wave transmissions suffer from limited sensitivity or nonlinear artifacts. To overcome these limitations, we introduce an ultrafast nonlinear imaging modality combining amplitude-modulated pulses, multiplane wave transmissions and selective coherent compounding. This technique achieves contrast imaging sensitivity comparable to much slower gold-standard amplitude modulation sequences and enables the acquisition of larger and deeper fields of view, while providing a much faster imaging framerate of 3.2kHz. Additionally, it enables simultaneous nonlinear and linear image formation, and allows concurrent monitoring of phenomena accessible only at ultrafast framerates, such as blood volume variations. We demonstrate the performance of this ultrafast amplitude modulation (uAM) technique by imaging gas vesicles, an emerging class of genetically encodable biomolecular contrast agents, in several in vitro and in vivo contexts. These demonstrations include the rapid discrimination of moving contrast agents and the real-time monitoring of phagolysosomal function in the mouse liver.

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.

Visualizing biomolecular and cellular processes inside intact living organisms is a major goal of chemical biology. However, existing molecular biosensors, based primarily on fluorescent emission, have limited utility in this context due to the scattering of light by tissue. In contrast, ultrasound can easily image deep tissue with high spatiotemporal resolution, but lacks the biosensors needed to connect its contrast to the activity of specific biomolecules such as enzymes. To overcome this limitation, we introduce the first genetically encodable acoustic biosensors - molecules that ‘light up’ in ultrasound imaging in response to protease activity. These biosensors are based on a unique class of air-filled protein nanostructures called gas vesicles, which we engineered to produce non-linear ultrasound signals in response to the activity of three different protease enzymes. We demonstrate the ability of these biosensors to be imaged in vitro, inside engineered probiotic bacteria, and in vivo in the mouse gastrointestinal tract.

Recent advances in molecular engineering and synthetic biology provide biomolecular and cell-based therapies with a high degree of molecular specificity, but limited spatiotemporal control. Here we show that biomolecules and cells can be engineered to deliver potent mechanical effects at specific locations inside the body through ultrasound-induced inertial cavitation. This capability is enabled by gas vesicles, a unique class of genetically encodable air-filled protein nanostructures. We show that low-frequency ultrasound can convert these biomolecules into micrometre-scale cavitating bubbles, unleashing strong local mechanical effects. This enables engineered gas vesicles to serve as remotely actuated cell-killing and tissue-disrupting agents, and allows genetically engineered cells to lyse, release molecular payloads and produce local mechanical damage on command. We demonstrate the capabilities of biomolecular inertial cavitation in vitro, in cellulo and in vivo, including in a mouse model of tumour-homing probiotic therapy.

Neurological and psychiatric disorders are often characterized by dysfunctional neural circuits in specific regions of the brain. Existing treatment strategies, including the use of drugs and implantable brain stimulators, aim to modulate the activity of these circuits. However, they are not cell-type-specific, lack spatial targeting or require invasive procedures. Here, we report a cell-type-specific and non-invasive approach based on acoustically targeted chemogenetics that enables the modulation of neural circuits with spatiotemporal specificity. The approach uses ultrasound waves to transiently open the blood-brain barrier and transduce neurons at specific locations in the brain with virally encoded engineered G-protein-coupled receptors. The engineered neurons subsequently respond to systemically administered designer compounds to activate or inhibit their activity. In a mouse model of memory formation, the approach can modify and subsequently activate or inhibit excitatory neurons within the hippocampus, with selective control over individual brain regions. This technology overcomes some of the key limitations associated with conventional brain therapies.

Gas vesicles are a unique class of gas-filled protein nanostructures whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and magnetic resonance imaging (MRI), detectable at sub-nanomolar concentrations. Here we provide a protocol for isolating gas vesicles from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and magnetic resonance imaging. Gas vesicles can be isolated from natural cyanobacterial and haloarchaeal host organisms or from E. coli expressing a heterologous gas vesicle gene cluster, and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants, or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, while dynamic light scattering and transmission electron microscopy are used to determine nanoparticle size and morphology, respectively. Gas vesicles can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between gas vesicle-bound and dissolved xenon – a technique currently implemented in vitro. Taking 3–8 days to prepare, these genetically encodable nanostructures enable multi-modal, noninvasive biological imaging with high sensitivity and potential for molecular targeting.

Hemodynamic functional ultrasound imaging (fUS) of neural activity provides a unique combination of spatial coverage, spatiotemporal resolution and compatibility with freely moving animals. However, deep and transcranial monitoring of brain activity and the imaging of dynamics in slow-flowing blood vessels remains challenging. To enhance fUS capabilities, we introduce biomolecular hemodynamic enhancers based on gas vesicles (GVs), genetically encodable ultrasound contrast agents derived from buoyant photosynthetic microorganisms. We show that intravenously infused GVs enhance ultrafast Doppler ultrasound contrast and visually-evoked hemodynamic contrast in transcranial fUS of the mouse brain. This hemodynamic contrast enhancement is smoother than that provided by conventional microbubbles, allowing GVs to more reliably amplify neuroimaging signals.

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 erasedin situwith 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 agentsin vivoin 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.

Neurological and psychiatric diseases often involve the dysfunction of specific neural circuits in particular regions of the brain. Existing treatments, including drugs and implantable brain stimulators, aim to modulate the activity of these circuits, but are typically not cell type-specific, lack spatial targeting or require invasive procedures. Here, we introduce an approach to modulating neural circuits noninvasively with spatial, cell-type and temporal specificity. This approach, called acoustically targeted chemogenetics, or ATAC, uses transient ultrasonic opening of the blood brain barrier to transduce neurons at specific locations in the brain with virally-encoded engineered G-protein-coupled receptors, which subsequently respond to systemically administered bio-inert compounds to activate or inhibit the activity of these neurons. We demonstrate this concept in mice by using ATAC to noninvasively modify and subsequently activate or inhibit excitatory neurons within the hippocampus, showing that this enables pharmacological control of memory formation. This technology allows a brief, noninvasive procedure to make one or more specific brain regions capable of being selectively modulated using orally bioavailable compounds, thereby overcoming some of the key limitations of conventional brain therapies.