Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Dutka, Przemysław; Metskas, Lauren Ann; Hurt, Robert C.; Salahshoor, Hossein; Malounda, Dina; Lu, George J.; Chou, Tsui‐Fen; Shapiro, Mikhail G.; Jensen, Grant J.

doi:10.1016/j.bpj.2022.11.427

Cited by 10 publications

(20 citation statements)

References 64 publications

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“…We selected the GV gene clusters obtained from these species as our starting points based on the previous use of the B. megaterium cluster as a bacterial ARG 2 and the use the A. flos-aquae cluster in reconstituted contrast agents and mammalian ARGs, 3,10,19 making it desirable to obtain their efficient bacterial expression. Starting without the benefit of the recently published structures and structural models of these proteins, 20,21 we chose an approach based on random mutagenesis and high-throughput acoustic screening of ARG mutants.…”

Section: Resultsmentioning

confidence: 99%

“…Similar to the mutations identified from the first-round libraries, these mutations cluster in the N-terminal linker domain, as well as the hinge and wall domains, and the C-terminal tail, but not the C-terminal stabilization domain. 20,21 Representative nonlinear US images of GvpA-T6A-L40A and GvpB-S9G-R31L-R85L, as well as the WT parents, are shown in Figure 4I-J. In addition to showing increased nonlinear contrast (Figure S5A-B), the top variants have slightly higher collapse pressure than their WT parents when normalized for nonlinear contrast (Figure S5C-D).…”

Section: Resultsmentioning

confidence: 99%

“…Such tuning would potentially make the more compact A. flos-aquae and B. megaterium -derived ARGs competitive with the larger Serratia -derived ARGs, which currently provide the best in vivo imaging performance. 4 To further accelerate ARG development, we need a deeper understanding of how mutations to GvpA/GvpB affect both their structures 20,21 and the protein-protein interactions in which they participate during GV assembly 30–33 , as well as biochemical methods to characterize intermediate steps that cannot be assayed by ultrasound, such as GV nucleation (Figure 5F).…”

Section: Discussionmentioning

confidence: 99%

“…6,7 GVs scatter US due to the difference in the density and compressibility of their gaseous interior relative to a surrounding aqueous medium. 8 GVs have been the subject of intense study [7][8][9][10][11][12][13][14] , development, 4 and application 5,[15][16][17][18][19][20][21][22][23] in recent years. 1,24,25 ARGs have received considerable attention due to their ability to enable noninvasive, long-term, real-time imaging of gene expression in both bacterial and mammalian cells deep inside living organisms: in particular, ARGs have been used to image tumor growth 3,4 and colonization by therapeutic bacteria, 4 protease activity, 5 phagolysosomal function, 9 and intracellular Ca 2+ dynamics.…”

Section: Introductionmentioning

confidence: 99%

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Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening

Hurt,

Jin,

Soufi

et al. 2024

Preprint

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A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm.1Within the past decade, reporter genes for US have been introduced2,3and engineered4,5to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs).6Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semi-automated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologs of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening

Hurt,

Jin,

Soufi

et al. 2024

Preprint

Self Cite

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“…To determine the high-resolution structure of the uroplakin complex, we isolated the AUM from porcine urinary bladders, leveraging its resistance to sarkosyl 25 , and employed cryo-electron microscopy techniques. Initially, we used cryo-electron tomography to observe the AUM; however, the obtained structure was of low resolution as a result of the irradiation-induced distortion of the specimen 26 , 27 . Subsequently, we performed single particle analysis on AUM samples tilted up to 55° (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane

Yanagisawa,

Kita,

Oda

et al. 2023

Commun Biol

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The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

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Truly Tiny Acoustic Biomolecules for Ultrasound Imaging and Therapy

Ling,

Gungoren,

Yao

et al. 2024

Advanced Materials

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Nanotechnology offers significant advantages for medical imaging and therapy, including enhanced contrast and precision targeting. However, integrating these benefits into ultrasonography has been challenging due to the size and stability constraints of conventional bubble‐based agents. Here we describe bicones, truly tiny acoustic contrast agents based on gas vesicles, a unique class of air‐filled protein nanostructures naturally produced in buoyant microbes. We show that these sub‐80 nm particles can be effectively detected both in vitro and in vivo, infiltrate tumors via leaky vasculature, deliver potent mechanical effects through ultrasound‐induced inertial cavitation, and are easily engineered for molecular targeting, prolonged circulation time, and payload conjugation.This article is protected by copyright. All rights reserved

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Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Cited by 10 publications

References 64 publications

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening

Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane

Truly Tiny Acoustic Biomolecules for Ultrasound Imaging and Therapy

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