Bioorthogonal labeling enables in situ fluorescence imaging of expressed gas vesicle nanostructures DOI Open Access

Erik Schrunk,

Przemysław Dutka, Robert C. Hurt

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2023, Номер unknown

Опубликована: Дек. 1, 2023

ABSTRACT Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nano-cages, and other macromolecular assemblies being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures’ sub-cellular assembly localization. However, traditional fluorescent protein fusions not tolerated by GVs’ primary constituent protein, making optical microscopy a challenge. Here, we introduce method fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes upon binding six-amino acid tetracysteine (TC) tag. We engineered GV subunit GvpA, display TC tag, showed that bearing TC-tagged GvpA can successfully assembled visualized in HEK 293T cells. used fluorescence images of tagged study size distance distributions within This labeling approach will enable research provide greater understanding could adapted similar nanostructures.

Язык: Английский

Genetically encoded multimeric tags for subcellular protein localization in cryo-EM DOI Creative Commons
Herman K.H. Fung, Yuki Hayashi, Veijo T. Salo

и другие.

Nature Methods, Год журнала: 2023, Номер 20(12), С. 1900 - 1908

Опубликована: Ноя. 6, 2023

Abstract Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization macromolecules interest tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The exhibit recognizable structural signatures, enabling automated detection cryo-ET data by convolutional neural networks. coupling GEMs to green fluorescent protein-tagged is triggered addition small-molecule ligand, allowing time-controlled minimize disturbance protein function. We demonstrate applicability subcellular-level endogenous and overexpressed across different organelles human cells using cryo-correlative fluorescence imaging. describe means quantifying specificity efficiency, systematic optimization rare abundant targets, with emphasis assessing potential effects

Язык: Английский

Процитировано

26
Cryo-electron tomography pipeline for plasma membranes DOI Creative Commons

Willy W. Sun,

Dennis J. Michalak, Kem A. Sochacki

и другие.

Nature Communications, Год журнала: 2025, Номер 16(1)

Опубликована: Янв. 20, 2025

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing plasma membrane and its associated proteins. Here, we present a correlative cryo-electron pipeline optimally suited to image large ultra-thin areas of isolated basal apical membranes. The allows for angstrom-scale determination with subtomogram averaging employs genetically encodable rapid chemically-induced electron microscopy visible tag marking specific proteins complex efficient, distributable, low-cost enables targeted structural studies identified mammalian cells.

Язык: Английский

Процитировано

1
Cryo-electron tomography pipeline for plasma membranes. DOI Creative Commons

Willy W. Sun,

Dennis J. Michalak, Kem A. Sochacki

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июнь 28, 2024

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing plasma membrane and its associated proteins. Here, we present a correlative cryo-electron pipeline optimally suited to image large ultra-thin areas of isolated basal apical membranes. The allows for angstrom-scale determination with sub-tomogram averaging employs genetically-encodable rapid chemically-induced electron microscopy visible tag marking specific proteins complex cell fast, efficient, distributable, low-cost enables targeted structural studies identified cells.

Язык: Английский

Процитировано

5
Multiplexed volumetric CLEM enabled by scFvs provides insights into the cytology of cerebellar cortex DOI Creative Commons
Xiaomeng Han, Xiaotang Lu, Peter H. Li

и другие.

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Авг. 5, 2024

Abstract Mapping neuronal networks is a central focus in neuroscience. While volume electron microscopy (vEM) can reveal the fine structure of (connectomics), it does not provide molecular information to identify cell types or functions. We developed an approach that uses fluorescent single-chain variable fragments (scFvs) perform multiplexed detergent-free immunolabeling and volumetric-correlated-light-and-electron-microscopy on same sample. generated eight scFvs targeting brain markers. Six probes were imaged cerebellum female mouse, using confocal with spectral unmixing, followed by vEM The results excellent ultrastructure superimposed multiple fluorescence channels. Using this approach, we documented poorly described type, two mossy fiber terminals, subcellular localization one type ion channel. Because be derived from existing monoclonal antibodies, hundreds such enable overlays for connectomic studies.

Язык: Английский

Процитировано

4
Techniques for Selective Labeling of Molecules and Subcellular Structures for Cryo-Electron Tomography DOI

Eugene P Kazakov,

Igor I. Kireev, S. A. Golyshev

и другие.

Biochemistry (Moscow), Год журнала: 2025, Номер 90(2), С. 173 - 187

Опубликована: Фев. 1, 2025

Язык: Английский

Процитировано

0
Volume electron microscopy for genetically and molecularly defined neural circuits DOI Creative Commons
Nobuhiko Ohno, Fuyuki Karube, Fumino Fujiyama

и другие.

Neuroscience Research, Год журнала: 2024, Номер unknown

Опубликована: Июнь 1, 2024

The brain networks responsible for adaptive behavioral changes are based on the physical connections between neurons. Light and electron microscopy have long been used to study neural projections Volume has recently expanded its scale of analysis due methodological advances, resulting in complete wiring maps neurites a large volume tissues even entire nervous systems growing number species. However, structural approaches frequently suffer from inherent limitations which elements images identified solely by morphological criteria. Recently, an increasing tools technologies developed characterize cells cellular components context molecules gene expression. These advancements include newly probes visualization microscopic as well correlative integration methods same across multiple modalities. Such advance our understanding interactions specific neurons circuits may help elucidate novel aspects basal ganglia network involving dopamine expected reveal mechanisms processing that modulate functions.

Язык: Английский

Процитировано

3
Characteristics of genetic tags for correlative light and electron microscopy DOI Creative Commons
Kimberly E. Beatty,

Clàudia López

Current Opinion in Chemical Biology, Год журнала: 2023, Номер 76, С. 102369 - 102369

Опубликована: Июль 13, 2023

Язык: Английский

Процитировано

6
Encapsulins: Nanotechnology’s future in a shell DOI

Amy Quinton,

Harry Benjamin McDowell,

Egbert Hoiczyk

и другие.

Advances in applied microbiology, Год журнала: 2023, Номер unknown, С. 1 - 48

Опубликована: Янв. 1, 2023

Язык: Английский

Процитировано

6
Magnetic and Fluorescent Dual-Labeled Genetically Encoded Targeted Nanoparticles for Malignant Glioma Cell Tracking and Drug Delivery DOI Creative Commons
А. Н. Габашвили, Nelly S. Chmelyuk,

Vera V. Oda

и другие.

Pharmaceutics, Год журнала: 2023, Номер 15(10), С. 2422 - 2422

Опубликована: Окт. 4, 2023

Human glioblastoma multiforme (GBM) is a primary malignant brain tumor, radically incurable disease characterized by rapid growth resistance to classical therapies, with median patient survival of about 15 months. For decades, plethora approaches have been developed make GBM therapy more precise and improve the diagnosis this pathology. Targeted delivery mediated use various molecules (monoclonal antibodies, ligands overexpressed tumor receptors) one promising methods achieve goal. Here we present novel genetically encoded nanoscale dual-labeled system based on Quasibacillus thermotolerans (Qt) encapsulins exploiting biologically inspired designs iron-containing nanoparticles as cargo, conjugated human fluorescent labeled transferrin (Tf) acting vector. It known that expression receptors (TfR) in glioma cells significantly higher compared non-tumor cells, which enables targeting resulting nanocarrier. The selectivity binding obtained nanosystem was studied qualitative quantitative assessment accumulation intracellular iron, well magnetic particle quantification method laser scanning confocal microscopy. Used unambiguously demonstrated transferrin-conjugated were captured much efficiently than benign cells. bioinspired nanoplatform can be supplemented chemotherapeutic drug or genotherapeutic agent used for targeted therapeutic Additionally, observed cell-assisted biosynthesis could an attractive way narrow size distribution particles applications.

Язык: Английский

Процитировано

4
Bioorthogonal Labeling Enables In Situ Fluorescence Imaging of Expressed Gas Vesicle Nanostructures DOI Creative Commons

Erik Schrunk,

Przemysław Dutka, Robert C. Hurt

и другие.

Bioconjugate Chemistry, Год журнала: 2024, Номер 35(3), С. 333 - 339

Опубликована: Фев. 12, 2024

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nanocages, and other macromolecular assemblies, being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures' subcellular assembly localization. However, traditional fluorescent protein fusions not tolerated by GVs' primary constituent protein, making optical microscopy a challenge. Here, we introduce method fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes upon reaction six-amino acid tetracysteine (TC) tag. We engineered GV subunit GvpA, display TC tag showed that bearing TC-tagged GvpA can successfully assembled visualized in HEK 293T cells. Importantly, this was achieved replacing only fraction of tagged version. used fluorescence images study size distance distributions within This fractional labeling approach will enable research provide greater understanding could adapted similar nanostructures.

Язык: Английский

Процитировано

1