Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure

Fang, Tao; Lu, Xiaotang; Berger, Daniel R.; Gmeiner, Christina; Cho, Julia; Schalek, Richard; Ploegh, Hidde L.; Lichtman, Jeff W.

doi:10.1038/s41592-018-0177-x

Cited by 97 publications

(104 citation statements)

References 23 publications

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“…The latter technical constraints also affect EM immunolabeling techniques [10][11][12][13] which may furthermore be complicated by limited epitope accessibility. The fully genetic iron storage protein ferritin generates contrast via its electron-dense iron core [14][15][16] , but its small size complicates differentiation of individual ferritin particles from cellular structures.…”

Section: Multi-coloredmentioning

confidence: 99%

Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters

Sigmund

Pettinger

Kube

et al. 2019

Preprint

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Multi-coloredgene reporters such as fluorescent proteins are indispensable for biomedical research, but equivalent tools for electron microscopy (EM), a gold standard for deciphering mechanistic details of cellular processes 1,2 and uncovering the network architecture of cell-circuits 3,4 , are still sparse and not easily multiplexable. Semi-genetic EM reporters are based on the precipitation of exogenous chemicals 5-9 which may limit spatial precision and tissue penetration and can affect ultrastructure due to fixation and permeabilization.The latter technical constraints also affect EM immunolabeling techniques 10-13 which may furthermore be complicated by limited epitope accessibility. The fully genetic iron storage protein ferritin generates contrast via its electron-dense iron core [14][15][16] , but its small size complicates differentiation of individual ferritin particles from cellular structures. To enable multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM), we here introduce the encapsulin system of Quasibacillus thermotolerans (Qt) as a fully genetic iron-biomineralizing nanocompartment. We reveal by cryo-electron reconstructions that the Qt monomers (QtEnc) self-assemble to nanospheres with T=4 icosahedral symmetry and an~44 nm diameter harboring two putative pore regions at the fivefold and threefold axes. We furthermore show that the native cargo (QtIMEF) auto-targets to the inner surface of QtEnc and exhibits ferroxidase activity leading to efficient iron sequestration inside mammalian cells. We then demonstrate that QtEnc can be robustly differentiated from the non-intermixing encapsulin of Myxococcus xanthus 17 (Mx,~32 nm) via a deep-learning model, thus enabling automated multiplexed EM gene reporter imaging in mammalian cells. Encapsulins are a class of proteinaceous spherical nanocompartments naturally occurring in bacteria and archaea, so far described as icosahedral structures with either T=1 (60 subunits,~18 nm diameter) or T=3 (180 subunits,~30 nm) symmetry, which can encapsulate cargo proteins with a wide range of functions 18-21 . It has also been shown that foreign cargos such as fluorescent proteins or enzymes can be genetically targeted into the encapsulin lumen in bacterial and mammalian hosts [22][23][24][25][26] .

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Section: Multi-coloredmentioning

confidence: 99%

Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters

Sigmund

Pettinger

Kube

et al. 2019

Preprint

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“…It has been suggested that nAbs preferentially bind to target proteins via a convex paratope (Wurch et al, 2012), which could contribute to the inability of the nAbs against the targets with the exception of Homer1 to detect aldehyde-fixed and/or denatured target proteins. While nAbs have widespread use in binding to native protein, which enhances their utility as chaperones for crystallography , some generated against GFP and other proteins have been used as immunolabels for immunohistochemistry (Fang et al, 2018;Yamagata and Sanes, 2018) and immunoblots (Bruce and McNaughton, 2017) In summary, we have generated a series of validated nAbs against neuronal proteins selectively expressed in specific subcellular compartments in brain neurons. These nAbs represent a valuable toolbox of reagents available to the neuroscience community for diverse applications.…”

Section: Discussionmentioning

confidence: 99%

“…nAbs are ideally suited for use as intracellular antibodies (intrabodies) in living cells (Beghein et al, 2016;Bertier et al, 2017;Lafaye et al, 2009;Schumacher et al, 2018;Staus et al, 2014;Van Audenhove and Gettemans, 2016), as they fold efficiently and remain stable under a wide range of conditions, including the reducing cytoplasmic environment (Boldicke et al, 2005;Gahrtz and Conrad, 2009;Goenaga et al, 2007;Lynch et al, 2008). In addition to their potential utility as intrabodies, nAbs also have advantages as immunolabeling reagents, as their small size (≈1/10 of conventional IgG antibodies) improves penetration of the cell or tissue samples (Fang et al, 2018;Perruchini et al, 2009). Importantly, nAbs improve imaging resolution by reducing the distance between the immunolabeling signal and the target protein from the 10-20 nm obtained with conventional primary and secondary antibodies down to 2-4 nm (Beghein and Gettemans, 2017;Pleiner et al, 2015;Ries et al, 2012;Szymborska et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…Their ability to precisely target specific proteins in living cells, and/or label them in post vivo samples with a high degree of efficacy and spatial resolution make nAbs attractive for numerous biomedical research applications, including in neuroscience research (Sudhof, 2018). However, their uses in neuroscience have largely been limited to samples exogenously expressing GFP-tagged proteins [e.g., (Chamma et al, 2016;Ekstrand et al, 2014;Fang et al, 2018;Joensuu et al, 2016;Modi et al, 2018;Tang et al, 2013)], or in studies targeting proteins expressed in non-neuronal brain cells (Fang et al, 2018), although a set of recent studies have employed nAbs against neuronal targets [e.g., (Maidorn et al, 2019;Schenck et al, 2017;Scholler et al, 2017;Schoonaert et al, 2017). Mammalian brain neurons are distinguished from other cells by extreme molecular and structural complexity that is intimately linked to the array of intra-and inter-cellular signaling events that underlie brain function.…”

Section: Introductionmentioning

confidence: 99%

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A toolbox of nanobodies developed and validated for diverse neuroscience research applications

Jiang¹,

Lee²,

Kirmiz³

et al. 2019

Preprint

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Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications.Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.3

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“…Moreover, sample penetration of full antibodies is a problem when staining thick biological samples such as tissues, biopsies or whole organisms 17,19 . For the optimal labelling of these thick samples, protocols have been established, but they are often laborious and require time-consuming incubations up to weeks 20 or artifact-prone epitope retrieval protocols 21 .…”

Section: Mainmentioning

confidence: 99%

Circumvention of common labeling artifacts using secondary nanobodies

Sograte‐Idrissi

Duque-Alfonso

Alevra

et al. 2019

Preprint

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The most common procedure to reveal the location of specific (sub)cellular elements in biological samples is via immunostaining followed by optical imaging. This is typically performed with target-specific primary antibodies (1.Abs), which are revealed by fluorophore-conjugated secondary antibodies (2.Abs). However, at high resolution this methodology can induce a series of artifacts due to the large size of antibodies, their bivalency, and their polyclonality. Here we use STED and DNA-PAINT super-resolution microscopy or light sheet microscopy on cleared tissue to show how monovalent secondary reagents based on camelid single-domain antibodies (nanobodies; 2.Nbs) attenuate these artifacts. We demonstrate that monovalent 2.Nbs have four additional advantages: 1) they increase localization accuracy with respect to 2.Abs; 2) they allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; 3) they penetrate thick tissues efficiently; and 4) they avoid the artificial clustering seen with 2.Abs both in live and in poorly fixed samples. Altogether, this suggests that 2.Nbs are a valuable alternative to 2.Abs, especially when super-resolution imaging or staining of thick tissue samples are involved.

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Nanobody immunostaining for correlated light and electron microscopy with preservation of ultrastructure

Cited by 97 publications

References 23 publications

Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters

Iron-sequestering nanocompartments as multiplexed Electron Microscopy gene reporters

A toolbox of nanobodies developed and validated for diverse neuroscience research applications

Circumvention of common labeling artifacts using secondary nanobodies

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