Directed evolution of APEX2 for electron microscopy and proximity labeling

Lam, Stephanie S; Martell, Jeffrey D.; Kamer, Kimberli J.; Deerinck, Thomas J.; Ellisman, Mark H.; Mootha, Vamsi K.; Ting, Alice Y.

doi:10.1038/nmeth.3179

Cited by 1,155 publications

(1,361 citation statements)

References 41 publications

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“…For example, a protein can be fused to a fluorescent protein in a variety of different colors to facilitate localization and co-localization studies. Tags developed for electron microscopy, such as APEX2 or miniSOG 11,12 , allow ultrastructural localization of the tagged protein. Tags for biochemistry, such as the TAP tag, and ProtC-TEV-ProtA (PTP) tag 7,13 allow purification of complexes associated with the protein for identification of binding partners or in vitro biochemical assays.…”

Section: Discussionmentioning

confidence: 99%

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

Dyer

Dean

Sunter

2016

JoVE

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Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

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Section: Discussionmentioning

confidence: 99%

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

Dyer

Dean

Sunter

2016

JoVE

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“…Dual-purpose fluorescent and electron-dense labels for EM (SOG, miniSOG, APEX2) are genetically encoded tags for correlative light and electron microscopy (Shu et al, 2011;Lam et al, 2015), which could be applied to the ON&LCN. Through-focus scanning optical microscopy (Attota et al, 2013), confocal soft X-ray scanning transmission microscopy (Spath et al, 2015) and intravital microscopy (Masedunskas et al, 2012;Alexander et al, 2013) -subcellular imaging in living animals -are techniques which may add to the repertoire for osteocyte investigation.…”

Section: Novel Imaging Methodsmentioning

confidence: 99%

High-resolution 3D imaging of osteocytes and computational modelling in mechanobiology: insights on bone development, ageing, health and disease

Goggin

Zygalakis

Oreffo³

et al. 2016

eCM

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Osteocytes are involved in mechanosensation and mechanotransduction in bone and hence, are key to bone adaptation in response to development, ageing and disease. Thus, detailed knowledge of the three-dimensional (3D) structure of the osteocyte network (ON) and the surrounding lacuno-canalicular network (LCN) is essential. Enhanced understanding of the ON&LCN will contribute to a better understanding of bone mechanics on cellular and sub-cellular scales, for instance through improved computational models of bone mechanotransduction. Until now, the location of the ON within the hard bone matrix and the sub-µm dimensions of the ON&LCN have posed significant challenges for 3D imaging. This review identifies relevant microstructural phenotypes of the ON&LCN in health and disease and summarises how light microscopy, electron microscopy and X-ray imaging techniques have been used in studies of osteocyte anatomy, pathology and mechanobiology to date. In this review, we assess the requirements for ON&LCN imaging and examine the state of the art in the fields of imaging and computational modelling as well as recent advances in high-resolution 3D imaging. Suggestions for future investigations using volume electron microscopy are indicated and we present new data on the ON&LCN using serial block-face scanning electron microscopy. A correlative approach using these high-resolution 3D imaging techniques in conjunction with in silico modelling in bone mechanobiology will increase understanding of osteocyte function and, ultimately, lead to improved pathways for diagnosis and treatment of bone diseases such as osteoporosis.Keywords: Osteocyte, 3D imaging, microscopy, b i o m e c h a n i c s , l a c u n o -c a n a l i c u l a r n e t w o r k , mechanobiology, mechanosensation, mechanotransduction, osteoporosis. IntroductionThrough bone adaptation, the skeleton adapts continuously to changed mechanical loading patterns due to development, growth, ageing, disease, disuse or exercise, by removing existing and adding new bone tissue. It has been recognised that osteocytes are the key cells which orchestrate bone adaptation (Tatsumi et al., 2007). Osteocytes are ovoid cells approximately 10 µm long, surrounded by a pericellular matrix (PCM). The osteocytes and their processes form the osteocyte network (ON), which is housed within the lacuno-canalicular network (LCN), a system of voids and channels in the calcified bone matrix (Fig. 1). Neve et al., 2012). Knowledge of the three-dimensional (3D) structure of the ON&LCN would inform conclusions about the function and malfunction of osteocytes for different bone states in development, ageing, disease, disuse or exercise. More specifically, enhanced knowledge would improve the predictive power and accuracy of computational models, which attempt to elucidate the mechanisms of mechanotransduction using 3D geometries for the ON&LCN. Recent finite element and fluid-structure interaction models have used relatively low resolution image data and made a priori assumptions about the ...

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“…Advances include 3D EM, large scale 2D EM and improved methods and reagents for correlated light microscopy and electron microscopy (CLEM) to compare other modes of microscopic analysis directly to the EM level [8][9][10] . Large-scale 2D EM is particularly suitable to quantify or identify (novel) disease features for human pathology, study animal models for disease and tissue culture models.…”

Section: Introductionmentioning

confidence: 99%

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Kuipers¹,

Kalicharan²,

Wolters³

et al. 2016

JoVE

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Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae [1][2][3][4][5][6][7] . Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on onehole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 -50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture [1][2][3][4][5] . Here, zebrafish brains were analyzed in a noninvasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM) 8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

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Directed evolution of APEX2 for electron microscopy and proximity labeling

Cited by 1,155 publications

References 41 publications

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

High-throughput Gene Tagging in <em>Trypanosoma brucei</em>

High-resolution 3D imaging of osteocytes and computational modelling in mechanobiology: insights on bone development, ageing, health and disease

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

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