Single-molecule visualization in cells with genetically encoded tags for electron microscopy (EM) has been a long-awaited but unimplemented tool for cell biologists.Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated cysteine-rich tags. We next exploited this mechanism to develop an approach for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies, and mitochondrial matrix. Thus, our implementation of genetically encoded tags for EM should allow cell biologists to address an enormous range of biological questions at single-molecule level in diverse cellular ultrastructural contexts without using antibodies.
Single-molecule visualization in cells with genetically encoded tags for electron microscopy (EM) has been a long-awaited but unimplemented tool for cell biologists.Here, we report an approach for directly synthesizing EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags for single-molecule visualization in cells. We first uncovered an auto-nucleation suppression mechanism that allows specific synthesis of AuNPs on isolated cysteine-rich tags. We next exploited this mechanism to develop an approach for single-molecule detection of proteins in prokaryotic cells and achieved an unprecedented labeling efficiency. We then expanded it to more complicated eukaryotic cells and successfully detected the proteins targeted to various organelles, including the membranes of endoplasmic reticulum (ER) and nuclear envelope, ER lumen, nuclear pores, spindle pole bodies, and mitochondrial matrix. Thus, our implementation of genetically encoded tags for EM should allow cell biologists to address an enormous range of biological questions at single-molecule level in diverse cellular ultrastructural contexts without using antibodies.need for antibodies and should permit cell biologists to quantitatively characterize the roles of proteins of interest in cells via genetic manipulation. RESULTSDesign of fixative-resistant cysteine-rich tags for electron microscopy An ideal tag for generating an EM-visible electron-dense label in cells would be small, fixative-resistant, and enable precise and sensitive detection. We considered the known limitations of MT in EM tag applications when we designed our tags 12 , primarily the need for multiple copies of MT to form EM-detectable clusters 12-17 and the reactivity of native MT tags with aldehyde-fixatives. To create more robust tags, first, we engineered mouse MT-1 protein (61 amino acid) into an aldehyde-fixative-resistant variant, MTn, by replacing the 21 aldehyde-reactive residues (e.g., Lys, Ser, Thr, Gln) 25 with aldehyde-inert residues (e.g., Ala) ( Fig.1b, Supplementary Table 1). We also developed a smaller variant, MTα, that consists of only the alpha domain of MTn.Additionally, to exploit the natural fixative-resistance of disulfide bonds, we employed another cysteine-rich tag, AFP, which we developed from an insect antifreeze protein, tmAFP 24 . AFP tags form highly stable structures via disulfide bonds in oxidizing cellular compartments ( Fig.1c, Supplementary Table 1). The design of these tags should, in theory, ensure the formation of relatively stable structures that protect them from fixative cross-linking denaturation. Moreover, upon exposure to nucleophiles during the synthesis of AuNPs, these tags can unfold readily (Fig.1d); this property is superior to tightly-packed cysteine-rich proteins (e.g., BSA and transferrin) that require harsh conditions to allow gold cluster formation 26,27 . To validate whether our engineered tags function as designed in cells, the genes for these tags were synthesized and used to construct tag-fused maltose-binding protein (MBP) that was...
We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 [1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in yeast cells expressing metallothionein (MTn) tags (Figure 1). This approach should be useful for EM visualization of single-molecule in yeast cells, and easier adapted for bacterial cells.
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