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Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT.

ABSTRACT: The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super-resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of circa 3?nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

SUBMITTER: Schlichthaerle T

PROVIDER: S-EPMC6771475 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT.

Schlichthaerle Thomas T Strauss Maximilian T MT Schueder Florian F Auer Alexander A Nijmeijer Bianca B Kueblbeck Moritz M Jimenez Sabinina Vilma V Thevathasan Jervis V JV Ries Jonas J Ellenberg Jan J Jungmann Ralf R

Angewandte Chemie (International ed. in English) 20190821 37

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super-resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC prot ...[more]

PMID: 31314157

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Project description:The nuclear pore complex (NPC) facilitates transport between nucleus and cytoplasm. The protein constituents of the NPC, termed nucleoporins (Nups), are conserved across a wide diversity of eukaryotes. In apparent exception to this, no nucleoporin genes have been identified in nucleomorph genomes. Nucleomorphs, nuclear remnants of once free-living eukaryotes, took up residence as secondary endosymbionts in cryptomonad and chlorarachniophyte algae. As these genomes are highly reduced, Nup genes may have been lost, or relocated to the host nucleus. However, Nup genes are often poorly conserved between species, so absence may be an artifact of low sequence similarity. We therefore constructed an evolutionary bioinformatic screen to establish whether the apparent absence of Nup genes in nucleomorph genomes is due to genuine absence or the inability of current methods to detect homologues. We searched green plant (Arabidopsis and rice), green alga (Chlamydomonas reinhardtii) and red alga (Cyanidioschyzon merolae) genomes, plus two nucleomorph genomes (Bigelowiella natans and Guillardia theta) with profile hidden Markov models (HMMs) from curated alignments of known vertebrate/yeast Nups. Since the plant, algal and nucleomorph genomes all belong to the kingdom Plantae, and are evolutionarily distant from the outgroup (vertebrate/yeast) training set, we use the plant and algal genomes as internal positive controls for the sensitivity of the searches in nucleomorph genomes. We find numerous Nup homologues in all plant and free-living algal species, but none in either nucleomorph genome. BLAST searches using identified plant and algal Nups also failed to detect nucleomorph homologues. We conclude that nucleomorph Nup genes have either been lost, being replaced by host Nup genes, or, that nucleomorph Nup genes have been transferred to the host nucleus twice independently; once in the evolution of the red algal nucleomorph and once in the green algal nucleomorph.

Dataset Information

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT.

Publications

Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically-Encoded Probes for DNA-PAINT.

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