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

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

A quantitative map of nuclear pore assembly reveals two distinct mechanisms

Understanding how the nuclear pore complex (NPC) assembles is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during evolution of eukaryotes1–4. While we know that at least two NPC assembly pathways exist, one during exit from mitosis and one during nuclear growth in interphase, we currently lack a quantitative map of their molecular events. Here, we use fluorescence correlation spectroscopy (FCS) calibrated live imaging of endogenously fluorescently-tagged nucleoporins to map the changes in composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways employ strikingly different molecular mechanisms, inverting the order of addition of two large structural components, the central ring complex and nuclear filaments. Our dynamic stoichiometry data allows us to perform the first computational simulation that predicts the structure of postmitotic NPC assembly intermediates

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements

Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3

Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 RS-domain variants retain their splice regulatory activity, which reveals that aberrant cellular localization is the main driver of their pathological phenotype. A genome-wide CRISPR knockout screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, and is disrupted by pathogenic variants. Relocalization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20's nuclear localization in RBM20-DCM patients

3D super-resolution fluorescence microscopy maps the variable molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of over 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of 3D super-resolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of over ten thousand individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ

Nuclear pore assembles via structurally and molecularly distinct mechanisms after mitosis and during interphase

The nuclear pore complex (NPC) is the largest non-polymeric protein complex in eukaryotic cells and spans the double membrane of the nucleus (nuclear envelope; NE) to mediate nucleocytoplasmic transport. In mammalian cells, NPCs are assembled in two cell cycle stages, during nuclear assembly after mitosis and nuclear growth in interphase. How the NPC and the double nuclear membrane reassemble concomitantly in late mitosis, and how the NPC newly assembles in the closed NE in interphase, has been unclear. By correlating live imaging with three-dimensional electron microscopy, we have recently revealed that nuclear pores assemble via structurally distinct mechanisms in mitosis and interphase; during mitotic exit, pore assembly proceeds by radial dilation of small membrane openings, while in interphase, assembly induces a novel asymmetric inside-out fusion of the inner with the outer nuclear membrane. To understand the molecular maturation processes of these two distinct NPC assembly pathways, we created genome-edited GFP knock-in cells for nucleoporins of all major NPC substructures, i.e. the cytoplasmic filaments, the cytoplasmic/nucleoplasmic rings, the inner rings, and the nuclear basket. By FCS-calibrated three-dimensional live cell imaging, we monitored the concentration changes of these GFP-tagged nucleoporins in different regions of the NE where postmitotic and interphase assembly can be spatially distinguished for the first hour after mitotic exit. Quantitative kinetic analysis of the concentration changes showed that the molecular assembly order and maturation kinetics are distinct for postmitotic and interphase assembly, demonstrating that NPC assembly is not only a structurally but also molecularly different process between mitosis and interphas

Chemogenetic control of nanobodies

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules

A quantitative map of human Condensins provides new insights into mitotic chromosome architecture

The two Condensin complexes in human cells are essential for mitotic chromosome structure. We used homozygous genome editing to fluorescently tag Condensin I and II subunits and mapped their absolute abundance, spacing, and dynamic localization during mitosis by fluorescence correlation spectroscopy (FSC)–calibrated live-cell imaging and superresolution microscopy. Although ∼35,000 Condensin II complexes are stably bound to chromosomes throughout mitosis, ∼195,000 Condensin I complexes dynamically bind in two steps: prometaphase and early anaphase. The two Condensins rarely colocalize at the chromatid axis, where Condensin II is centrally confined, but Condensin I reaches ∼50% of the chromatid diameter from its center. Based on our comprehensive quantitative data, we propose a three-step hierarchical loop model of mitotic chromosome compaction: Condensin II initially fixes loops of a maximum size of ∼450 kb at the chromatid axis, whose size is then reduced by Condensin I binding to ∼90 kb in prometaphase and ∼70 kb in anaphase, achieving maximum chromosome compaction upon sister chromatid segregation

Photoactivation of silicon rhodamines via a light-induced protonation

hotoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore’s outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy

3D super-resolution fluorescence microscopy maps the variable molecular architecture of the Nuclear Pore Complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of more than 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of three-dimensional (3D) superresolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of more than 10,000 individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ