MINFLUX nanoscopy delivers multicolor nanometer 3D-resolution in (living) cells

Mapping Intimacies ◽

10.1101/734251 ◽

2019 ◽

Cited By ~ 1

Author(s):

Klaus C. Gwosch ◽

Jasmin K. Pape ◽

Francisco Balzarotti ◽

Philipp Hoess ◽

Jan Ellenberg ◽

...

Keyword(s):

Protein Complexes ◽

Three Dimensional ◽

Living Cells ◽

Three Dimensions ◽

Nanometer Scale ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Fluorescent Marker ◽

Excitation Beam ◽

Pore Complexes

The ultimate goal of biological superresolution fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. Here, we show that, by localizing individual switchable fluorophores with a probing doughnut-shaped excitation beam, MINFLUX nanoscopy provides 1–3 nanometer resolution in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing doughnut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. MINFLUX imaging of nuclear pore complexes of a mammalian cell shows that this true nanometer scale resolution is obtained in three dimensions and in two color channels. Relying on fewer detected photons than popular camera-based localization, MINFLUX nanoscopy is poised to open a new chapter in the imaging of protein complexes and distributions in fixed and living cells.

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The dynamic three-dimensional organization of the diploid yeast genome

10.1101/091827 ◽

2016 ◽

Author(s):

Seungsoo Kim ◽

Ivan Liachko ◽

Donna G Brickner ◽

Kate Cook ◽

William S Noble ◽

...

Keyword(s):

Nuclear Periphery ◽

Three Dimensional ◽

Culture Conditions ◽

Yeast Genome ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Chromosome Conformation ◽

Homolog Pairing ◽

Pore Complexes ◽

Eukaryotic Genomes

AbstractThe budding yeast Saccharomyces cerevisiae is a long-standing model for the three-dimensional organization of eukaryotic genomes. Even in this well-studied model, it is unclear how homolog pairing in diploids and environment-induced gene relocalization influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged Saccharomyces hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation, we observe significant homolog proximity that increased in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the HAS1 alleles specifically under galactose induction and saturated growth, mediated by association with nuclear pore complexes at the nuclear periphery. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization.

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Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

The Journal of Cell Biology ◽

10.1083/jcb.201411003 ◽

2015 ◽

Vol 208 (3) ◽

pp. 283-297 ◽

Cited By ~ 39

Author(s):

Monika Gaik ◽

Dirk Flemming ◽

Alexander von Appen ◽

Panagiotis Kastritis ◽

Norbert Mücke ◽

...

Keyword(s):

Structural Model ◽

Three Dimensional ◽

Nucleocytoplasmic Transport ◽

Structural Basis ◽

Integrative Approach ◽

Asymmetric Structure ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Pore Complexes ◽

And Function

Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery.

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The Effect of FG-Nup Phosphorylation on NPC Selectivity: A One-Bead-Per-Amino-Acid Molecular Dynamics Study

International Journal of Molecular Sciences ◽

10.3390/ijms20030596 ◽

2019 ◽

Vol 20 (3) ◽

pp. 596 ◽

Cited By ~ 3

Author(s):

Ankur Mishra ◽

Wouter Sipma ◽

Liesbeth Veenhoff ◽

Erik Van der Giessen ◽

Patrick Onck

Keyword(s):

Molecular Dynamics ◽

Amino Acid ◽

Intrinsically Disordered Proteins ◽

Protein Complexes ◽

Disordered Proteins ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Intrinsically Disordered ◽

Dynamics Simulations ◽

Pore Complexes

Nuclear pore complexes (NPCs) are large protein complexes embedded in the nuclear envelope separating the cytoplasm from the nucleoplasm in eukaryotic cells. They function as selective gates for the transport of molecules in and out of the nucleus. The inner wall of the NPC is coated with intrinsically disordered proteins rich in phenylalanine-glycine repeats (FG-repeats), which are responsible for the intriguing selectivity of NPCs. The phosphorylation state of the FG-Nups is controlled by kinases and phosphatases. In the current study, we extended our one-bead-per-amino-acid (1BPA) model for intrinsically disordered proteins to account for phosphorylation. With this, we performed molecular dynamics simulations to probe the effect of phosphorylation on the Stokes radius of isolated FG-Nups, and on the structure and transport properties of the NPC. Our results indicate that phosphorylation causes a reduced attraction between the residues, leading to an extension of the FG-Nups and the formation of a significantly less dense FG-network inside the NPC. Furthermore, our simulations show that upon phosphorylation, the transport rate of inert molecules increases, while that of nuclear transport receptors decreases, which can be rationalized in terms of modified hydrophobic, electrostatic, and steric interactions. Altogether, our models provide a molecular framework to explain how extensive phosphorylation of FG-Nups decreases the selectivity of the NPC.

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Structure and Assembly of the Nuclear Pore Complex

Annual Review of Biophysics ◽

10.1146/annurev-biophys-052118-115308 ◽

2019 ◽

Vol 48 (1) ◽

pp. 515-536 ◽

Cited By ~ 40

Author(s):

Bernhard Hampoelz ◽

Amparo Andres-Pons ◽

Panagiotis Kastritis ◽

Martin Beck

Keyword(s):

Posttranslational Modifications ◽

Current Knowledge ◽

Protein Complexes ◽

Hierarchical Organization ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Multiple Copies ◽

Architectural Principles ◽

Protein Components ◽

Pore Complexes

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange. They are exceptionally large protein complexes that fuse the inner and outer nuclear membranes to form channels across the nuclear envelope. About 30 different protein components, termed nucleoporins, assemble in multiple copies into an intricate cylindrical architecture. Here, we review our current knowledge of the structure of nucleoporins and how those come together in situ. We delineate architectural principles on several hierarchical organization levels, including isoforms, posttranslational modifications, nucleoporins, and higher-order oligomerization of nucleoporin subcomplexes. We discuss how cells exploit this modularity to faithfully assemble NPCs.

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Postmitotic nuclear pore assembly proceeds by radial dilation of small ER membrane openings

10.1101/141150 ◽

2017 ◽

Author(s):

Shotaro Otsuka ◽

Anna M. Steyer ◽

Martin Schorb ◽

Jean-Karim Hériché ◽

M. Julius Hossain ◽

...

Keyword(s):

Time Course ◽

Three Dimensional ◽

Membrane Topology ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Ring Complex ◽

Pore Complexes ◽

Transport Channels ◽

Membrane Sealing ◽

Nuclear Pore Assembly

AbstractThe nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

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The dynamic three-dimensional organization of the diploid yeast genome

eLife ◽

10.7554/elife.23623 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 28

Author(s):

Seungsoo Kim ◽

Ivan Liachko ◽

Donna G Brickner ◽

Kate Cook ◽

William S Noble ◽

...

Keyword(s):

Nuclear Periphery ◽

Three Dimensional ◽

Culture Conditions ◽

Yeast Genome ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Yeast Saccharomyces Cerevisiae ◽

Chromosome Conformation ◽

Homolog Pairing ◽

Pore Complexes

The budding yeast Saccharomyces cerevisiae is a long-standing model for the three-dimensional organization of eukaryotic genomes. However, even in this well-studied model, it is unclear how homolog pairing in diploids or environmental conditions influence overall genome organization. Here, we performed high-throughput chromosome conformation capture on diverged Saccharomyces hybrid diploids to obtain the first global view of chromosome conformation in diploid yeasts. After controlling for the Rabl-like orientation using a polymer model, we observe significant homolog proximity that increases in saturated culture conditions. Surprisingly, we observe a localized increase in homologous interactions between the HAS1-TDA1 alleles specifically under galactose induction and saturated growth. This pairing is accompanied by relocalization to the nuclear periphery and requires Nup2, suggesting a role for nuclear pore complexes. Together, these results reveal that the diploid yeast genome has a dynamic and complex 3D organization.

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Nuclear Envelope Dynamics During Mitosis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103188 ◽

1988 ◽

Vol 46 ◽

pp. 224-225

Author(s):

Brian Burke

Keyword(s):

Nuclear Envelope ◽

Membrane Vesicles ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Animal Cells ◽

Interphase Chromosome ◽

Lamin B ◽

Chromosome Architecture ◽

Complex Membrane ◽

Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

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