Biology subjects

Gunkel, P.

Publications and source records attributed to Gunkel, P..

5 recordsLinked to original sources

bioRxiv2026

Hierarchical TPR:ZC3HC1 interactions drive stepwise assembly of nuclear basket extensions projecting into vertebrate cell nuclei

The nuclear basket (NB) is a fibrillar scaffold anchored to the nuclear pore complex (NPC), with protein TPR constituting its structural backbone. Recently, ZC3HC1 was identified as an additional structural NB component interconnecting TPR subpopulations. However, it had remained unclear how ZC3HC1 and TPR engage each other and how the resulting TPR:ZC3HC1 ensembles are arranged relative to the NB. Here, we uncover a hierarchy of sequential interactions between distinct ZC3HC1 and TPR domains, resulting in NB-appended ZC3HC1:TPR ensembles that extend beyond the NB dimensions traditionally considered prototypic. Consistent with a modular organisational principle, we furthermore find copy numbers of NPC/NB-associated TPR to be far higher than commonly anticipated. Together with sufficient ZC3HC1 copy numbers, our data define a model in which alternating recruitment of ZC3HC1 and TPR extends the NB's spatial and functional reach into the nuclear interior.

cell biology↗

bioRxiv2022

Nuclear basket protein ZC3HC1 and its yeast homolog Pml39p feature anevolutionary conserved bimodular construction essential for initial bindingto NPC-anchored homologs of scaffold protein TPR

Proteins ZC3HC1 and TPR are construction elements of the nuclear pore complex (NPC)-attached nuclear basket (NB). NB-location of ZC3HC1 depends on TPR already occurring NPC-anchored, whereas additional TPR polypeptides are appended to the NB by ZC3HC1. The current study examined the molecular properties of ZC3HC1 that enable it to bind to the NB and TPR. We report the identification and definition of a nuclear basket-interaction domain (NuBaID) of HsZC3HC1 comprising two similarly built modules, both essential for the binding to the NBs NPC-anchored HsTPR. Furthermore, we describe such a bimodular construction as evolutionarily conserved and exemplify the kinship of HsZC3HC1 by the NB- and DdTPR-interacting homolog of Dictyostelium discoideum and by characterizing protein Pml39 as the ZC3HC1 homolog in Saccharomyces cerevisiae. Among several properties shared by the different species homologs, we unveil the integrity of the bimodular NuBaID of ScPml39p as being essential for binding to the yeasts NBs and its TPR homologs ScMlp1p and ScMlp2p, and we further present Pml39p as enabling interlinkage of Mlp1p subpopulations. In addition to phyla-specific features, we delineate the three species common NuBaID as the characterizing structural entity of a one-of-a-kind protein found not in all but likely most taxa of the eukaryotic realm.

cell biology↗

bioRxiv2022

MINSTED nanoscopy enters the Angstrom localization range

We report all-optical, room-temperature localization of fluorophores with precision in the [A]ngstrom range. These precisions are attained in a STED microscope, by encircling the fluorophore with the low-intensity edge of the STED donut beam, while constantly increasing the absolute donut power. Individual fluorophores bound to a DNA strand are localized with{sigma} = 4.7 [A], corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Since our experiments yield a localization precision{sigma} = 2.3 [A], estimated for 10,000 detected photons, we anticipate that MINSTED will open up entirely new areas of application in the study of macromolecular complexes in cells.

biophysics↗

bioRxiv2022

ZC3HC1 is a structural element of the nuclear basket effecting interlinkage of TPR polypeptides

The nuclear basket (NB), anchored to the nuclear pore complex (NPC), is commonly thought of as built solely of protein TPR polypeptides, the latter thus regarded as the NBs only scaffold-forming components. In the current study, we report ZC3HC1 as a second building element of the NB. Recently described as an NB-appended protein omnipresent in vertebrates, we now show that ZC3HC1, both in vivo and in vitro, enables in a step-wise fashion the recruitment of TPR subpopulations to the NB and their linkage to already NPC-anchored TPR polypeptides. We further demonstrate that the degron-mediated rapid elimination of ZC3HC1 results in the prompt detachment of the ZC3HC1-appended TPR polypeptides from the NB and their release back into the nucleoplasm again, underscoring the role of ZC3HC1 as a natural structural element of the NB. Finally, we show that ZC3HC1 can keep TPR polypeptides positioned even at sites remote from the NB, in line with ZC3HC1 functioning as a protein connecting TPR polypeptides.

cell biology↗

bioRxiv2020

Structure of human RNA Polymerase III

ABSTRACTIn eukaryotes, RNA Polymerase (Pol) III is the enzyme specialised for the transcription of the entire pool of tRNAs and several other short, essential, untranslated RNAs. Pol III is a critical determinant of cellular growth and lifespan across the eukaryotic kingdom. Upregulation of Pol III transcription is often observed in cancer cells and causative Pol III mutations have been described in patients affected by severe neurodevelopmental disorders and hypersensitivity to viral infection.Harnessing CRISPR-Cas9 genome editing in HeLa cells, we isolated endogenous human Pol III and obtained a cryo-EM reconstruction at 4.0 Å. The structure of human Pol III allowed us to map the reported genetic mutations and rationalise them. Mutations causing neurodevelopmental defects cluster in hotspots that affect the stability and/or biogenesis of Pol III, thereby resulting in loss-of-function of the enzyme. Mutations affecting viral sensing are located in the periphery of the enzyme in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing activity.Furthermore, integrating x-ray crystallography and SAXS data, we describe the structure of the RPC5 C-terminal extension, which is absent in lower eukaryotes and not visible in our EM map. Surprisingly, experiments in living cells highlight a role for the RPC5 C-terminal extension in the correct assembly and stability of the human Pol III enzyme, thus suggesting an added layer of regulation during the biogenesis of Pol III in higher eukaryotes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗