Biology subjects

Görlich, D.

Publications and source records attributed to Görlich, D..

4 recordsLinked to original sources

bioRxiv2025

Governed by surface amino acid composition: HIV capsid passage through the NPC barrier

Nuclear transport receptors (NTRs) carry cargo across the permeability barrier of nuclear pore complexes (NPCs) - an FG phase condensed from disordered but cohesive FG repeats. This phase repels normal macromolecules but allows NTR passage. When HIV infects non-dividing cells, its capsid is transported into nuclei not like cargo but crosses NPCs like NTRs. We now uncovered the molecular determinants of the capsids NTR behavior: The FG-binding pocket is insufficient. Hexameric and pentameric capsomers contribute. The highly exposed outer capsid surface is key. It lacks FG-repulsive charged residues (K,D,E) that are very abundant on normal protein surfaces. FG-attractive residues dominate the capsid surface. Introducing FG-repulsive ones impedes FG phase-partitioning, NPC-targeting and NPC-passage of assembled capsids. Capsids are thus made FG phase-soluble by myriads of transient FG-attractive interactions originating from individual surface sidechains. We discuss that CPSF6 releases the capsid from NPCs by switching its surface from FG-attractive to FG-repulsive.

cell biology↗

bioRxiv2023

Nucleoporin-binding nanobodies that either track or inhibit nuclear pore complex assembly

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact NPCs from frog to human. We further report a simplified assay that directly tracks postmitotic NPC assembly by added labeled anti-Nup nanobodies. In interphase, NPCs are inserted into a pre-existing nuclear envelope. This makes it difficult to monitor this process as newly-assembled NPCs must be distinguished from pre-existing ones. We solved this problem by inserting Xenopus NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We also asked whether anti-nucleoporin (Nup) nanobodies could serve as NPC assembly inhibitors. A first generation, selected from immune libraries against Xenopus Nups, comprised only bright stainers of intact NPCs but no inhibitors, perhaps because the immune response was biased towards non-conserved and, thus, functionally-irrelevant epitopes. To overcome this bias, we selected for crossreactivity between Xenopus and human Nups and obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup-Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93--solenoid in recruiting Nup358 and the Nup214{middle dot}88{middle dot}62 complex, and for Nup155 and the Nup98 autoproteolytic domain in NPC-scaffold assembly. The latter suggests an assembly checkpoint linking pore formation to permeability barrier assembly.

cell biology↗

bioRxiv2022

A nanobody toolbox to investigate localisation and dynamics of Drosophila titins

Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understand protein function. However, to date this is still a challenging task, as accessibility of large antibodies to dense tissues is often limited and fluorescent proteins inserted close to a domain of interest may affect function of the tagged protein. These complications are particularly present in the muscle sarcomere, arguably one of the most protein dense structures in nature, which makes studying morphogenesis at molecular resolution challenging. Here, we have employed an efficient pipeline to generate a nanobody toolbox specifically recognising various domains of two large Drosophila titin homologs, Sallimus and Projectin. We demonstrate the superior labelling qualities of our nanobodies compared to conventional antibodies in intact muscle tissue. Applying our nanobody toolbox to larval muscles revealed a gigantic Sallimus isoform stretched more than 2 {micro}m to bridge the sarcomeric I-band. Furthermore, N- and C-terminal nanobodies against Projectin identified an unexpected polar orientation of Projectin covering the myosin filaments in larval muscles. Finally, expression of a Sallimus nanobody in living larval muscles confirmed the high affinity binding of nanobodies to target epitopes in living tissue and hence demonstrated their power to reveal the in vivo dynamics of sarcomeric protein domains. Together, our toolbox substantiates the multiple advantages of nanobodies to study sarcomere biology. It may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.

developmental biology↗

bioRxiv2022

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nano-architecture in flight muscles

Sarcomeres are the force producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super- resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A- band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

developmental biology↗