Barrier properties of Nup98 FG phases ruled by FG motif identity and inter-FG spacer length.

Nup98 FG repeat domains comprise hydrophobic FG motifs linked through uncharged spacers. FG motifs capture nuclear transport receptors (NTRs) during nuclear pore complex (NPC) passage, confer inter-repeat cohesion, and condense the domains into a selective phase with NPC-typical barrier properties. We show that shortening inter-FG spacers enhances cohesion, increases phase density, and tightens such barrier - all consistent with a sieve-like phase. Phase separation tolerates mutating the Nup98-typical GLFG motifs, provided domain-hydrophobicity remains preserved. NTR-entry, however, is sensitive to (certain) deviations from canonical FG motifs, suggesting co-evolutionary adaptation. Unexpectedly, we observed that arginines promote FG-phase-entry apparently also by hydrophobic interactions/ hydrogen-bonding and not just through cation-π interactions. Although incompatible with NTR·cargo complexes, a YG phase displays remarkable transport selectivity, particularly for engineered GFPNTR-variants. GLFG to FSFG mutations make the FG phase hypercohesive, precluding NTR-entry. Extending spacers relaxes this hypercohesion. Thus, antagonism between cohesion and NTR·FG interactions is key to transport selectivity.

Nanobodies combined with DNA-PAINT super-resolution reveal a staggered titin nanoarchitecture 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.

From ants to humans, the muscles that set an organism in motion are formed of bundles of fiber-like cells which can shorten and lengthen at will. At the microscopic level, changes in muscle cell lengths are underpinned by contractile filaments formed of multiple repeats of a basic unit, known as the sarcomere. Each unit is bookended by intricate 'Z-discs' and features an 'M-band' in its center. Three protein types give a sarcomere its ability to shorten and expand at will: two types of filaments (myosin and actin), which can slide on one another; and a spring-like molecule known as titin, which ensures that the unit does not fall apart by mechanically connecting myosin and actin. More specifically, actin filaments are anchored to the Z-discs and extend towards the M-band, while myosin filaments are centered around the M-band and extend towards the Z-discs. As myosin and actin slide alongside each other, the overlap between the two types of filaments increases or decreases and the whole unit changes its length. In vertebrates, one gigantic molecule of titin spans from the Z-disc to the M-band, linking together actin and myosin filaments and determining the length of the sarcomere. In insects and other invertebrates, however, this single molecule is replaced by two titin proteins known as Projectin and Sallimus. Understanding how these titins work together remains unclear and difficult to study. Traditional approaches are unable to precisely label titin in an environment teaming with other molecules, and they cannot offer the nanometer resolution required to dissect sarcomere organization. As a response, Schueder, Mangeol et al. combined super-resolution microscopy and a new toolbox of labelling molecules known as nanobodies to track the position of Sallimus and Projectin in the flight muscles of fruit flies. These experiments revealed that the two proteins are arranged in tandem along the length of the sarcomere, forming a structure that measures about 350 nm. Sallimus is anchored in the Z-disc and it runs alongside actin until it reaches the end of a myosin filament; there, it overlaps with Projectin for about 10 nm. Projectin then stretches for 250 nm along the length of the beginning myosin filament. These findings confirm the importance of titin in dictating the length of a sarcomere; they suggest that, in invertebrates, this role is split between two proteins, each possibly ruling over a section of the sarcomere. In addition, the work by Schueder, Mangeol et al. demonstrate the value of combining nanobodies and super-resolution microscopy to study complex structures in tissues.

Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies.

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.

A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins.

We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane-sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes; thus it should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine-glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTRs) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport.

Promiscuous Dimerization Between the Caenorhabditis elegans IF Proteins and a Hypothesis to Explain How Multiple IFs Persist Over Evolutionary Time.

Our previous calculations of ionic interactions indicated that the Caenorhabditis elegans intermediate filament (IF) IFA proteins, in addition to IFA/IFB-1 heterodimers, may also form homodimers. In order to prove the significance of these calculations, we analysed the dimerization potential of the IFA chains in blot overlays. Unexpectedly, we found here that the dimerization of the IFA-1 protein was of both homotypic and heterotypic nature, and involved all proteins immobilized on the membrane (IFA-1, IFA-2, IFA-4, IFB-1, IFB-2, IFC-1, IFC-2, IFD-1, IFD-2 and IFP-1). A similar interaction profile, though less complex, was observed for two biotinylated proteins (IFA-2 and IFA-4). These and previous results indicate that the IFA proteins are able to form many different heteropolymeric and homopolymeric complexes in the C. elegans tissue, but that only those triggered by the IFA-specific IFB-1 protein result in mature IFs. Moreover, the calculations of the possible ionic interactions between the individual rod sequences as well as their various deletion variants indicated a special role in this process for the middle part of the C. elegans IF coil 1B segment that is deleted in all vertebrate cytoplasmic IFs. We hypothesized here, therefore, that the striking promiscuity of the C. elegans IFs originally involved a nuclear lamin which, due to a two-heptad-long rod deletion, prevented formation of a functional lamin/cIF dimer. This, in concert with an efficient dimerization and a strict tissue-specific co-expression, may allow expansion and maintenance of the multiple Caenorhabditis IFs. A possible implication for evolution of chordate IFs proteins is also discussed.

Surface Properties Determining Passage Rates of Proteins through Nuclear Pores.

Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport through an FG domain-controlled barrier. We now explore how surface-features of a mobile species determine its NPC passage rate. Negative charges and lysines impede passage. Hydrophobic residues, certain polar residues (Cys, His), and, surprisingly, charged arginines have striking translocation-promoting effects. Favorable cation-π interactions between arginines and FG-phenylalanines may explain this apparent paradox. Application of these principles to redesign the surface of GFP resulted in variants that show a wide span of transit rates, ranging from 35-fold slower than wild-type to ∼500 times faster, with the latter outpacing even naturally occurring nuclear transport receptors (NTRs). The structure of a fast and particularly FG-specific GFPNTR variant illustrates how NTRs can expose multiple regions for binding hydrophobic FG motifs while evading non-specific aggregation. Finally, we document that even for NTR-mediated transport, the surface-properties of the "passively carried" cargo can strikingly affect the translocation rate.

Assembly studies of six intestinal intermediate filament (IF) proteins B2, C1, C2, D1, D2, and E1 in the nematode C. elegans.

The dimerisation properties of six intestine-expressed intermediate filament (IF) proteins (B2, C1, C2, D1, D2, E1) were analysed in blot overlay assay on membranes containing all of the eleven recombinant C. elegans IF proteins (A1, A2, A3, A4, B1, B2, C1, C2, D1, D2, and E1). The interactions detected in the blot assays exclusively comprise intestine-expressed IF proteins and the protein A4, which is found in the dauer larva intestine. About 86% of these interactions are heterotypic, while the remaining interactions relate to C1, C2, and D2 homodimers. These multiple modes of interaction were also supported by calculations of the numbers of possible interchain ionic interactions derived from the individual rod sequences. The results predict that the six B2, C1, C2, D1, D2, and E1 IF proteins are able to form as many as eleven different heteropolymeric and three homopolymeric IFs in the C. elegans intestine. This simple model of the intestinal IF meshwork enables us to speculate that our previously reported triple RNAi worms arrested or decreased their growth because of feeding reduction due to morphological defects of the mechanically compromised intestine.

Unusual ultrastructures of the Branchiostoma IF protein C2 containing heptads in the tail.

Branchiostoma intermediate filament (IF) protein C2 contains a long tail domain consisting of several degenerate repeats which display a heptad repeat pattern. This unique tail sequence is predicted to constitute a long coiled coil domain in C2, which is separated from the rod by a glycine-rich linker L3. The recombinant IF protein C2 shows, in electron microscopy (EM), parallel rodlike dimers of 66.7 nm decorated by a larger globule on one side and a smaller globule on the other side. In contrast, the length of the tailless C2 dimers, decorated by only one small globule, is about 26 nm shorter. These results indicate that both the rod domain and the newly predicted coiled coil segment 3 participate in the formation of a double-stranded coiled coil dimer. Moreover, the two to four C2 dimers are able to associate via their globular tail domain into multiarm oligomers, an ability not seen by the tailless C2 mutant or the other currently known protostomic and vertebrate IFs.

A rod domain sequence in segment 1B triggers dimerisation of the two small Branchiostoma IF proteins B2 and A3.

Previously, we cloned two Branchiostoma IF proteins A3 and B2 and demonstrated that both can form heteropolymeric IF based on a coiled coil dimer consisting of one B2 and one A3 polypeptide. In this study we continued in the characterisation of the B2/A3 heterodimer by searching for the sequences that play an important role in the triggering of the B2/A3 heterodimer. Using a series of deletion and chimeric B2, A3 and B1 constructs and the overlay assay as a tool, we were able to identify a part of the B2 sequence (segment 1A, linker L1 and the N-terminal part of segment 1B) which retains the ability of the full length protein B2 to specifically recognize A3 in blot overlays. Moreover, inspection of this A3-competent B2 fragment identified a short sequence in segment 1B which shares with the currently known trigger-like motif of cortexillin and other coiled coil proteins potential to form multiple inter-chain ionic interactions. Thus, a common and essential feature of trigger sequences with different primary structures found so far in IF and other coiled coil proteins seems to be their ability to form multiple inter-chain ionic interactions which brings the chains close to one another and allows coiled coil formation to propagate accordingly.

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis.

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.

In vivo and in vitro evidence that the four essential intermediate filament (IF) proteins A1, A2, A3 and B1 of the nematode Caenorhabditis elegans form an obligate heteropolymeric IF system.

The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.

The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity.

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.

Tissue-specific co-expression and in vitro heteropolymer formation of the two small branchiostoma intermediate filament proteins A3 and B2.

The two small intermediate filament (IF) proteins A3 and B2 of the cephalochordate Amphioxus were investigated. Blot overlays indicated a heterotypic interaction pattern of the recombinant proteins. While the individual proteins formed only aggregates, the stoichiometric mixture formed obligatory heteropolymeric filaments. Mutant proteins with a single cysteine residue in equivalent positions gave rise to filaments that oxidize to the disulfide-linked heterodimer, which can again form IF. Thus the A3/B2 filaments, which are expressed in the intestinal epithelium, are based on a hetero coiled coil. This keratin-like assembly process of A3 plus B2 was unexpected, since previous evolutionary tree calculations performed by two laboratories on the various Amphioxus IF proteins identified keratin I and II orthologs but left the A/B group as a separate branch. We discuss obvious evolutionary aspects of the Amphioxus IF multigene family, including the previously made observation that B1, the closest relative of B2, forms homopolymeric IF in vitro and is, like vertebrate type III proteins, expressed in mesodermally derived tissues.