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Abstract Background Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP), amyotrophic lateral sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE) are associated with deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons. One complexity of this process lies in the ability of TDP-43 to form liquid-phase membraneless organelles in cells. Previous work has shown that the recombinant, purified, prion-like domain (PrLD) forms liquid droplets in vitro, but the behaviour of the complementary fragment is uncertain. Methods We have purified such a construct without the PrLD (PrLD-less TDP-43) and have induced its phase separation using a solution-jump method and an array of biophysical techniques to study the morphology, state of matter and structure of the TDP-43 assemblies. Results The fluorescent TMR-labelled protein construct, imaged using confocal fluorescence, formed rapidly (

Background: Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP), amyotrophic lateral sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE) are associated with deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons. One complexity of this process lies in the ability of TDP-43 to form liquid-phase membraneless organelles in cells. Previous work has shown that the recombinant, purified, prion-like domain (PrLD) forms liquid droplets in vitro, but the behaviour of the complementary fragment is uncertain. Methods: We have purified such a construct without the PrLD (PrLD-less TDP-43) and have induced its phase separation using a solution-jump method and an array of biophysical techniques to study the morphology, state of matter and structure of the TDP-43 assemblies. Results: The fluorescent TMR-labelled protein construct, imaged using confocal fluorescence, formed rapidly (< 1 min) round, homogeneous and 0.5–1.0 µm wide assemblies which then coalesced into larger, yet round, species. When labelled with AlexaFluor488, they initially exhibited fluorescence recovery after photobleaching (FRAP), showing a liquid behaviour distinct from full-length TDP-43 and similar to PrLD. The protein molecules did not undergo major structural changes, as determined with circular dichroism and intrinsic fluorescence spectroscopies. This process had a pH and salt dependence distinct from those of full-length TDP-43 and its PrLD, which can be rationalized on the grounds of electrostatic forces. Conclusions: Similarly to PrLD, PrLD-less TDP-43 forms liquid droplets in vitro through liquid–liquid phase separation (LLPS), unlike the full-length protein that rather undergoes liquid–solid phase separation (LSPS). These results offer a rationale of the complex electrostatic forces governing phase separation of full-length TDP-43 and its fragments. On the one hand, PrLD-less TDP-43 has a low pI and oppositively charged domains, and LLPS is inhibited by salts, which attenuate inter-domain electrostatic attractions. On the other hand, PrLD is positively charged due to a high isoionic point (pI) and LLPS is therefore promoted by salts and pH increases as they both reduce electrostatic repulsions. By contrast, full-length TDP-43 undergoes LSPS most favourably at its pI, with positive and negative salt dependences at lower and higher pH, respectively, depending on whether repulsive or attractive forces dominate, respectively. [ABSTRACT FROM AUTHOR]

The Balbiani body (Bb) is the first marker of polarity in vertebrate oocytes. The Bb is a conserved structure found in diverse animals including insects, fish, amphibians, and mammals. During early zebrafish oogenesis, the Bb assembles as a transient aggregate of mRNA, proteins, and membrane-bound organelles at the presumptive vegetal side of the oocyte. As the early oocyte develops, the Bb appears to grow slowly, until at the end of stage I of oogenesis it disassembles and deposits its cargo of localized mRNAs and proteins. In fish and frogs, this cargo includes the germ plasm as well as gene products required to specify dorsal tissues of the future embryo. We demonstrate that the Bb is a stable, solid structure that forms a size exclusion barrier similar to other biological hydrogels. Despite its central role in oocyte polarity, little is known about the mechanism behind the Bb's action. Analysis of the few known protein components of the Bb is insufficient to explain how the Bb assembles, translocates, and disassembles. We isolated Bbs from zebrafish oocytes and performed mass spectrometry to define the Bb proteome. We successfully identified 77 proteins associated with the Bb sample, including known Bb proteins and novel RNA-binding proteins. In particular, we identified Cirbpa and Cirbpb, which have both an RNA-binding domain and a predicted self-aggregation domain. In stage I oocytes, Cirbpa and Cirbpb localize to the Bb rather than the nucleus (as in somatic cells), indicating that they may have a specialized function in the germ line. Both the RNA-binding domain and the self-aggregation domain are sufficient to localize to the Bb, suggesting that Cirbpa and Cirbpb interact with more than just their mRNA targets within the Bb. We propose that Cirbp proteins crosslink mRNA cargo and proteinaceous components of the Bb as it grows. Beyond Cirbpa and Cirbpb, our proteomics dataset presents many candidates for further study, making it a valuable resource for building a comprehensive mechanism for Bb function at a protein level. [Display omitted] • The solid membraneless oocyte Balbiani body aggregate as a size exclusion barrier. • Balbiani body isolation, collection, and proteomics reveals 77+ proteins. • Cold Inducible RNA Binding Protein validated as Balbiani body resident protein. [ABSTRACT FROM AUTHOR]

Wall slip or, more usually, liquid-solid phase separation at the boundary wall when measuring the rheological properties of particulate suspensions is normally considered an undesirable source of error. However, exclusion of a structure consisting of multiple particulates at a planar boundary can, in turn, reveal the nature of that structure and the way it interacts with other elements in the dispersion. Using a system of surface-treated ground calcite particles, designed to control lyophilicity, dispersed, respectively, in two comparative liquids, hexadecane (dispersive surface tension component only) and linseed oil (both dispersive and polar surface tension components), the relative wettability of the particulate surface can be studied. The static state is viscoelastic, with the elastic component reflecting the network of interacting forces acting to structure the particles together and/or to trap liquid within the long-range particle-particle matrix. As strain is applied under plate-plate geometry, selected aggregate structures become size-excluded at the wall, leading to a loss of shear coupling with the bulk polydisperse suspension. At high strain, given optimal solids content, this results in a stochastic transition between two discrete stress data sets, i.e. that with full shear coupling and that with only partial coupling. Stress recovery is subsequently monitored as strain is step-wise reduced, and the progress toward loss of the stochastic transient phenomenon, together with its parallel change in magnitude, is used to describe the re-formation of primary agglomerates. Cessation of the phase separation indicates re-build of the close-to-static structure. Under certain conditions it is observed that the cessation may be accompanied by a secondary relaxation of state, indicating the build of a secondary but weaker structure, likened to the well-known dual-level flocculation in aqueous colloidal suspension. Rheo-optical observations using small angle light scattering illumination (SALS) are used to confirm a structure model switching from static (uncoupled with shear) to rotating (fully coupled to the boundary-defined shear) and finally uniformly sheared.

Living cells comprise diverse subcellular structures, such as cytoskeletal networks, which can regulate essential cellular activities through dynamic assembly and synergistic interactions with biomolecular condensates. Despite extensive efforts, reproducing viscoelastic networks for modulating biomolecular condensates in synthetic systems remains challenging. Here, a new aqueous two-phase system (ATPS) is proposed, which consists of poly(N-isopropylacrylamide) (PNIPAM) and dextran (DEX), to construct viscoelastic networks capable of being assembled and dissociated dynamically to regulate the self-assembly of condensates on-demand. Viscoelastic networks are generated using liquid-liquid phase-separated DEX droplets as templates and the following liquid-to-solid transition of the PNIPAM-rich phase. The resulting networks can dissolve liquid fused in sarcoma (FUS) condensates within 5 min. This work demonstrates rich phase-separation behaviors in a single ATPS through incorporating stimuli-responsive polymers. The concept can potentially be applied to other macromolecules through other stimuli to develop materials with rich phase behaviors and hierarchical structures., (© 2022 Wiley-VCH GmbH.)

• Tag modulates liquid-solid phase separation and AEX chromatography of rHPV 16 L1 vaccine. • Tag improves overall yield and does not abrogate VLP formation under optimized conditions. • Three step purification process for WT and tagged protein results in > 98% purity. The separation of heterogeneous protein mixtures has always been characterized by a trade-off between purity and yield. One way this issue has been addressed in the past is by recombinantly modifying protein to improve separations. Such modifications are mostly employed in the form of tags used specifically for affinity chromatography, though it is also possible to make changes to a protein that will have a sizeable impact on its hydrophobicity and charge/charge distribution. As such, it should also be possible to use protein tags to modulate phase separations and protein-resin binding kinetics when performing ion exchange chromatography. Here, we employed a three-step purification scheme on E. coli expressed, His-tagged, human papilloma virus 16 L1-based recombinant proteins (rHPV 16 L1) that consisted of an inclusion body (IB) wash step, a diethylaminoethyl (DEAE) anion exchange chromatography (AEX) step, and an immobilized metal affinity chromatography (IMAC) polishing step. Purification of the wild type rHPV 16 L1 protein (WT) was characterized by substantial losses during the IB wash but relatively high yield over the DEAE column. In contrast, purification of modified rHPV 16 L1, a chimeric version of the WT protein that had the last 34 amino acids replaced with an MHC class II multi-epitope insert derived from tetanus toxin and diphtheria toxin (WTΔC34-2TEp), was characterized by little to no losses in the IB wash but had a relatively low yield over the DEAE column. Since the fate of these proteins was to be used in vaccine formulations, it is important to note that the modifications made to the WTΔC34-2TEp protein had little to no effect on its ability to assemble into virus-like particles (VLPs). These results demonstrate that modifications of the WT protein via the recombinant insertion of immunofunctional polypeptides can modulate both phase-based separation and charge-based chromatographic processes. Additionally, incorporation of the specific, multi-epitope tag used in this study may prove to be beneficial in recombinant HPV vaccine development due to its potential to improve phase separation yield and vaccine immunogenicity without inhibiting VLP formation. [ABSTRACT FROM AUTHOR]