Engineered nanoparticles enable deep proteomics studies at scale by leveraging tunable nano-bio interactions.

SignificanceDeep profiling of the plasma proteome at scale has been a challenge for traditional approaches. We achieve superior performance across the dimensions of precision, depth, and throughput using a panel of surface-functionalized superparamagnetic nanoparticles in comparison to conventional workflows for deep proteomics interrogation. Our automated workflow leverages competitive nanoparticle-protein binding equilibria that quantitatively compress the large dynamic range of proteomes to an accessible scale. Using machine learning, we dissect the contribution of individual physicochemical properties of nanoparticles to the composition of protein coronas. Our results suggest that nanoparticle functionalization can be tailored to protein sets. This work demonstrates the feasibility of deep, precise, unbiased plasma proteomics at a scale compatible with large-scale genomics enabling multiomic studies.

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells.

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells.

The intestinal intermediate filament network responds to and protects against microbial insults and toxins.

The enrichment of intermediate filaments in the apical cytoplasm of intestinal cells is evolutionarily conserved, forming a sheath that is anchored to apical junctions and positioned below the microvillar brush border, which suggests a protective intracellular barrier function. To test this, we used Caenorhabditiselegans, the intestinal cells of which are endowed with a particularly dense intermediate filament-rich layer that is referred to as the endotube. We found alterations in endotube structure and intermediate filament expression upon infection with nematicidal B.thuringiensis or treatment with its major pore-forming toxin crystal protein Cry5B. Endotube impairment due to defined genetic mutations of intermediate filaments and their regulators results in increased Cry5B sensitivity as evidenced by elevated larval arrest, prolonged time of larval development and reduced survival. Phenotype severity reflects the extent of endotube alterations and correlates with reduced rescue upon toxin removal. The results provide in vivo evidence for a major protective role of a properly configured intermediate filament network as an intracellular barrier in intestinal cells. This notion is further supported by increased sensitivity of endotube mutants to oxidative and osmotic stress.

Proteomic mapping by rapamycin-dependent targeting of APEX2 identifies binding partners of VAPB at the inner nuclear membrane.

Vesicle-associated membrane protein-associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and nonspecific hits. Furthermore, we identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.

Nuclear pore complex tethers to the cytoskeleton.

The nuclear envelope is tethered to the cytoskeleton. The best known attachments of all elements of the cytoskeleton are via the so-called LINC complex. However, the nuclear pore complexes, which mediate the transport of soluble and membrane bound molecules, are also linked to the microtubule network, primarily via motor proteins (dynein and kinesins) which are linked, most importantly, to the cytoplasmic filament protein of the nuclear pore complex, Nup358, by the adaptor BicD2. The evidence for such linkages and possible roles in nuclear migration, cell cycle control, nuclear transport and cell architecture are discussed.

BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability.

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations.

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson-Gilford progeria syndrome fibroblasts.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal premature ageing disease in children. HGPS is one of several progeroid syndromes caused by mutations in the LMNA gene encoding the nuclear structural proteins lamins A and C. In classic HGPS the mutation G608G leads to the formation of a toxic lamin A protein called progerin. During post-translational processing progerin remains farnesylated owing to the mutation interfering with a step whereby the farnesyl moiety is removed by the enzyme ZMPSTE24. Permanent farnesylation of progerin is thought to be responsible for the proteins toxicity. Farnesyl is generated through the mevalonate pathway and three drugs that interfere with this pathway and hence the farnesylation of proteins have been administered to HGPS children in clinical trials. These are a farnesyltransferase inhibitor (FTI), statin and a bisphosphonate. Further experimental studies have revealed that other drugs such as N-acetyl cysteine, rapamycin and IGF-1 may be of use in treating HGPS through other pathways. We have shown previously that FTIs restore chromosome positioning in interphase HGPS nuclei. Mis-localisation of chromosomes could affect the cells ability to regulate proper genome function. Using nine different drug treatments representing drug regimes in the clinic we have shown that combinatorial treatments containing FTIs are most effective in restoring specific chromosome positioning towards the nuclear periphery and in tethering telomeres to the nucleoskeleton. On the other hand, rapamycin was found to be detrimental to telomere tethering, it was, nonetheless, the most effective at inducing DNA damage repair, as revealed by COMET analyses.

Entry into the nuclear pore complex is controlled by a cytoplasmic exclusion zone containing dynamic GLFG-repeat nucleoporin domains.

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic movement. The central channel contains proteins with phenylalanine-glycine (FG) repeats, or variations (GLFG, glycine-leucine-phenylalanine-glycine). These are 'intrinsically disordered' and often represent weak interaction sites that become ordered upon interaction. We investigated this possibility during nuclear transport. Using electron microscopy of S. cerevisiae, we show that NPC cytoplasmic filaments form a dome-shaped structure enclosing GLFG domains. GLFG domains extend out of this structure and are part of an 'exclusion zone' that might act as a partial barrier to entry of transport-inert proteins. The anchor domain of a GLFG nucleoporin locates exclusively to the central channel. By contrast, the localisation of the GLFG domains varied between NPCs and could be cytoplasmic, central or nucleoplasmic and could stretch up to 80 nm. These results suggest a dynamic exchange between ordered and disordered states. In contrast to diffusion through the NPC, transport cargoes passed through the exclusion zone and accumulated near the central plane. We also show that movement of cargo through the NPC is accompanied by relocation of GLFG domains, suggesting that binding, restructuring and movement of these domains could be part of the translocation mechanism.

Yeast dynamin Vps1 and amphiphysin Rvs167 function together during endocytosis.

Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Rvs167-GFP localization is reduced in cells lacking vps1 suggesting that Vps1 influences Rvs167 association with the endocytic complex. Unlike classical dynamins, Vps1 does not have a proline-arginine domain that could interact with SH3 domain-containing proteins. Thus, while Rvs167 has an SH3 domain, it is not clear how an interaction would be mediated. Here, we demonstrate an interaction between Rvs167 SH3 domain and the single type I SH3-binding motif in Vps1. Mutant Vps1 that cannot bind Rvs167 rescues all membrane fusion/fission functions associated with Vps1 except for endocytic function, demonstrating the specificity and mechanistic importance of the interaction. In vitro, an Rvs161/Rvs167 heterodimer can disassemble Vps1 oligomers. Overall, the data support the idea that Vps1 and the amphiphysins function together to mediate scission during endocytosis in yeast.

A gradient of matrix-bound FGF-2 and perlecan is available to lens epithelial cells.

Fibroblast growth factors play a key role in regulating lens epithelial cell proliferation and differentiation via an anteroposterior gradient that exists between the aqueous and vitreous humours. FGF-2 is the most important for lens epithelial cell proliferation and differentiation. It has been proposed that the presentation of FGF-2 to the lens epithelial cells involves the lens capsule as a source of matrix-bound FGF-2. Here we used immunogold labelling to measure the matrix-bound FGF-2 gradient on the inner surface of the lens capsule in flat-mounted preparations to visualize the FGF-2 available to lens epithelial cells. We also correlated FGF-2 levels with levels of its matrix-binding partner perlecan, a heparan sulphate proteoglycan (HSPG) and found the levels of both to be highest at the lens equator. These also coincided with increased levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in lens epithelial cells that localised to condensed chromosomes of epithelial cells that were Ki-67 positive. The gradient of matrix-bound FGF-2 (anterior pole: 3.7 ± 1.3 particles/µm2; equator: 8.2 ± 1.9 particles/µm2; posterior pole: 4 ± 0.9 particles/µm2) and perlecan (anterior pole: 2.1 ± 0.4 particles/µm2; equator: 5 ± 2 particles/µm2; posterior pole: 1.9 ± 0.7 particles/µm2) available at the inner lens capsule surface was measured for the bovine lens. These data support the anteroposterior gradient hypothesis and provide the first measurement of the gradient for an important morphogen and its HSPG partner, perlecan, at the epithelial cell-lens capsule interface.

The major inducible small heat shock protein HSP20-3 in the tardigrade Ramazzottius varieornatus forms filament-like structures and is an active chaperone.

The tardigrade Ramazzottius varieornatus has remarkable resilience to a range of environmental stresses. In this study, we have characterised two members of the small heat shock protein (sHSP) family in R. varieornatus, HSP20-3 and HSP20-6. These are the most highly upregulated sHSPs in response to a 24 h heat shock at 35 0C of adult tardigrades with HSP20-3 being one of the most highly upregulated gene in the whole transcriptome. Both R. varieornatus sHSPs and the human sHSP, CRYAB (HSPB5), were produced recombinantly for comparative structure-function studies. HSP20-3 exhibited a superior chaperone activity than human CRYAB in a heat-induced protein aggregation assay. Both tardigrade sHSPs also formed larger oligomers than CRYAB as assessed by size exclusion chromatography and transmission electron microscopy of negatively stained samples. Whilst both HSP20-3 and HSP20-6 formed particles that were variable in size and larger than the particles formed by CRYAB, only HSP20-3 formed filament-like structures. The particles and filament-like structures formed by HSP20-3 appear inter-related as the filament-like structures often had particles located at their ends. Sequence analyses identified two unique features; an insertion in the middle region of the N-terminal domain (NTD) and preceding the critical-sequence identified in CRYAB, as well as a repeated QNTN-motif located in the C-terminal domain of HSP20-3. The NTD insertion is expected to affect protein-protein interactions and subunit oligomerisation. Removal of the repeated QNTN-motif abolished HSP20-3 chaperone activity and also affected the assembly of the filament-like structures. We discuss the potential contribution of HSP20-3 to protein condensate formation.

Inhibition of PDIs Downregulates Core LINC Complex Proteins, Promoting the Invasiveness of MDA-MB-231 Breast Cancer Cells in Confined Spaces In Vitro.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

Nesprin interchain associations control nuclear size.

Nesprins-1/-2/-3/-4 are nuclear envelope proteins, which connect nuclei to the cytoskeleton. The largest nesprin-1/-2 isoforms (termed giant) tether F-actin through their N-terminal actin binding domain (ABD). Nesprin-3, however, lacks an ABD and associates instead to plectin, which binds intermediate filaments. Nesprins are integrated into the outer nuclear membrane via their C-terminal KASH-domain. Here, we show that nesprin-1/-2 ABDs physically and functionally interact with nesprin-3. Thus, both ends of nesprin-1/-2 giant are integrated at the nuclear surface: via the C-terminal KASH-domain and the N-terminal ABD-nesprin-3 association. Interestingly, nesprin-2 ABD or KASH-domain overexpression leads to increased nuclear areas. Conversely, nesprin-2 mini (contains the ABD and KASH-domain but lacks the massive nesprin-2 giant rod segment) expression yields smaller nuclei. Nuclear shrinkage is further enhanced upon nesprin-3 co-expression or microfilament depolymerization. Our findings suggest that multivariate intermolecular nesprin interactions with the cytoskeleton form a lattice-like filamentous network covering the outer nuclear membrane, which determines nuclear size.

Filaments assembly of ectopically expressed Caenorhabditis elegans lamin within Xenopus oocytes.

Lamins are the major components of the nuclear lamina, a filamentous layer underlying the inner nuclear membrane and attached to the peripheral chromatin. Lamins are required for maintaining nuclear shape and are involved in most nuclear activities. Here, we studied the 3D organization of the nuclear lamina formed upon the expression of Caenorhabditis elegans lamin (Ce-lamin) within the nucleus of a Xenopus laevis oocyte. We show that Ce-lamin forms an intricate 3D meshwork of 5-6 nm lamin protofilaments. The diverse protofilament interactions and organization may shed light upon the unique mechano-elastic properties of the nuclear lamina scaffold supporting the nuclear envelope. The Q159K Hutchinson-Gilford Progeria Syndrome-linked mutation alters interactions between protofilaments within the lamina, leading to the formation of more bundled arrays of less isotropically-oriented protofilaments. Using this system, we show for the first time the organization of lamin proteins that were translated and assembled within the environment of a living cell.

Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex.

Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import.

A role for the dynamin-like protein Vps1 during endocytosis in yeast.

Dynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has raised questions over the general applicability of the current yeast model of endocytosis, and has also precluded studies using well-developed methods in yeast, to further our understanding of the mechanism of dynamin function during endocytosis. Here, we investigate the yeast dynamin-like protein Vps1 and demonstrate a transient burst of localisation to sites of endocytosis. Using live-cell imaging of endocytic reporters in strains lacking vps1, and also electron microscopy and biochemical approaches, we demonstrate a role for Vps1 in facilitating endocytic invagination. Vps1 mutants were generated, and analysis in several assays reveals a role for the C-terminal self-assembly domain in endocytosis but not in other membrane fission events with which Vps1 has previously been associated.

Scanning Electron Microscopy (SEM) and Immuno-SEM of Nuclear Pore Complexes from Amphibian Oocytes, Mammalian Cell Cultures, Yeast, and Plants.

Scanning electron microscopy (SEM) can be used to image nuclear pore complex (NPC) surface structure of from a number of organisms and model systems. With a field emission SEM , this is a medium resolution technique where details of the organization of various components can be directly imaged. Some components, such as the NPC baskets and cytoplasmic filaments, are difficult to visualize in any other way. Protein components can be identified by immunogold labeling. Any surface that can be exposed can potentially be studied by SEM . Several overlapping protocols for SEM sample preparation and immunogold labeling of NPCs are given here. Various parameters for sample preparation, fixation, immunogold labeling, drying, metal coating, and imaging are detailed which have been optimized for different types of specimens and desired endpoints.

NPC Structure in Model Organisms: Transmission Electron Microscopy and Immunogold Labeling Using High-Pressure Freezing/Freeze Substitution of Yeast, Worms, and Plants.

The nuclear pore complex (NPC) is a large elaborate structure embedded within the nuclear envelope, and intimately linked to the cytoskeleton, nucleoskeleton, and chromatin. Many different cargoes pass through its central channel and along the membrane at its periphery. The NPC is dismantled and reassembly, fully or partially, every cell cycle. In post-mitotic cells it consists of a combination of hyper-stable and highly dynamic proteins. Because of its size, dynamics, heterogeneity and integration, it is not possible to understand its structure and molecular function by any one, or even several, methods. For decades, and to this day, thin section transmission electron microscopy (TEM) has been a central tool for understanding the NPC, its associations, dynamics and role in transport as it can uniquely answer questions concerning fine structural detail within a cellular context. Using immunogold labeling specific components can also be identified within the ultrastructural context. Model organisms such as Saccharomyces cerevisiae are also central to NPC studies but have not been used extensively in structural work. This is because the cell wall presents difficulties with structural preservation and processing for TEM. In recent years, high-pressure freezing and freeze substitution have overcome these problems, as well as opened up methods to combine immunogold labeling with detailed structural analysis. Other model organisms such as the worm Caenorhabditis elegans and the plant Arabidopsis thaliana have been underused for similar reasons, but with similar solutions, which we present here. There are also many advantages to using these methods, adapted for use in mammalian systems, due to the instant nature of the initial fixation, to capture rapid processes such as nuclear transport, and preservation of dynamic membranes.

Imaging Fluorescent Nuclear Pore Complex Proteins in C. elegans.

C. elegans is a well-characterized and relatively simple model organism, making it attractive for studying nuclear pore complex proteins in cell and developmental biology. C. elegans is transparent and highly amendable to genetic manipulation. Therefore, it is possible to generate fluorescently tagged proteins and combine this with various light microscopy techniques to study protein behavior in space and time. Here, we provide protocols to prepare both fixed and live C. elegans for confocal and light sheet microscopy. This enables the analysis of nuclear pore complex proteins from embryonic stages to the aging adult.

Enhanced Competition at the Nano-Bio Interface Enables Comprehensive Characterization of Protein Corona Dynamics and Deep Coverage of Proteomes.

Introducing engineered nanoparticles (NPs) into a biofluid such as blood plasma leads to the formation of a selective and reproducible protein corona at the particle-protein interface, driven by the relationship between protein-NP affinity and protein abundance. This enables scalable systems that leverage protein-nano interactions to overcome current limitations of deep plasma proteomics in large cohorts. Here the importance of the protein to NP-surface ratio (P/NP) is demonstrated and protein corona formation dynamics are modeled, which determine the competition between proteins for binding. Tuning the P/NP ratio significantly modulates the protein corona composition, enhancing depth and precision of a fully automated NP-based deep proteomic workflow (Proteograph). By increasing the binding competition on engineered NPs, 1.2-1.7× more proteins with 1% false discovery rate are identified on the surface of each NP, and up to 3× more proteins compared to a standard plasma proteomics workflow. Moreover, the data suggest P/NP plays a significant role in determining the in vivo fate of nanomaterials in biomedical applications. Together, the study showcases the importance of P/NP as a key design element for biomaterials and nanomedicine in vivo and as a powerful tuning strategy for accurate, large-scale NP-based deep proteomic studies.