Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates.

The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organisation and dynamics of chromatin compacted by gene-repressing factors are unknown. Using cryo-electron tomography, we solved the threedimensional structure of chromatin condensed by the Polycomb Repressive Complex 1 (PRC1) in a complex with CBX8. PRC1-condensed chromatin is porous and stabilised through multivalent dynamic interactions of PRC1 with chromatin. Mechanistically, positively charged residues on the internally disordered regions (IDRs) of CBX8 mask negative charges on the DNA to stabilize the condensed state of chromatin. Within condensates, PRC1 remains dynamic while maintaining a static chromatin structure. In differentiated mouse embryonic stem cells, CBX8-bound chromatin remains accessible. These findings challenge the idea of rigidly compacted polycomb domains and instead provides a mechanistic framework for dynamic and accessible PRC1-chromatin condensates.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.

Correlative AFM and fluorescence imaging demonstrate nanoscale membrane remodeling and ring-like and tubular structure formation by septins.

Septins are ubiquitous cytoskeletal filaments that interact with the inner plasma membrane and are essential for cell division in eukaryotes. In cellular contexts, septins are often localized at micrometric Gaussian curvatures, where they assemble onto ring-like structures. The behavior of budding yeast septins depends on their specific interaction with inositol phospholipids, enriched at the inner leaflet of the plasma membrane. Septin filaments are built from the non-polar self-assembly of short rods into filaments. However, the molecular mechanisms regulating the interplay with the inner plasma membrane and the resulting interaction with specific curvatures are not fully understood. In this report, we have imaged dynamical molecular assemblies of budding yeast septins on PIP2-containing supported lipid bilayers using a combination of high-speed AFM and correlative AFM-fluorescence microscopy. Our results clearly demonstrate that septins are able to bind to flat supported lipid bilayers and thereafter induce the remodeling of membranes. Short septin rods (octamers subunits) can indeed destabilize supported lipid bilayers and reshape the membrane to form 3D structures such as rings and tubes, demonstrating that long filaments are not necessary for septin-induced membrane buckling.

Synergistic role of nucleotides and lipids for the self-assembly of Shs1 septin oligomers.

Budding yeast septins are essential for cell division and polarity. Septins assemble as palindromic linear octameric complexes. The function and ultra-structural organization of septins are finely governed by their molecular polymorphism. In particular, in budding yeast, the end subunit can stand either as Shs1 or Cdc11. We have dissected, here, for the first time, the behavior of the Shs1 protomer bound to membranes at nanometer resolution, in complex with the other septins. Using electron microscopy, we have shown that on membranes, Shs1 protomers self-assemble into rings, bundles, filaments or two-dimensional gauzes. Using a set of specific mutants we have demonstrated a synergistic role of both nucleotides and lipids for the organization and oligomerization of budding yeast septins. Besides, cryo-electron tomography assays show that vesicles are deformed by the interaction between Shs1 oligomers and lipids. The Shs1-Shs1 interface is stabilized by the presence of phosphoinositides, allowing the visualization of micrometric long filaments formed by Shs1 protomers. In addition, molecular modeling experiments have revealed a potential molecular mechanism regarding the selectivity of septin subunits for phosphoinositide lipids.

Automated cryo-lamella preparation for high-throughput in-situ structural biology.

Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity.

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology.

Membrane reshaping by micrometric curvature sensitive septin filaments.

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.

Derivation of original RESP atomic partial charges for MD simulations of the LDAO surfactant with AMBER: applications to a model of micelle and a fragment of the lipid kinase PI4KA.

In this paper, we describe the derivation and the validation of original RESP atomic partial charges for the N, N-dimethyl-dodecylamine oxide (LDAO) surfactant. These charges, designed to be fully compatible with all the AMBER force fields, are at first tested against molecular dynamics simulations of pure LDAO micelles and with a fragment of the lipid kinase PIK4A (DI) modeled with the QUARK molecular modeling server. To model the micelle, we used two distinct AMBER force fields (i.e. Amber99SB and Lipid14) and a variety of starting conditions. We find that the micelle structural properties (such as the shape, size, the LDAO headgroup hydration, and alkyl chain conformation) slightly depend on the force field but not on the starting conditions and more importantly are in good agreement with experiments and previous simulations. We also show that the Lipid14 force field should be used instead of the Amber99SB one to better reproduce the C(sp3)C(sp3)C(sp3)C(sp3) conformation in the surfactant alkyl chain. Concerning the simulations with LDAO-DI protein, we carried out different runs at two NaCl concentrations (i.e. 0 and 300 mM) to mimic, in the latter case, the experimental conditions. We notice a small dependence of the simulation results with the LDAO parameters and the salt concentration. However, we find that in the simulations, three out of four tryptophans of the DI protein are not accessible to water in agreement with our fluorescence spectroscopy experiments reported in the paper.

Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV.

The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway.

Definition and expression in E. coli of large fragments from the human lipid kinase phosphatidylinositol 4-kinase type III alpha, and purification of a 1100-residue N-terminal module.

The eukaryotic lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KA in higher eukaryotes) is a ubiquitous enzyme that synthesizes the plasma membrane pool of phosphatidylinositol 4-phosphate. This important phosphoinositide has key roles in different signalization pathways, vesicular traffic and cellular compartment identity. Moreover, human PI4K4A is an essential factor for hepatitis C virus replication. PI4KA is a large protein (2102 residues for human PI4KA) with the kinase domain making up the ca 400 C-terminal residues. There is essentially no structural information about the 1500N-terminal residues and no clue as to the function of most of this region of PI4KA. In this report, we use computational methods in order to delineate fragments of human PI4KA amenable to soluble production in Escherichia coli. We clone and express these fragments as GST-fusions and evaluate the soluble fraction of each protein. Finally, we produce and purify to homogeneity a 1100-residue PI4KA N-terminal fragment. Our results further suggest that PI4KA can be described as a two-module protein. They open the way to structural characterization of the N-terminal regulatory module of PI4KA.

Mapping of functional domains of the lipid kinase phosphatidylinositol 4-kinase type III alpha involved in enzymatic activity and hepatitis C virus replication.

UNLABELLED: The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE: The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIIIα mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication.