Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3.

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

A Tripartite Complex HIV-1 Tat-Cyclophilin A-Capsid Protein Enables Tat Encapsidation That Is Required for HIV-1 Infectivity.

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

Structure and mechanics of the human nuclear pore complex basket using correlative AFM-fluorescence superresolution microscopy.

Nuclear pore complexes (NPCs) are the only gateways between the nucleus and cytoplasm in eukaryotic cells. They restrict free diffusion to molecules below 5 nm while facilitating the active transport of selected cargoes, sometimes as large as the pore itself. This versatility implies an important pore plasticity. Recently, cryo-EM and AI-based protein modeling of human NPC revealed with acute precision how most constituents are arranged. But the basket, a fish trap-like structure capping the nucleoplasmic side of the pore, remains poorly resolved. Here by atomic force microscopy (AFM) coupled to single molecule localization microscopy (SMLM) we revealed that the basket is very soft and explores a large conformational landscape: apart from its canonical basket shape, it dives into the central pore channel or opens, with filaments reaching to the pore sides. Our observations highlight how this structure can adapt and let morphologically diverse cargoes shuttle through NPCs.

Mechanical signatures of human colon cancers.

Besides the standard parameters used for colorectal cancer (CRC) management, new features are needed in clinical practice to improve progression-free and overall survival. In some cancers, the microenvironment mechanical properties can contribute to cancer progression and metastasis formation, or constitute a physical barrier for drug penetration or immune cell infiltration. These mechanical properties remain poorly known for colon tissues. Using a multidisciplinary approach including clinical data, physics and geostatistics, we characterized the stiffness of healthy and malignant colon specimens. For this purpose, we analyzed a prospective cohort of 18 patients with untreated colon adenocarcinoma using atomic force microscopy to generate micrometer-scale mechanical maps. We characterized the stiffness of normal epithelium samples taken far away or close to the tumor area and selected tumor tissue areas. These data showed that normal epithelium was softer than tumors. In tumors, stroma areas were stiffer than malignant epithelial cell areas. Among the clinical parameters, tumor left location, higher stage, and RAS mutations were associated with increased tissue stiffness. Thus, in patients with CRC, measuring tumor tissue rigidity may have a translational value and an impact on patient care.

A Novel Approach to the Facile Growth and Organization of Photothermal Prussian Blue Nanocrystals on Different Surfaces.

We report here a novel "one-pot" approach for the controlled growth and organization of Prussian blue nanostructures on three different surfaces: pure Au0, cysteamine-functionalized Au0, and SiO2-supported lipid bilayers with different natures of lipids. We demonstrate that fine control over the size, morphology, and the degree and homogeneity of the surface coverage by Prussian Blue (PB) nanostructures may be achieved by manipulating different parameters, which are the precursor concentration, the nature of the functional groups or the nature of lipids on the surfaces. This allows the growth of isolated PB nanopyramids and nanocubes or the design of thin dense films over centimeter square surfaces. The formation of unusual Prussian blue nanopyramids is discussed. Finally, we demonstrate, by using experimental techniques and theoretical modeling, that PB nanoparticles deposited on the gold surface exhibit strong photothermal properties, permitting a rapid temperature increase up to 90 °C with a conversion of the laser power of almost 50% for power source heat.

CD82 and Gangliosides Tune CD81 Membrane Behavior.

Tetraspanins are a family of transmembrane proteins that form a network of protein-protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

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.

Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization.

Many transmembrane receptors have a desensitized state, in which they are unable to respond to external stimuli. The family of microbial rhodopsin proteins includes one such group of receptors, whose inactive or dark-adapted (DA) state is established in the prolonged absence of light. Here, we present high-resolution crystal structures of the ground (light-adapted) and DA states of Archaerhodopsin-3 (AR3), solved to 1.1 Å and 1.3 Å resolution respectively. We observe significant differences between the two states in the dynamics of water molecules that are coupled via H-bonds to the retinal Schiff Base. Supporting QM/MM calculations reveal how the DA state permits a thermodynamic equilibrium between retinal isomers to be established, and how this same change is prevented in the ground state in the absence of light. We suggest that the different arrangement of internal water networks in AR3 is responsible for the faster photocycle kinetics compared to homologs.

TAT-RasGAP317-326 kills cells by targeting inner-leaflet-enriched phospholipids.

TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.

Author Correction: Synchronous, Crosstalk-free Correlative AFM and Confocal Microscopies/Spectroscopies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Compression, Rupture, and Puncture of Model Membranes at the Molecular Scale.

Elastic properties of biological membranes are involved in a large number of membrane functionalities and activities. Conventionally characterized in terms of Young's modulus, bending stiffness and stretching modulus, membrane mechanics can be assessed at high lateral resolution by means of atomic force microscopy (AFM). Here we show that the mechanical response of biomimetic model systems such as supported lipid bilayers (SLBs) is highly affected by the size of the AFM tip employed as a membrane indenter. Our study is focused on phase-separated fluid-gel lipid membranes at room temperature. In a small tip radius regime (≈ 2 nm) and in the case of fluid phase membranes, we show that the tip can penetrate through the membrane minimizing molecular vertical compression and in absence of molecular membrane rupture. In this case, AFM indentation experiments cannot assess the vertical membrane Young's modulus. In agreement with the data reported in the literature, in the case of larger indenters (>2 nm) SLBs can be compressed leading to an evaluation of Young's modulus and membrane maximal withstanding force before rupture. We show that such force increases with the indenter in agreement with the existing theoretical frame. Finally, we demonstrate that the latter has no influence on the number of molecules involved in the rupture process that is observed to be constant and rather dependent on the indenter chemical composition.

Synchronous, Crosstalk-free Correlative AFM and Confocal Microscopies/Spectroscopies.

Microscopies have become pillars of our characterization tools to observe biological systems and assemblies. Correlative and synchronous use of different microscopies relies on the fundamental assumption of non-interference during images acquisitions. In this work, by exploring the correlative use of Atomic Force Microscopy and confocal-Fluorescence-Lifetime Imaging Microscopy (AFM-FLIM), we quantify cross-talk effects occurring during synchronous acquisition. We characterize and minimize optomechanical forces on different AFM cantilevers interfering with normal AFM operation as well as spurious luminescence from the tip and cantilever affecting time-resolved fluorescence detection. By defining non-interfering experimental imaging parameters, we show accurate real-time acquisition and two-dimensional mapping of interaction force, fluorescence lifetime and intensity characterizing morphology (AFM) and local viscosity (FLIM) of gel and fluid phases separation of supported lipid model membranes. Finally, as proof of principle by means of synchronous force and fluorescence spectroscopies, we precisely tune the lifetime of a fluorescent nanodiamond positioned on the AFM tip by controlling its distance from a metallic surface. This opens up a novel pathway of quench sensing to image soft biological samples such as membranes since it does not require tip-sample mechanical contact in contrast with conventional AFM in liquid.

Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM.

Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell. Specifically assembly and release of HIV-1 particles require specific cellular components, which are recruited to the egress site by the viral protein Gag. We previously demonstrated that HIV-1 assembly alters both partitioning and dynamics of the tetraspanins CD9 and CD81, which are key players in many infectious processes, forming enriched areas where the virus buds. In this study we correlated super resolution microscopy mapping of tetraspanins with membrane topography delineated by atomic force microscopy (AFM) in Gag-expressing cells. We revealed that CD9 is specifically trapped within the nascent viral particles, especially at buds tips, suggesting that Gag mediates CD9 and CD81 depletion from the plasma membrane. In addition, we showed that CD9 is organized as small membrane assemblies of few tens of nanometers that can coalesce upon Gag expression.

Imaging Artificial Membranes Using High-Speed Atomic Force Microscopy.

Supported lipid bilayers represent a very attractive way to mimic biological membranes, especially to investigate molecular mechanisms associated with the lateral segregation of membrane components. Observation of these model membranes with high-speed atomic force microscopy (HS-AFM) allows the capture of both topography and dynamics of membrane components, with a spatial resolution in the nanometer range and image capture time of less than 1 s. In this context, we have developed new protocols adapted for HS-AFM to form supported lipid bilayers on small mica disks using the vesicle fusion or Langmuir-Blodgett methods. In this chapter we describe in detail the protocols to fabricate supported artificial bilayers as well as the main guidelines for HS-AFM imaging of such samples.

Sequence-dependent catalytic regulation of the SpoIIIE motor activity ensures directionality of DNA translocation.

Transport of cellular cargo by molecular motors requires directionality to ensure proper biological functioning. During sporulation in Bacillus subtilis, directionality of chromosome transport is mediated by the interaction between the membrane-bound DNA translocase SpoIIIE and specific octameric sequences (SRS). Whether SRS regulate directionality by recruiting and orienting SpoIIIE or by simply catalyzing its translocation activity is still unclear. By using atomic force microscopy and single-round fast kinetics translocation assays we determined the localization and dynamics of diffusing and translocating SpoIIIE complexes on DNA with or without SRS. Our findings combined with mathematical modelling revealed that SpoIIIE directionality is not regulated by protein recruitment to SRS but rather by a fine-tuned balance among the rates governing SpoIIIE-DNA interactions and the probability of starting translocation modulated by SRS. Additionally, we found that SpoIIIE can start translocation from non-specific DNA, providing an alternative active search mechanism for SRS located beyond the exploratory length defined by 1D diffusion. These findings are relevant in vivo in the context of chromosome transport through an open channel, where SpoIIIE can rapidly explore DNA while directionality is modulated by the probability of translocation initiation upon interaction with SRS versus non-specific DNA.

In-plane molecular organization of hydrated single lipid bilayers: DPPC:cholesterol.

Understanding the physical properties of cholesterol-phospholipid systems is essential to gain a better knowledge of the function of each membrane constituent. We present a novel, simple and user-friendly setup that allows for the straightforward grazing incidence X-ray diffraction characterization of hydrated individual supported lipid bilayers. This configuration minimizes the scattering from the liquid and allows the detection of the extremely weak diffracted signal of the membrane, enabling the differentiation of the coexisting domains in DPPC:cholesterol single bilayers.

Tetraspanin Tspan9 regulates platelet collagen receptor GPVI lateral diffusion and activation.

The tetraspanins are a superfamily of four-transmembrane proteins, which regulate the trafficking, lateral diffusion and clustering of the transmembrane proteins with which they interact. We have previously shown that tetraspanin Tspan9 is expressed on platelets. Here we have characterised gene-trap mice lacking Tspan9. The mice were viable with normal platelet numbers and size. Tspan9-deficient platelets were specifically defective in aggregation and secretion induced by the platelet collagen receptor GPVI, despite normal surface GPVI expression levels. A GPVI activation defect was suggested by partially impaired GPVI-induced protein tyrosine phosphorylation. In mechanistic experiments, Tspan9 and GPVI co-immunoprecipitated and co-localised, but super-resolution imaging revealed no defects in collagen-induced GPVI clustering on Tspan9-deficient platelets. However, single particle tracking using total internal reflection fluorescence microscopy showed that GPVI lateral diffusion was reduced by approximately 50% in the absence of Tspan9. Therefore, Tspan9 plays a fine-tuning role in platelet activation by regulating GPVI membrane dynamics.

Automatic detection of diffusion modes within biological membranes using back-propagation neural network.

BACKGROUND: Single particle tracking (SPT) is nowadays one of the most popular technique to probe spatio-temporal dynamics of proteins diffusing within the plasma membrane. Indeed membrane components of eukaryotic cells are very dynamic molecules and can diffuse according to different motion modes. Trajectories are often reconstructed frame-by-frame and dynamic properties often evaluated using mean square displacement (MSD) analysis. However, to get statistically significant results in tracking experiments, analysis of a large number of trajectories is required and new methods facilitating this analysis are still needed. RESULTS: In this study we developed a new algorithm based on back-propagation neural network (BPNN) and MSD analysis using a sliding window. The neural network was trained and cross validated with short synthetic trajectories. For simulated and experimental data, the algorithm was shown to accurately discriminate between Brownian, confined and directed diffusion modes within one trajectory, the 3 main of diffusion encountered for proteins diffusing within biological membranes. It does not require a minimum number of observed particle displacements within the trajectory to infer the presence of multiple motion states. The size of the sliding window was small enough to measure local behavior and to detect switches between different diffusion modes for segments as short as 20 frames. It also provides quantitative information from each segment of these trajectories. Besides its ability to detect switches between 3 modes of diffusion, this algorithm is able to analyze simultaneously hundreds of trajectories with a short computational time. CONCLUSION: This new algorithm, implemented in powerful and handy software, provides a new conceptual and versatile tool, to accurately analyze the dynamic behavior of membrane components.