Centre for Microscopy and Microanalysis

The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10-15% of the cell surface at 37 degrees C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 microm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.

There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration.

Lipid droplets (LDs) are independent organelles that are composed of a lipid ester core and a surface phospholipid monolayer. Recent studies have revealed many new proteins, functions, and phenomena associated with LDs. In addition, a number of diseases related to LDs are beginning to be understood at the molecular level. It is now clear that LDs are not an inert store of excess lipids but are dynamically engaged in various cellular functions, some of which are not directly related to lipid metabolism. Compared to conventional membrane organelles, there are still many uncertainties concerning the molecular architecture of LDs and how each function is placed in a structural context. Recent findings and remaining questions are discussed.

Ras proteins must be localized to the inner surface of the plasma membrane to be biologically active. The motifs that effect Ras plasma membrane targeting consist of a C-terminal CAAX motif plus a second signal comprising palmitoylation of adjacent cysteine residues or the presence of a polybasic domain. In this study, we examined how Ras proteins access the cell surface after processing of the CAAX motif is completed in the endoplasmic reticulum (ER). We show that palmitoylated CAAX proteins, in addition to being localized at the plasma membrane, are found throughout the exocytic pathway and accumulate in the Golgi region when cells are incubated at 15 degrees C. In contrast, polybasic CAAX proteins are found only at the cell surface and not in the exocytic pathway. CAAX proteins which lack a second signal for plasma membrane targeting accumulate in the ER and Golgi. Brefeldin A (BFA) significantly inhibits the plasma membrane accumulation of newly synthesized, palmitoylated CAAX proteins without inhibiting their palmitoylation. BFA has no effect on the trafficking of polybasic CAAX proteins. We conclude that H-ras and K-ras traffic to the cell surface through different routes and that the polybasic domain is a sorting signal diverting K-Ras out of the classical exocytic pathway proximal to the Golgi. Farnesylated Ras proteins that lack a polybasic domain reach the Golgi but require palmitoylation in order to traffic further to the cell surface. These data also indicate that a Ras palmitoyltransferase is present in an early compartment of the exocytic pathway.

We have examined the expression of caveolin in MDCK cells under conditions that vary cellular cholesterol concentration. Caveolin mRNA levels dropped to one-sixth of control levels after treatment with simvastatin, an inhibitor of cholesterol synthesis, or beta-trimethyl cyclodextrin (CD), a cholesterol sequestering drug. Both simvastatin and CD treatment decreased total cellular cholesterol levels to about 50% of control values. The potent activator of the sterol regulatory element, 25-hydroxycholesterol, showed no direct regulation of caveolin mRNA levels. Caveolin protein concentration was also decreased to 50% of control values in cholesterol-depleted cells, giving rise to a severe attenuation of caveolin expression detected by indirect immunofluorescence labeling. Quantitative electron microscopy showed a total loss of morphologically recognizable invaginated caveolae after these cholesterol depletion treatments. When the number of invaginated caveolae per cell was expressed as a function of the cellular cholesterol content, a threshold phenomenon was observed, suggesting that caveolae only form when the steady state cellular cholesterol is above 50% of control values. These findings indicate that caveolins, and caveolae, may play an important part in cellular cholesterol homeostasis.

We have used transient terahertz photoconductivity measurements to assess the efficacy of two-temperature growth and core-shell encapsulation techniques on the electronic properties of GaAs nanowires. We demonstrate that two-temperature growth of the GaAs core leads to an almost doubling in charge-carrier mobility and a tripling of carrier lifetime. In addition, overcoating the GaAs core with a larger-bandgap material is shown to reduce the density of surface traps by 82%, thereby enhancing the charge conductivity.

Modern wide field-of-view atom probes permit observation of a wide range of crystallographic features that can be used to calibrate the tomographic reconstruction of the analyzed volume. In this study, methodologies to determine values of the geometric parameters involved in the tomographic reconstruction of atom probe data sets are presented and discussed. The influence of the tip to electrode distance and specimen temperature on these parameters is explored. Significantly, their influence is demonstrated to be very limited, indicating a relatively wide regime of experimental parameters space for sound atom probe tomography (APT) experiments. These methods have been used on several specimens and material types, and the results indicate that the reconstruction parameters are specific to each specimen. Finally, it is shown how an accurate calibration of the reconstruction enables improvements to the quality and reliability of the microscopy and microanalysis capabilities of the atom probe.

A highly defective CeO₂ supported RuO₂ as an electrocatalyst shows improved OER activity and stability in alkaline media. This improvement likely originates from the highly oxidative oxygen species O₂²⁻/O⁻ formed in the defective CeO₂, which can easily migrate from CeO₂ and “spillover” to the surface of RuO₂ during the OER process.

Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.

Caveolae are an abundant feature of the plasma membrane in many cells. Until recently, they were generally considered to be membrane invaginations whose formation primarily driven by integral membrane proteins called caveolins. However, the past decade has seen the emergence of the cavin family of peripheral membrane proteins as essential coat components and regulators of caveola biogenesis. In this Commentary, we summarise recent data on the role of cavins in caveola formation, highlighting structural studies that provide new insights into cavin coat assembly. In mammals, there are four cavin family members that associate through homo- and hetero-oligomerisation to form distinct subcomplexes on caveolae, which can be released into the cell in response to stimuli. Studies from several labs have provided a better understanding of cavin stoichiometry and the molecular basis for their oligomerisation, as well as identifying interactions with membrane phospholipids that may be important for caveola function. We propose a model in which coincident, low-affinity electrostatically controlled protein-protein and protein-lipid interactions allow the formation of caveolae, generating a meta-stable structure that can respond to plasma membrane stress by release of cavins.

This review comprehensively summarizes the recent progress of fiber-based thermoelectric materials and devices for solid, portable, and wearable electronics.

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.

Abstract A model is presented to explain why tubular halloysite rolls in preference to tetrahedral rotation to correct misfit of the octahedral and tetrahedral sheets. It is shown that the rolling mechanism operates as it encounters significantly less resistance from Si-Si repulsion in comparison to tetrahedral rotation to correct the same amount of misfit. The model explains the observed and experimental rolling of planar kaolinites to form tubular halloysite upon hydration and exfoliation.

The Transmembrane 4 Superfamily member, PETA-3/CD151, is ubiquitously expressed by endothelial cells in vivo. In cultured human umbilical vein endothelial cells PETA-3 is present on the plasma membrane and predominantly localises to regions of cell-cell contact. Additionally, this protein is abundant within an intracellular compartment which accounts for up to 66% of the total PETA-3 expressed. Intracellular PETA-3 showed colocalisation with transferrin receptor and CD63 suggesting an endosomal/lysosomal localisation which was supported by immuno-electronmicroscopy studies. Co-immunoprecipitation experiments investigating possible interactions of PETA-3 with other molecules demonstrated associations with several integrin chains including beta1, beta3, beta4, (alpha)2, (alpha)3, (alpha)5, (alpha)6 and provide the first report of Transmembrane 4 Superfamily association with the (alpha)6beta4 integrin. Using 2-colour confocal microscopy, we demonstrated similar localisation of PETA-3 and integrin chains within cytoplasmic vesicles and endothelial cell junctions. In order to assess the functional implications of PETA-3/integrin associations, the effect of anti-PETA-3 antibodies on endothelial function was examined. Anti-PETA-3 mAb inhibited endothelial cell migration and modulated in vitro angiogenesis, but had no detectable effect on neutrophil transendothelial migration. The broad range of integrin associations and the presence of PETA-3 with integrins both on the plasma membrane and within intracellular vesicles, suggests a primary role for PETA-3 in regulating integrin trafficking and/or function.

Several studies have suggested crosstalk between different clathrin-independent endocytic pathways. However, the molecular mechanisms and functional relevance of these interactions are unclear. Caveolins and cavins are crucial components of caveolae, specialized microdomains that also constitute an endocytic route. Here we show that specific caveolar proteins are independently acting negative regulators of clathrin-independent endocytosis. Cavin-1 and Cavin-3, but not Cavin-2 or Cavin-4, are potent inhibitors of the clathrin-independent carriers/GPI-AP enriched early endosomal compartment (CLIC/GEEC) endocytic pathway, in a process independent of caveola formation. Caveolin-1 (CAV1) and CAV3 also inhibit the CLIC/GEEC pathway upon over-expression. Expression of caveolar protein leads to reduction in formation of early CLIC/GEEC carriers, as detected by quantitative electron microscopy analysis. Furthermore, the CLIC/GEEC pathway is upregulated in cells lacking CAV1/Cavin-1 or with reduced expression of Cavin-1 and Cavin-3. Inhibition by caveolins can be mimicked by the isolated caveolin scaffolding domain and is associated with perturbed diffusion of lipid microdomain components, as revealed by fluorescence recovery after photobleaching (FRAP) studies. In the absence of cavins (and caveolae) CAV1 is itself endocytosed preferentially through the CLIC/GEEC pathway, but the pathway loses polarization and sorting attributes with consequences for membrane dynamics and endocytic polarization in migrating cells and adult muscle tissue. We also found that noncaveolar Cavin-1 can act as a modulator for the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. This work provides new insights into the regulation of noncaveolar clathrin-independent endocytosis by specific caveolar proteins, illustrating multiple levels of crosstalk between these pathways. We show for the first time a role for specific cavins in regulating the CLIC/GEEC pathway, provide a new tool to study this pathway, identify caveola-independent functions of the cavins and propose a novel mechanism for inhibition of the CLIC/GEEC pathway by caveolin.

Cd-Free CZTS solar cell with above 10% efficiency was achieved by an Al₂O₃passivation layer prepared by ALD.

doped state, and the intensive crystal imperfections such as dislocations, lattice distortions, and strains, play key roles in keeping low thermal conductivity. This study fills in the gaps of the existing knowledge concerning the doping mechanisms of Cu in SnSe systems, and provides a new strategy to achieve high thermoelectric performance in SnSe-based thermoelectric materials.

The integration of different metal-organic frameworks (MOFs) into one system has led to the recent combinatorial innovation of various MOF-on-MOF hybrids; however control over their site-specific growth beyond MOF analogues remains challenging. In this work, a site-specific epitaxial-growth strategy is developed to synthesize MOF-on-MOF heterostructures comprised of two MOFs with totally different compositions. A guest MOF (ZIF-8) is epitaxially grown on the specific {110} facets of a host MOF (MIL-125). Moreover, the position of ZIF-8 growth on MIL-125 is also selectable by using MIL-125 hosts with {110} facets exposed on either the corner or side surface. Consequently, two MIL-125@ZIF-8 heterostructures with elaborately designed different architectures are synthesized. Benefiting from the high adsorption capacity of ZIF-8 and the photocatalytic activity of MIL-125, the MIL-125@ZIF-8 heterostructures demonstrate synergistically enhanced photocatalytic performance compared to single MOF subunits. Moreover, the corner growth leads to higher activity than the side growth of the MIL-125@ZIF-8 heterostructures. Our contribution paves the way for the rational design of composite MOFs with tunable compositions and nanostructures using the crystal engineering approach.

Solute clustering is a technologically important microstructural process in Al alloys. Exerting control over this process to enhance the alloy properties and reduce the energy costs of production is a major scientific and technological focus, with automotive sheet applications serving as a key driver. In this work, we detail changes in the state of clustering arising from the insertion of a thermomechanical pre-ageing process, via a coil-cooling step in a commercial production line, immediately after solution treatment. This pre-ageing step effectively mitigates the negative effects of the natural ageing on the mechanical properties that would otherwise occur after solution treatment and ahead of the final paint-bake step. Our work was focussed on a short duration/low temperature single-step pre-ageing process on a Cu-lean grade of AA6016. Using a combination of atom probe tomography and first principles density functional theory simulations, we are able to reveal and explain the atomic-scale microstructure arising from this thermomechanical process. Mg–Si co-clusters ≥ 20 solute atoms were found responsible for the excellent properties in the material exposed to the pre-ageing plus paint-baking process. Our simulations revealed that vacancies can effectively stabilise single-species Si clusters enabling them to attract further solute and serving as a pathway to formation of the larger Mg–Si co-clusters that are so beneficial to alloy properties. Our simulations also revealed that the nearest neighbour configurations are a critical aspect to the stability of the clusters.

Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets.