Alison J. Beckett

Transport of Fibroblast Growth Factor 2 in the Pericellular Matrix Is Controlled by the Spatial Distribution of Its Binding Sites in Heparan Sulfate

A single-molecule imaging study reveals that heparan sulfate chains in the pericellular matrix present a structured network of binding sites that controls FGF2 transport.

The role of palmitoylation in regulating Ras localization and function

Ras GTPases are important regulators of pathways controlling proliferation, differentiation and transformation. Three ubiquitously expressed almost identical Ras genes are not functionally redundant; this has been attributed to their distinctive trafficking and localization profiles. A palmitoylation cycle controls the correct compartmentalization of H-Ras and N-Ras. We review recent data that reveal how this cycle can be regulated by membrane organization to influence the spatiotemporal signalling of Ras.

Raft protein clustering alters N-Ras membrane interactions and activation pattern

ABSTRACT The trafficking, membrane localization, and lipid raft association of Ras proteins, which are crucial oncogenic mediators, dictate their isoform-specific biological responses. Accordingly, their spatiotemporal dynamics are tightly regulated. While extensively studied for H- and K-Ras, such information on N-Ras, an etiological oncogenic factor, is limited. Here, we report a novel mechanism regulating the activation-dependent spatiotemporal organization of N-Ras, its modulation by biologically relevant stimuli, and isoform-specific effects on signaling. We combined patching/immobilization of another membrane protein with fluorescence recovery after photobleaching (patch-FRAP) and FRAP beam size analysis to investigate N-Ras membrane interactions. Clustering of raft-associated proteins, either glycosylphosphatidylinositol-anchored influenza virus hemagglutinin (HA-GPI) or fibronectin receptors, selectively enhanced the plasma membrane-cytoplasm exchange of N-Ras–GTP (preferentially associated with raft domains) in a cholesterol-dependent manner. Electron microscopy (EM) analysis showed N-Ras–GTP localization in cholesterol-sensitive clusters, from which it preferentially detached upon HA-GPI cross-linking. HA-GPI clustering enhanced the Golgi compartment (GC) accumulation and signaling of epidermal growth factor (EGF)-stimulated N-Ras–GTP. Notably, the cross-linking-mediated enhancement of N-Ras–GTP exchange and GC accumulation depended strictly on depalmitoylation. We propose that the N-Ras activation pattern (e.g., by EGF) is altered by raft protein clustering, which enhances N-Ras–GTP raft localization and depalmitoylation, entailing its exchange and GC accumulation following repalmitoylation. This mechanism demonstrates a functional signaling role for the activation-dependent differential association of Ras isoforms with raft nanodomains.

3D-CLEM reveals that a major portion of mitotic chromosomes is not Chromatin

Summary Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%–47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.

Highly Stable and Conductive Microcapsules for Enhancement of Joule Heating Performance

Nanocarbons show great promise for establishing the next generation of Joule heating systems, but suffer from the limited maximum temperature due to precociously convective heat dissipation from electrothermal system to surrounding environment. Here we introduce a strategy to eliminate such convective heat transfer by inserting highly stable and conductive microcapsules into the electrothermal structures. The microcapsule is composed of encapsulated long-chain alkanes and graphene oxide/carbon nanotube hybrids as core and shell material, respectively. Multiform carbon nanotubes in the microspheres stabilize the capsule shell to resist volume-change-induced rupture during repeated heating/cooling process, and meanwhile enhance the thermal conductance of encapsulated alkanes which facilitates an expeditious heat exchange. The resulting microcapsules can be homogeneously incorporated in the nanocarbon-based electrothermal structures. At a dopant of 5%, the working temperature can be enhanced by 30% even at a low voltage and moderate temperature, which indicates a great value in daily household applications. Therefore, the stable and conductive microcapsule may serve as a versatile and valuable dopant for varieties of heat generation systems.

Oceanic Sharks Clean at Coastal Seamount

Interactions between pelagic thresher sharks (Alopias pelagicus) and cleaner wrasse were investigated at a seamount in the Philippines. Cleaning associations between sharks and teleosts are poorly understood, but the observable interactions seen at this site may explain why these mainly oceanic sharks regularly venture into shallow coastal waters where they are vulnerable to disturbance from human activity. From 1,230 hours of observations recorded by remote video camera between July 2005 and December 2009, 97 cleaner-thresher shark events were analyzed, 19 of which were interrupted. Observations of pelagic thresher sharks interacting with cleaners at the seamount were recorded at all times of day but their frequency declined gradually from morning until evening. Cleaners showed preferences for foraging on specific areas of a thresher sharks body. For all events combined, cleaners were observed to conduct 2,757 inspections, of which 33.9% took place on the sharks pelvis, 23.3% on the pectoral fins, 22.3% on the caudal fin, 8.6% on the body, 8.3% on the head, 2.1% on the dorsal fin, and 1.5% on the gills respectively. Cleaners did not preferentially inspect thresher sharks by time of day or by shark sex, but there was a direct correlation between the amount of time a thresher shark spent at a cleaning station and the number of inspections it received. Thresher shark clients modified their behavior by “circular-stance-swimming,” presumably to facilitate cleaner inspections. The cleaner-thresher shark association reflected some of the known behavioral trends in the cleaner-reef teleost system since cleaners appeared to forage selectively on shark clients. Evidence is mounting that in addition to acting as social refuges and foraging grounds for large visiting marine predators, seamounts may also support pelagic ecology by functioning as cleaning stations for oceanic sharks and rays.

The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells.

The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca2+ concentration ([Ca2+]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as ‘initiating’ organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca2+ influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca2+ influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca2+ entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca2+ pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca2+ elevation and endocytic vacuole formation.

Oncogenic K-ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status.

Summary Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110&agr;, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation – by activation of protein kinase C (PKC) – increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110&agr;) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide – for the first time – evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis

ABSTRACT Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light–SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle. Summary: Spatial analysis of microtubule organisation in mitotic cells by using 3D imaging and mathematical modelling.

Bacterial viability on chemically modified silicon nanowire arrays

The global threat of antimicrobial resistance is driving an urgent need for novel antimicrobial strategies. Functional surfaces are essential to prevent spreading of infection and reduce surface contamination. In this study we have fabricated and characterized multiscale-functional nanotopographies with three levels of functionalization: (1) nanostructure topography in the form of silicon nanowires, (2) covalent chemical modification with (3-aminopropyl)triethoxysilane, and (3) incorporation of chlorhexidine digluconate. Cell viability assays were carried out on two model microorganisms E. coli and S. aureus over these nanotopographic surfaces. Using SEM we have identified two growth modes producing distinctive multicellular structures, i.e. in plane growth for E. coli and out of plane growth for S. aureus. We have also shown that these chemically modified SiNWs arrays are effective in reducing the number of planktonic and surface-attached microorganisms.