Science and society: Frozen delight

In the April, 1985 issue of Bio Essays, F. Vella presented an evaluation of the recent GPEP report, concerning medical school education in the United States. Here, Hilliard Jason and Jane Westberg present an additional discussion of the issues.     

Tragedy and delight: the ethics of decelerated ageing

Biogerontology is sometimes viewed as similar to other forms of biomedical research in that it seeks to understand and treat a pathological process. Yet the prospect of treating ageing is extraordinary in terms of the profound changes to the human condition that would result. Recent advances in biogerontology allow a clearer view of the ethical issues and dilemmas that confront humanity with respect to treating ageing. For example, they imply that organismal senescence is a disease process with a broad spectrum of pathological consequences in late life (causing or exascerbating cardiovascular disease, cancer, neurodegenerative disease and many others). Moreover, in laboratory animals, it is possible to decelerate ageing, extend healthy adulthood and reduce the age-incidence of a broad spectrum of ageing-related diseases. This is accompanied by an overall extension of lifespan, sometimes of a large magnitude. Discussions of the ethics of treating ageing sometimes involve hand-wringing about detrimental consequences (e.g. to society) of marked life extension which, arguably, would be a form of enhancement technology. Yet given the great improvements in health that decelerated ageing could provide, it would seem that the only possible ethical course is to pursue it energetically. Thus, decelerated ageing has an element of tragic inevitability: its benefits to health compel us to pursue it, despite the transformation of human society, and even human nature, that this could entail.     

Cryptococcus neoformans: gastronomic delight of a soil ameba.

During 7 days of incubation in vitro the trophozoite stage of the free-living soil amoeba, Acanthamoeba polyphaga, phagocytized and killed 78-97% of the cells of three strains of Cryptococcus neoformans. With one strain, incubation time was increased to nine days and 99% of the yeast cells were killed. It was calculated that during 4-9 days of incubation a single trophozoite phagocytized and killed a daily average of 84 yeast cells. The lethal effect of A. polyphaga on C. neoformans may represent a biological control mechanism in nature. Some of the surviving cells of C. neoformans developed into colonies containing pseudohyphae; these pseudolhyphal forms may be a biological 'escape hatch'.     

Destruction's our delight: Controlling apoptosis during mitotic arrest

Comment on: Harley ME, et al. EMBO J. 2010; 29:2407-20.     

Chlamydia trachomatis Transports NAD via the Npt1 ATP/ADP Translocase

Obligate intracellular bacteria comprising the order Chlamydiales lack the ability to synthesize nucleotides de novo and must acquire these essential compounds from the cytosol of the host cell. The environmental protozoan endosymbiont Protochlamydia amoebophila UWE25 encodes five nucleotide transporters with specificities for different nucleotide substrates, including ATP, GTP, CTP, UTP, and NAD. In contrast, the human pathogen Chlamydia trachomatis encodes only two nucleotide transporters, the ATP/ADP translocase C. trachomatis Npt1 (Npt1_Ct) and the nucleotide uniporter Npt2_Ct, which transports GTP, UTP, CTP, and ATP. The notable absence of a NAD transporter, coupled with the lack of alternative nucleotide transporters on the basis of bioinformatic analysis of multiple C. trachomatis genomes, led us to re-evaluate the previously characterized transport properties of Npt1_Ct. Using [adenylate-³²P]NAD, we demonstrate that Npt1_Ct expressed in Escherichia coli enables the transport of NAD with an apparent K_m and V_max of 1.7 μM and 5.8 nM mg⁻¹ h⁻¹, respectively. The K_m for NAD transport is comparable to the K_m for ATP transport of 2.2 μM, as evaluated in this study. Efflux and substrate competition assays demonstrate that NAD is a preferred substrate of Npt1_Ct compared to ATP. These results suggest that during reductive evolution, the pathogenic chlamydiae lost individual nucleotide transporters, in contrast to their environmental endosymbiont relatives, without compromising their ability to obtain nucleotides from the host cytosol through relaxation of transport specificity. The novel properties of Npt1_Ct and its conservation in chlamydiae make it a potential target for the development of antimicrobial compounds and a model for studying the evolution of transport specificity.     

Phosphate Translocator of Isolated Guard-Cell Chloroplasts from Pisum sativum L. Transports Glucose-6-Phosphate

Chloroplasts were isolated from ruptured guard-cell protoplasts of the Argenteum mutant of Pisum sativum L. and purified by centrifugation through a Percoll layer. The combined volume of the intact plastids and the uptake of phosphate were determined by silicone oil-filtering centrifugation, using tritiated water and [14C]sorbitol as membrane-permeating and nonpermeating markers and [32P]phosphate as tracer for phosphate. The affinities of the phosphate translocator for organic phosphates were assessed by competition with inorganic phosphate. The affinities for dihydroxyacetone phosphate, 3-phosphoglycerate (PGA), and phosphoenolpyruvate were in the same order as those reported for mesophyll chloroplasts of several species. However, the guard-cell phosphate translocator had an affinity for glucose-6-phosphate that was as high as that for PGA. Guard-cell chloroplasts share this property with amyloplasts from the root of pea (H.W. Heldt, U.I. Flugge, S. Borchert [1991] Plant Physiol 95: 341-343). An ability to import glucose-6-phosphate enables guard-cell chloroplasts to synthesize starch despite the reported absence of a fructose-1,6-bisphosphatase activity in the plastids, which would be required if only C3 phosphates could enter through the translocator.     

Effect of Cytochalasin B on the Development of Membrane Transports in Sea Urchin Eggs after Fertilization

Investigations were made on the role of the cytoskeleton in the onset of ionic events following fertilization of sea urchin eggs. Events which depend upon phosphoinositide metabolism, such as the cortical reantion and acid release are affected by cytochalasin B (CB) after fertilization but not after activation of eggs with the ionophore A23187. These findings suggest that the sequence of events following sperm-egg attachment depends on the cytoskeleton. CB also inhibits the Na⁺ pump and alanine uptake when added before insemination and during the following 30 min. These results argue for a role of the egg cortex cytoskeleton in activation of the Na⁺ pump by fertilization. We propose that the inhibitory effect of CB on the development of amino-acid uptake after fertilization may result from an increase in the Na⁺ content of the egg resulting from Na⁺ pump suppression rather than from direct blockage of the carrier.     

The Motor Protein Myosin-X Transports VE-Cadherin along Filopodia To Allow the Formation of Early Endothelial Cell-Cell Contacts

Vascular endothelium (VE), the monolayer of endothelial cells that lines the vascular tree, undergoes damage at the basis of some vascular diseases. Its integrity is maintained by VE-cadherin, an adhesive receptor localized at cell-cell junctions. Here, we show that VE-cadherin is also located at the tip and along filopodia in sparse or subconfluent endothelial cells. We observed that VE-cadherin navigates along intrafilopodial actin filaments. We found that the actin motor protein myosin-X is colocalized and moves synchronously with filopodial VE-cadherin. Immunoprecipitation and pulldown assays confirmed that myosin-X is directly associated with the VE-cadherin complex. Furthermore, expression of a dominant-negative mutant of myosin-X revealed that myosin-X is required for VE-cadherin export to cell edges and filopodia. These features indicate that myosin-X establishes a link between the actin cytoskeleton and VE-cadherin, thereby allowing VE-cadherin transportation along intrafilopodial actin cables. In conclusion, we propose that VE-cadherin trafficking along filopodia using myosin-X motor protein is a prerequisite for cell-cell junction formation. This mechanism may have functional consequences for endothelium repair in pathological settings.The endothelium is composed of a monolayer of endothelial cells that lines the vascular tree. Hemodynamic forces, immune-mediated mechanisms, or drug ingestion can injure the endothelium (35). These types of damage are frequently accompanied by a loss of endothelium integrity, an increase in vascular permeability, and possibly by a detachment of endothelial cells from vascular walls (14). These alterations can be circumvented by initiating rapid repair mechanisms that reestablish endothelium integrity and consequently reduce the extent of vascular diseases. The molecular mechanisms at the basis of the endothelium repair process remain elusive, but it can be assumed that the reconstitution of endothelium integrity requires cell-cell junction rebuilding.In the endothelium, intercellular adherence is maintained by tight and adherens junctions. Adherens junctions are particularly crucial in controlling the formation and maintenance of interendothelial adhesion and constitute dynamic structures that undergo remodeling in migrating as well as resting cells (31). They are essentially composed of vascular endothelial-cadherin (VE-Cad) (22), an adhesive receptor able to elaborate homophilic/homotypic interactions via its extracellular domain and to recruit, through its cytoplasmic tail, partners such as α-, β-, and γ-catenins and p120 (1). Catenins, in turn, promote the association of the adherens junction with the actin cytoskeleton, another player regulating vascular endothelial barrier function, by molecular mechanisms that are incompletely defined (8). Although there is a general agreement about the critical role played by actin filaments in the maintenance of mature cell-cell junctions (27, 41), their precise role in the elaboration of premature adherens junctions is poorly understood. Some studies indicate that cells form intercellular junctions by a dynamic process driven by actin polymerization (38). It was proposed but, to our knowledge not firmly demonstrated, that cell-cell junction formation is initiated by the production of filopodia emanating from neighboring cells (3, 30, 39, 42). Filopodia lead to the elaboration of puncta, which correspond to microdomains where cadherin molecules concentrate together with their intracellular partners (3). These puncta spatially coincide with cell membrane attachment sites for actin filaments (2). The mechanism by which puncta are elaborated remains to be elucidated.Filopodia are highly dynamic structures filled with bundles of linear actin filaments (15). Their protrusion is driven by actin polymerization taking place at filament barbed ends that are mainly located at filopodium tips (24). The precise mechanisms of the nucleation and elongation of filopodia are controversial. In fact, two mechanisms for their formation have been proposed, each using different sets of actin-regulating proteins. According to the “convergent elongation model,” filopodia are continuously initiated by the elongation of preexisting lamellipodial actin filaments (34). This remodeling of actin filaments should require the branching activity of Arp2/3 (29), the F-actin-bundling activity of fascin along filopodium shafts and the anticapping activity of Ena/VASP at the barbed ends of actin filaments (4). In the opposing model, it was proposed that some members of the formin family such as Dia2 perform all these activities (17). Indeed, in vitro, Dia2 nucleates linear actin filaments, accelerates actin polymerization, and protects barbed ends from capping proteins, thus slowing actin depolymerization (7, 17). Additionally, new players such as myosin-X (MyoX), able to induce filopodium formation, have been recently discovered.Here, using cryo-electron microscopy (cryo-EM), we show that VE-cadherin is not exclusively located at cell-cell junctions but is also present along and at the tip of filopodia in sparse endothelial cells. By video microscopy, we observed that VE-cadherin migrates along filopodia in forward and backward movements. We hypothesized that motor proteins of the myosin family may be involved in the VE-cadherin transportation along filopodia. We considered MyoX as a potential candidate for promoting VE-cadherin trafficking.Myosins participate in a range of diverse cellular processes such as cell migration, membrane trafficking, and formation of cellular protrusions. They share conserved structural features such as a motor domain located at their N termini that can bind to actin filaments and hydrolyze ATP to produce movement and force. At their C termini, members of the unconventional myosin family such as myosin-VII, -X, -XII, and -XV exhibit a myosin tail homology 4 domain (MyTH4) followed by a FERM (band 4.1 protein, ezrin, radixin, and moesin) domain that confers upon them with the ability to perform unique cellular functions (6). A fascinating feature of MyoX is that it uses its motor activity to move along the intrafilopodial actin filaments. This probably allows MyoX to carry cargoes along filopodia. Potential cargoes are the β-chains of integrins, recently reported to directly interact with the FERM domain of MyoX (43), and Mena/VASP, which is synchronously transported with MyoX toward the tip or the base of filopodia (36). In addition to its motor and transport functions, MyoX also promotes the formation of filopodia (5, 9, 37). Hence, MyoX overexpression stimulates filopodium growth (5), whereas its knockdown decreases filopodium formation (9, 28, 37).Herein, we discovered that MyoX is colocalized with VE-cadherin in filopodia and moves synchronously with it. Using immunoprecipitation (IP) experiments and pulldown assays, we demonstrated that MyoX interacts with the VE-Cad-catenin complex. Our data thus support a role of MyoX in the transportation of VE-cadherin along intrafilopodial actin. The forward MyoX-mediated transport facilitates the accumulation of VE-Cad at the tips of filopodia, where VE-Cad can interact with partners of adjacent cells, thus establishing preliminary cell-cell contacts. Formation of these early cell-cell contacts can be inhibited by blocking MyoX transport capacity. At filopodium tips, VE-Cad linked to MyoX, but not engaged in homophilic interactions, may also be transported backwards to the cell body by the actin retrograde flow. Once at the lamellipodium edge, VE-Cad can be picked up again by newly formed filopodia. Our data suggest that MyoX-mediated transport of the VE-Cad-catenin complex along filopodia is a key event required for the early steps of formation of cell-cell contacts, a process that may be of functional importance in endothelium repair and angiogenesis.     

Alternative Electron Transports Participate in the Maintenance of Violaxanthin De-Epoxidase Activity of Ulva sp. under Low Irradiance

The xanthophyll cycle (Xc), which involves violaxanthin de-epoxidase (VDE) and the zeaxanthin epoxidase (ZEP), is one of the most rapid and efficient responses of plant and algae to high irradiance. High light intensity can activate VDE to convert violaxanthin (Vx) to zeaxanthin (Zx) via antheraxanthin (Ax). However, it remains unclear whether VDE remains active under low light or dark conditions when there is no significant accumulation of Ax and Zx, and if so, how the ΔpH required for activation of VDE is built. In this study, we used salicylaldoxime (SA) to inhibit ZEP activity in the intertidal macro-algae Ulva sp. (Ulvales, Chlorophyta) and then characterized VDE under low light and dark conditions with various metabolic inhibitors. With inhibition of ZEP by SA, VDE remained active under low light and dark conditions, as indicated by large accumulations of Ax and Zx at the expense of Vx. When PSII-mediated linear electron transport systems were completely inhibited by SA and DCMU, alternative electron transport systems (i.e., cyclic electron transport and chlororespiration) could maintain VDE activity. Furthermore, accumulations of Ax and Zx decreased significantly when SA, DCMU, or DBMIB together with an inhibitor of chlororespiration (i.e., propyl gallate (PG)) were applied to Ulva sp. This result suggests that chlororespiration not only participates in the build-up of the necessary ΔpH, but that it also possibly influences VDE activity indirectly by diminishing the oxygen level in the chloroplast.     

E. coli Transports Aggregated Proteins to the Poles by a Specific and Energy-Dependent Process

Aggregation of proteins due to failure of quality control mechanisms is deleterious to both eukaryotes and prokaryotes. We found that in Escherichia coli, protein aggregates are delivered to the pole and form a large polar aggregate (LPA). The formation of LPAs involves two steps: the formation of multiple small aggregates and the delivery of these aggregates to the pole to form an LPA. Formation of randomly distributed aggregates, their delivery to the poles, and LPA formation are all energy-dependent processes. The latter steps require the proton motive force, activities of the DnaK and DnaJ chaperones, and MreB. About 90 min after their formation, the LPAs are dissolved in a process that is dependent upon ClpB, DnaK, and energy. Our results confirm and substantiate the notion that the formation of LPAs allows asymmetric inheritance of the aggregated proteins to a small number of daughter cells, enabling their rapid elimination from most of the bacterial population. Moreover, the results show that the processing of aggregated proteins by the protein quality control system is a multi-step process with distinct spatial and temporal controls.