Palisade structure in intact vaccinia virions.

Vaccinia virus assembly in the cytoplasm of infected cells involves the formation of a biconcave viral core inside the maturing viral particle. The boundary of the core is defined by a pseudohexagonal palisade layer, composed of trimers projecting from an inner wall. To understand the assembly of this complex core architecture, we obtained a subnanometer structure of the palisade trimer by cryo-electron tomography and subtomogram averaging of purified intact virions. Using AlphaFold2 structure predictions, we determined that the palisade is formed from trimers of the proteolytically processed form of the viral protein A10. In addition, we found that each A10 protomer associates with an α-helix (residues 24-66) of A4. Cellular localization assays outside the context of infection demonstrate that the A4 N-terminus is necessary and sufficient to interact with A10. The interaction between A4 and A10 provides insights into how the palisade layer might become tightly associated with the viral membrane during virion maturation. Reconstruction of the palisade layer reveals that, despite local hexagonal ordering, the A10/A4 trimers are widely spaced, suggesting that additional components organize the lattice. This spacing would, however, allow the adoption of the characteristic biconcave shape of the viral core. Finally, we also found that the palisade incorporates multiple copies of a hexameric portal structure. We suggest that these portals are formed by E6, a viral protein that is essential for virion assembly and required to release viral mRNA from the core early in infection.IMPORTANCEPoxviruses such as variola virus (smallpox) and monkeypox cause diseases in humans. Other poxviruses, including vaccinia and modified vaccinia Ankara, are used as vaccine vectors. Given their importance, a greater structural understanding of poxvirus virions is needed. We now performed cryo-electron tomography of purified intact vaccinia virions to study the structure of the palisade, a protein lattice that defines the viral core boundary. We identified the main viral proteins that form the palisade and their interaction surfaces and provided new insights into the organization of the viral core.

A succession of two viral lattices drives vaccinia virus assembly.

During its cytoplasmic replication, vaccinia virus assembles non-infectious spherical immature virions (IV) coated by a viral D13 lattice. Subsequently, IV mature into infectious brick-shaped intracellular mature virions (IMV) that lack D13. Here, we performed cryo-electron tomography (cryo-ET) of frozen-hydrated vaccinia-infected cells to structurally characterise the maturation process in situ. During IMV formation, a new viral core forms inside IV with a wall consisting of trimeric pillars arranged in a new pseudohexagonal lattice. This lattice appears as a palisade in cross-section. As maturation occurs, which involves a 50% reduction in particle volume, the viral membrane becomes corrugated as it adapts to the newly formed viral core in a process that does not appear to require membrane removal. Our study suggests that the length of this core is determined by the D13 lattice and that the consecutive D13 and palisade lattices control virion shape and dimensions during vaccinia assembly and maturation.

Ageing across the great divide: tissue transformation, organismal growth and temperature shape telomere dynamics through the metamorphic transition.

Telomere attrition is considered a useful indicator of cellular and whole-organism ageing rate. While approximately 80% of animal species undergo metamorphosis that includes extensive tissue transformations (involving cell division, apoptosis, de-differentiation and de novo formation of stem cells), the effect on telomere dynamics is unknown. We measured telomeres in Xenopus laevis developing from larvae to adults under contrasting environmental temperatures. Telomere dynamics were linked to the degree of tissue transformation during development. Average telomere length in gut tissue increased dramatically during metamorphosis, when the gut shortens by 75% and epithelial cells de-differentiate into stem cells. In the liver (retained from larva) and hindlimb muscle (newly formed before metamorphosis), telomeres gradually shortened until adulthood, likely due to extensive cell division. Tail muscle telomere lengths were constant until tail resorption, and those in heart (retained from larva) showed no change over time. Telomere lengths negatively correlated with larval growth, but for a given growth rate, telomeres were shorter in cooler conditions, suggesting that growing in the cold is more costly. Telomere lengths were not related to post-metamorphic growth rate. Further research is now needed to understand whether telomere dynamics are a good indicator of ageing rate in species undergoing metamorphosis.

Recruitment of clathrin to intracellular membranes is sufficient for vesicle formation.

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.

Comment on Dimou et al. Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans. J. Fungi 2021, 7, 560.

Contrary to the opinion recently offered by Dimou et al., our previously published biochemical, subcellular and genetic data supported our contention that AN11127 corresponds to the A. nidulans gene encoding Sec12, which is the guanine nucleotide exchange factor (GEF) specific for SAR1. We add here additional bioinformatics evidence that fully disprove the otherwise negative evidence reported by Dimou et al., highlighting the dangers associated with the lax interpretation of genomic data. On the positive side, we establish guidelines for the identification of this key secretory gene in other species of Ascomycota and Basidiomycota, including species of medical and applied interest.

Viral use and subversion of membrane organization and trafficking.

Membrane trafficking is an essential cellular process conserved across all eukaryotes, which regulates the uptake or release of macromolecules from cells, the composition of cellular membranes and organelle biogenesis. It influences numerous aspects of cellular organisation, dynamics and homeostasis, including nutrition, signalling and cell architecture. Not surprisingly, malfunction of membrane trafficking is linked to many serious genetic, metabolic and neurological disorders. It is also often hijacked during viral infection, enabling viruses to accomplish many of the main stages of their replication cycle, including entry into and egress from cells. The appropriation of membrane trafficking by viruses has been studied since the birth of cell biology and has helped elucidate how this integral cellular process functions. In this Review, we discuss some of the different strategies viruses use to manipulate and take over the membrane compartments of their hosts to promote their replication, assembly and egress.

Tracking exocytosis of a GPI-anchored protein in Aspergillus nidulans.

Secretion of the glycosylphosphatidylinositol-anchored protein (GPI-AP) EglC was investigated in the filamentous fungus Aspergillus nidulans, exploiting a sucrose-inducible promoter to conditionally express the protein in cells blocked at different steps of exocytosis. EglC is delivered to the cell surface in a polarized fashion, but appears to redistribute rapidly toward apico-distal regions. Inactivation of SarASar1 mediating COPII vesicle biogenesis resulted in the accumulation of EglC in the endoplasmic reticulum (ER) but, rather than concentrating in ER-exit-sites, the reporter labeled the ER uniformly. Abnormal posttranslational modifications of EglC were detected in sarAts and sed5ts mutants, suggesting that blocking COPII biogenesis or traffic in the ER/Golgi interface might affect GPI remodeling. EglC delivery to the plasma membrane requires, besides Golgi function, the TRAPPII complex mediating the biogenesis of RAB11 secretory vesicles at the TGN, but is unaffected by the absence of RAB5, the key regulator of early endosome biogenesis/maturation. Thus, unlike the soluble extracellular enzyme inulinase, EglC is directly delivered from the TGN to the plasma membrane without involvement of endosomes. We conclude that in A. nidulans, GPI-APs follow a direct secretory pathway from the ER to the plasma membrane.

Identification of the guanine nucleotide exchange factor for SAR1 in the filamentous fungal model Aspergillus nidulans.

In spite of its basic and applied interest, the regulation of ER exit by filamentous fungi is insufficiently understood. In previous work we isolated a panel of conditional mutations in sarA encoding the master GTPase SarASAR1 in A. nidulans and demonstrated its key role in exocytosis and hyphal morphogenesis. However, the SAR1 guanine nucleotide exchange factor (GEF), Sec12, has not been characterized in any filamentous fungus, largely due to the fact that SEC12 homologues share little amino acid sequence identity beyond a GGGGxxxxGϕxN motif involved in guanine nucleotide exchange. Here we demonstrate that AN11127 encodes A. nidulans Sec12, which is an essential protein that localizes to the ER and that, when overexpressed, rescues the growth defect resulting from a hypomorphic sarA6ts mutation at 37 °C. Using purified, bacterially expressed proteins we demonstrate that the product of AN11127 accelerates nucleotide exchange on SarASAR1, but not on its closely related GTPase ArfAARF1, as expected for a bona fide GEF. The unequivocal characterization of A. nidulans Sec12 paves the way for the tailored modification of ER exit in a model organism that is closely related to industrial species of filamentous fungi.

COPI localizes to the early Golgi in Aspergillus nidulans.

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedVSed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypBSec7 and HypATrs120, implying that COPI budding predominates at the SedVSed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodVIC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.

Genetic dissection of the secretory route followed by a fungal extracellular glycosyl hydrolase.

Hyphal tip cells of Aspergillus nidulans are > 100 µm-long, which challenges intracellular traffic. In spite of the basic and applied interest of the secretory pathway of filamentous fungi, only recently has it been investigated in detail. We used InuA, an inducible and highly glycosylated inulinase, and mutations affecting different intracellular membranous compartments, to investigate the route by which the enzyme traffics to the extracellular medium. InuA is core-N-glycosylated in the ER and hyperglycosylated during transit across the Golgi. Hyperglycosylation was prevented by ts mutations in sarASAR1 impeding ER exit, and in sedVSED5 and rabORAB1 dissipating the early Golgi, but not by mutations in the TGN regulators hypATRS120 and hypBSEC7 , implicating the early Golgi in cargo glycosylation. podB1ts (cog2ts ) affecting the COG complex also prevents glycosylation, without disassembling early Golgi cisternae. That InuA exocytosis is prevented by inactivation of any of the above genes shows that it follows a conventional secretory pathway. However, ablation of RabBRAB5 regulating early endosomes (EEs), but not of RabSRAB7 , its equivalent in late endosomes, also prevents InuA accumulation in the medium, indicating that EEs are specifically required for InuA exocytosis. This work provides a framework to understand the secretion of enzyme cargoes by industrial filamentous fungi.

Endocytic recycling via the TGN underlies the polarized hyphal mode of life.

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1µm/min) and the long intracellular distances (>100 µm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.

Conditional inactivation of Aspergillus nidulans†sarA(SAR1) uncovers the morphogenetic potential of regulating endoplasmic reticulum (ER) exit.

In the genetic model Aspergillus nidulans, hyphal growth is exquisitely dependent on exocytic traffic. Following mutagenic PCR and gene replacement, we characterized thermosensitive mutations in sarA(SAR1) encoding a key regulator of endoplasmic reticulum (ER) exit. Six sarA(ts) alleles permitting relatively normal growth at 30°C prevented it at 42°C. This growth phenotype correlated with markedly reduced SarA levels at high temperature, suggesting that these alleles cause temperature-dependent SarA misfolding. sarA8 results in Ser substitution for conserved P-loop Gly27. sarA5 (Trp185Cys) and sarA6 (Ser186Pro) substitutions underscore the importance of the C-terminal α-helix on SarA(Sar1) function/stability. sarA6 markedly diminishing growth at 37°C was useful for microscopy experiments in which ER exit was impaired by shifting the incubation temperature. Early and late Golgi cisternae, labeled with the integral membrane syntaxins SedV(Sed5) and TlgB(Tlg2) , respectively, were rapidly dissipated by sarA6. However, whereas SedV(Sed5) was shifted toward the ER, TlgB(Tlg2) relocalized to a haze, underscoring the asymmetry of Golgi organization. This rapid Golgi dissipation that takes place after blocking anterograde COPII traffic is consistent with the cisternal maturation model. Incubation of sarA6 cells at 37°C led to the formation of apical balloons resembling specialized fungal structures. The formation of these balloons highlights the morphogenetic consequences of impairing ER exit.

GBF/Gea mutant with a single substitution sustains fungal growth in the absence of BIG/Sec7.

Golgi Arf1-guanine nucleotide exchange factors (GEFs) belong to two subfamilies: GBF/Gea and BIG/Sec7. Both are conserved across eukaryotes, but the physiological role of each is not well understood. Aspergillus nidulans has a single member of the early Golgi GBF/Gea-subfamily, geaA, and the late Golgi BIG/Sec7-subfamily, hypB. Both geaA and hypB are essential. hypB5 conditionally blocks secretion. We sought extragenic hypB5 suppressors and obtained geaA1. geaA1 results in Tyr1022Cys within a conserved GBF/Gea-specific S(Y/W/F)(L/I) motif in GeaA. This mutation alters GeaA localization. Remarkably, geaA1 suppresses hypBΔ, indicating that a single mutant Golgi Arf1-GEF suffices for growth.

[Effects of a submarine discharge of urban waste on octocoral (Octocorallia: Alcyonacea) communities in Cuba]. / Efectos de un emisario submarino de aguas "residuales" urbanas sobre comunidades de octocorales (Octocorallia: Alcyonacea) en Cuba.

The composition and structure of octocoral communities on coral reefs close to a submarine outfall were studied at Reparto Flores, west of Havana City, Cuba. Octocoral community changes after the deployment of the submarine outfall in 2000 were monitored from June 2002 to September 2005, taking as baseline the data existing before its construction. The area also receives the influence of the polluted river Quibú that passes through a great part of the west side of the City. Sampling was done by means of SCUBA diving, counting and identifying colonies in situ within a 1 m2 frame that was randomly placed as many times as to warrant stabilized values of Shannon and Weaver's heterogeneity index H'. In agreement with the available hydrochemical information, changes in the diversity indexes (Shannon and Weaver's heterogeneity index H', Pielou's equitability index J', and Margalef's species richness index R1), the Herrera-Moreno's comparative pollution index (ICC), and density of some octocoral species at a depth of 10 m suggest a decrease in the influence of polluters from 1989 to 2005. Nevertheless, these indicators were affected in 2004 by a sudden intense but brief colonization of Briareum asbestinum, a species that is not typical of polluted places. At a depth of 20 m, a co-dominance of Plexaura kuekenthali and Eunicea clavigera (resistant and non resistant to pollution, respectively) and an increase of the comparative pollution index (ICC) was observed. The increase of P. kuekenthali, a pollution indicator, suggests a rise in the pollution effect 20 m in depth, because of the recent impact caused by the greater closeness of the outfall mouth 50 m deep. Results corroborate the hypothesis about the pollution indicator character of P. kuekenthali. However, this could not be explored for Eunicea flexuosa (also considered a pollution-indicator) due to an intensive illegal selective extraction for lucrative handicraft purposes, which led to a remarkable decrease in its density. B. asbestinum and E. clavigera were outlined as poorly resistant to pollution.

Efectos de un emisario submarino de aguas "residuales" urbanas sobre comunidades de octocorales (Octocorallia: Alcyonacea) en Cuba / Effects of a submarine discharge of urban waste on octocoral (Octocorallia: Alcyonacea) communities in Cuba

The composition and structure of octocoral communities on coral reefs close to a submarine outfall were studied at Reparto Flores, west of Havana City, Cuba. Octocoral community changes after the deployment of the submarine outfall in 2000 were monitored from June 2002 to September 2005, taking as baseline the data existing before its construction. The area also receives the influence of the polluted river Quibú that passes through a great part of the west side of the City. Sampling was done by means of SCUBA diving, counting and identifying colonies in situ within a 1 m2 frame that was randomly placed as many times as to warrant stabilized values of Shannon and Weaver's heterogeneity index H'. In agreement with the available hydrochemical information, changes in the diversity indexes (Shannon and Weaver's heterogeneity index H', Pielou's equitability index J', and Margalef's species richness index R1), the Herrera-Moreno's comparative pollution index (ICC), and density of some octocoral species at a depth of 10 m suggest a decrease in the influence of polluters from 1989 to 2005. Nevertheless, these indicators were affected in 2004 by a sudden intense but brief colonization of Briareum asbestinum, a species that is not typical of polluted places. At a depth of 20 m, a co-dominance of Plexaura kuekenthali and Eunicea clavigera (resistant and non resistant to pollution, respectively) and an increase of the comparative pollution index (ICC) was observed. The increase of P. kuekenthali, a pollution indicator, suggests a rise in the pollution effect 20 m in depth, because of the recent impact caused by the greater closeness of the outfall mouth 50 m deep. Results corroborate the hypothesis about the pollution indicator character of P. kuekenthali. However, this could not be explored for Eunicea flexuosa (also considered a pollution-indicator) due to an intensive illegal selective extraction for lucrative handicraft purposes, which...

Para profundizar en las respuestas de las comunidades de octocorales a la influencia de residuales urbanos se estudió su composición y estructura en sitios de arrecife cercanos a un emisario submarino urbano en el Reparto Flores, La Habana, Cuba. Desde junio del 2002 hasta septiembre del 2005, se efectuó el monitoreo de los cambios ocurridos en las comunidades después de la puesta en operación del emisario en el año 2000, tomando como línea base datos existentes antes del inicio de su construcción. El muestreo se realizó mediante buceo SCUBA, contando e identificando las colonias in situ dentro de cuadricula de 1 m2 de lado. Diversos índices sugieren una disminución de la influencia de la contaminación en la última década.El incremento de P. kuekenthali (indicadora de contaminación, , sugiere un aumento del efecto de la contaminación a 20 m de profundidad debido al impacto reciente de la mayor cercanía del desagüe del emisario que se encuentra a 50 m de profundidad. B. asbestinum y E. clavigera se perfilaron como poco resistentes a la contaminación.