Anadenanthera colubrina (Fabaceae) logs in the Atlantic Forest biome: first host plant for Thoracibidion lineatocolle (Col: Cerambycidae) and a new host for Temnopis megacephala (Col: Cerambycidae) / Troncos de Anadenanthera colubrina (Fabaceae) no bioma da Mata Atlântica: primeira planta hospedeira de Thoracibidion lineatocolle (Col: Cerambycidae) e uma nova planta hospedeira para Temnopis megacephala (Col: Cerambycidae)

Wood-boring beetles develop in live trees and dead wood, performing ecological services such as decomposition and regulation of forest resources. Species of the Cerambycidae family, widely distributed in the world, bore into the trunks of trees and dead wood in native and cultivated areas. The objective is to report the first host plant for Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) and a new host plant for Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) in the Brazilian Atlantic Forest biome. Three logs, with one-meter-long by 20 cm in diameter, were cut from the trunk of a healthy Anadenanthera colubrina (Fabaceae) tree in October 2013 and tied in the understory at 1.5m high in the Rio Doce State Park, Minas Gerais State, Brazil. The logs, exposed in the forest, were each removed after 40, 80 and 120 days and stored individually in a cardboard box in the "Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq)" in the Rio Doce State Park. A total of 94 individuals of T. lineatocolle and 228 of T. megacephala emerged from the A. colubrina logs. This is the first report of a host plant for T. lineatocolle and a new host plant for T. megacephala.

Besouros broqueadores se desenvolvem em árvores vivas e madeira morta, realizando serviços ecológicos como decomposição e regulação de recursos da floresta. Espécies da família Cerambycidae, amplamente distribuídas no mundo, perfuram o caule de árvores e madeira morta em áreas nativas e cultivadas. O objetivo é relatar a primeira planta hospedeira de Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) e uma nova planta hospedeira para Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) no bioma da Mata Atlântica brasileira. Três toras, com um metro de comprimento por 20 cm de diâmetro, foram cortadas de uma árvore sadia de Anadenanthera colubrina (Fabaceae) em outubro de 2013 e amarradas no sub-bosque a 1,5m de altura no Parque Estadual do Rio Doce, estado de Minas Gerais, Brasil. As toras, expostas na floresta, foram removidas, cada uma, após 40, 80 e 120 dias e armazenadas, individualmente, em caixas de papelão no “Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq)” no Parque Estadual do Rio Doce. Um total de 94 indivíduos de T. lineatocolle e 228 de T. megacephala emergiu das toras de A. colubrina. Esse é o primeiro registro de uma planta hospedeira para T. lineatocolle e o de uma nova planta hospedeira para T. megacephala.

Anadenanthera colubrina (Fabaceae) logs in the Atlantic Forest biome: first host plant for Thoracibidion lineatocolle (Col.: Cerambycidae) and a new host for Temnopis megacephala (Col.: Cerambycidae)

Abstract Wood-boring beetles develop in live trees and dead wood, performing ecological services such as decomposition and regulation of forest resources. Species of the Cerambycidae family, widely distributed in the world, bore into the trunks of trees and dead wood in native and cultivated areas. The objective is to report the first host plant for Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) and a new host plant for Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) in the Brazilian Atlantic Forest biome. Three logs, with one-meter-long by 20 cm in diameter, were cut from the trunk of a healthy Anadenanthera colubrina (Fabaceae) tree in October 2013 and tied in the understory at 1.5m high in the Rio Doce State Park, Minas Gerais State, Brazil. The logs, exposed in the forest, were each removed after 40, 80 and 120 days and stored individually in a cardboard box in the Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq) in the Rio Doce State Park. A total of 94 individuals of T. lineatocolle and 228 of T. megacephala emerged from the A. colubrina logs. This is the first report of a host plant for T. lineatocolle and a new host plant for T. megacephala.

Resumo Besouros broqueadores se desenvolvem em árvores vivas e madeira morta, realizando serviços ecológicos como decomposição e regulação de recursos da floresta. Espécies da família Cerambycidae, amplamente distribuídas no mundo, perfuram o caule de árvores e madeira morta em áreas nativas e cultivadas. O objetivo é relatar a primeira planta hospedeira de Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) e uma nova planta hospedeira para Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) no bioma da Mata Atlântica brasileira. Três toras, com um metro de comprimento por 20 cm de diâmetro, foram cortadas de uma árvore sadia de Anadenanthera colubrina (Fabaceae) em outubro de 2013 e amarradas no sub-bosque a 1,5m de altura no Parque Estadual do Rio Doce, estado de Minas Gerais, Brasil. As toras, expostas na floresta, foram removidas, cada uma, após 40, 80 e 120 dias e armazenadas, individualmente, em caixas de papelão no Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq) no Parque Estadual do Rio Doce. Um total de 94 indivíduos de T. lineatocolle e 228 de T. megacephala emergiu das toras de A. colubrina. Esse é o primeiro registro de uma planta hospedeira para T. lineatocolle e o de uma nova planta hospedeira para T. megacephala.

Anadenanthera colubrina (Fabaceae) logs in the Atlantic Forest biome: first host plant for Thoracibidion lineatocolle (Col: Cerambycidae) and a new host for Temnopis megacephala (Col: Cerambycidae) / Troncos de Anadenanthera colubrina (Fabaceae) no bioma da Mata Atlântica: primeira planta hospedeira de Thoracibidion lineatocolle (Col: Cerambycidae) e uma nova planta hospedeira para Temnopis megacephala (Col: Cerambycidae)

Wood-boring beetles develop in live trees and dead wood, performing ecological services such as decomposition and regulation of forest resources. Species of the Cerambycidae family, widely distributed in the world, bore into the trunks of trees and dead wood in native and cultivated areas. The objective is to report the first host plant for Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) and a new host plant for Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) in the Brazilian Atlantic Forest biome. Three logs, with one-meter-long by 20 cm in diameter, were cut from the trunk of a healthy Anadenanthera colubrina (Fabaceae) tree in October 2013 and tied in the understory at 1.5m high in the Rio Doce State Park, Minas Gerais State, Brazil. The logs, exposed in the forest, were each removed after 40, 80 and 120 days and stored individually in a cardboard box in the "Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq)" in the Rio Doce State Park. A total of 94 individuals of T. lineatocolle and 228 of T. megacephala emerged from the A. colubrina logs. This is the first report of a host plant for T. lineatocolle and a new host plant for T. megacephala.

Besouros broqueadores se desenvolvem em árvores vivas e madeira morta, realizando serviços ecológicos como decomposição e regulação de recursos da floresta. Espécies da família Cerambycidae, amplamente distribuídas no mundo, perfuram o caule de árvores e madeira morta em áreas nativas e cultivadas. O objetivo é relatar a primeira planta hospedeira de Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) e uma nova planta hospedeira para Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) no bioma da Mata Atlântica brasileira. Três toras, com um metro de comprimento por 20 cm de diâmetro, foram cortadas de uma árvore sadia de Anadenanthera colubrina (Fabaceae) em outubro de 2013 e amarradas no sub-bosque a 1,5m de altura no Parque Estadual do Rio Doce, estado de Minas Gerais, Brasil. As toras, expostas na floresta, foram removidas, cada uma, após 40, 80 e 120 dias e armazenadas, individualmente, em caixas de papelão no "Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq)" no Parque Estadual do Rio Doce. Um total de 94 indivíduos de T. lineatocolle e 228 de T. megacephala emergiu das toras de A. colubrina. Esse é o primeiro registro de uma planta hospedeira para T. lineatocolle e o de uma nova planta hospedeira para T. megacephala.

Anadenanthera colubrina (Fabaceae) logs in the Atlantic Forest biome: first host plant for Thoracibidion lineatocolle (Col.: Cerambycidae) and a new host for Temnopis megacephala (Col.: Cerambycidae).

Wood-boring beetles develop in live trees and dead wood, performing ecological services such as decomposition and regulation of forest resources. Species of the Cerambycidae family, widely distributed in the world, bore into the trunks of trees and dead wood in native and cultivated areas. The objective is to report the first host plant for Thoracibidion lineatocolle (Thomson, 1865) (Coleoptera: Cerambycidae) and a new host plant for Temnopis megacephala (Germar, 1824) (Coleoptera: Cerambycidae) in the Brazilian Atlantic Forest biome. Three logs, with one-meter-long by 20 cm in diameter, were cut from the trunk of a healthy Anadenanthera colubrina (Fabaceae) tree in October 2013 and tied in the understory at 1.5m high in the Rio Doce State Park, Minas Gerais State, Brazil. The logs, exposed in the forest, were each removed after 40, 80 and 120 days and stored individually in a cardboard box in the "Laboratório de Campo do Projeto de Ecologia de Longa Duração (PELD-CNPq)" in the Rio Doce State Park. A total of 94 individuals of T. lineatocolle and 228 of T. megacephala emerged from the A. colubrina logs. This is the first report of a host plant for T. lineatocolle and a new host plant for T. megacephala.

Targeting myelin lipid metabolism as a potential therapeutic strategy in a model of CMT1A neuropathy.

In patients with Charcot-Marie-Tooth disease 1A (CMT1A), peripheral nerves display aberrant myelination during postnatal development, followed by slowly progressive demyelination and axonal loss during adult life. Here, we show that myelinating Schwann cells in a rat model of CMT1A exhibit a developmental defect that includes reduced transcription of genes required for myelin lipid biosynthesis. Consequently, lipid incorporation into myelin is reduced, leading to an overall distorted stoichiometry of myelin proteins and lipids with ultrastructural changes of the myelin sheath. Substitution of phosphatidylcholine and phosphatidylethanolamine in the diet is sufficient to overcome the myelination deficit of affected Schwann cells in vivo. This treatment rescues the number of myelinated axons in the peripheral nerves of the CMT rats and leads to a marked amelioration of neuropathic symptoms. We propose that lipid supplementation is an easily translatable potential therapeutic approach in CMT1A and possibly other dysmyelinating neuropathies.

A t-SNARE of the endocytic pathway must be activated for fusion.

The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast. We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p. In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion. Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell. Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

Sphingomyelin-enriched microdomains at the Golgi complex.

Sphingomyelin- and cholesterol-enriched microdomains can be isolated as detergent-resistant membranes from total cell extracts (total-DRM). It is generally believed that this total-DRM represents microdomains of the plasma membrane. Here we describe the purification and detailed characterization of microdomains from Golgi membranes. These Golgi-derived detergent-insoluble complexes (GICs) have a low buoyant density and are highly enriched in lipids, containing 25% of total Golgi phospholipids including 67% of Golgi-derived sphingomyelin, and 43% of Golgi-derived cholesterol. In contrast to total-DRM, GICs contain only 10 major proteins, present in nearly stoichiometric amounts, including the alpha- and beta-subunits of heterotrimeric G proteins, flotillin-1, caveolin, and subunits of the vacuolar ATPase. Morphological data show a brefeldin A-sensitive and temperature-sensitive localization to the Golgi complex. Strikingly, the stability of GICs does not depend on its membrane environment, because, after addition of brefeldin A to cells, GICs can be isolated from a fused Golgi-endoplasmic reticulum organelle. This indicates that GIC microdomains are not in a dynamic equilibrium with neighboring membrane proteins and lipids. After disruption of the microdomains by cholesterol extraction with cyclodextrin, a subcomplex of several GIC proteins including the B-subunit of the vacuolar ATPase, flotillin-1, caveolin, and p17 could still be isolated by immunoprecipitation. This indicates that several of the identified GIC proteins localize to the same microdomains and that the microdomain scaffold is not required for protein interactions between these GIC proteins but instead might modulate their affinity.

Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles.

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

Megavesicles implicated in the rapid transport of intracisternal aggregates across the Golgi stack.

Engineered protein aggregates ranging up to 400 nm in diameter were selectively deposited within the cis-most cisternae of the Golgi stack following a 15 degrees C block. These aggregates are much larger than the standard volume of Golgi vesicles, yet they are transported across the stack within 10 min after warming the cells to 20 degrees C. Serial sectioning reveals that during the peak of anterograde transport, about 20% of the aggregates were enclosed in topologically free "megavesicles" which appear to pinch off from the rims of the cisternae. These megavesicles can explain the rapid transport of aggregates without cisternal progression on this time scale.

Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors.

It has recently been reported that N-ethylmaleimide-sensitive fusion ATPase (NSF) can fuse protein-free liposomes containing substantial amounts of 1,2-dioleoylphosphatidylserine (DOPS) and 1, 2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999). The authors impart physiological significance to this observation and propose to re-conceptualize the general role of NSF in fusion processes. We can confirm that isolated NSF can fuse liposomes of the specified composition. However, this activity of NSF is resistant to inactivation by N-ethylmaleimide and does not depend on the presence of alpha-SNAP (soluble NSF-attachment protein). Moreover, under the same conditions, either alpha-SNAP, other proteins apparently unrelated to vesicular transport (glyceraldehyde-3-phosphate dehydrogenase or lactic dehydrogenase) or even 3 mM magnesium ions can also cause lipid mixing. In contrast, neither NSF nor the other proteins nor magnesium had any significant fusogenic activity with liposomes composed of a biologically occurring mixture of lipids. A straightforward explanation is that the lipid composition chosen as optimal for NSF favors non-specific fusion because it is physically unstable when formed into liposomes. A variety of minor perturbations could then trigger coalescence.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.

Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.

Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes.

Neuronal maturation is a gradual process; first axons and dendrites are established as distinct morphological entities; next the different intracellular organization of these processes occurs; and finally the specialized plasma membrane domains of these two compartments are formed. Only when this has been accomplished does proper neuronal function take place. In this work we present evidence that the correct distribution of a class of axonal membrane proteins requires a mechanism which involves formation of protein-lipid (sphingomyelin/cholesterol) detergent-insoluble complexes (DIGs). Using biochemistry and immunofluorescence microscopy we now show that in developing neurons the randomly distributed Thy-1 does not interact with lipids into DIGs (in fully developed neurons the formation of such complexes is essential for the correct axonal targeting of this protein). Using lipid mass spectrometry and thin layer chromatography we show that the DIG lipid missing in the developing neurons is sphingomyelin, but not cholesterol or glucosylceramide. Finally, by increasing the intracellular levels of sphingomyelin in the young neurons the formation of Thy-1/DIGs was induced and, consistent with a role in sorting, proper axonal distribution was facilitated. These results emphasize the role of sphingomyelin in axonal, and therefore, neuronal maturation.

Receptor-induced polymerization of coatomer.

Coatomer, the coat protein complex of COPI vesicles, is involved in the budding of these vesicles, but the underlying mechanism is unknown. Toward a better understanding of this process, the interaction between coatomer and the cytoplasmic domain of a major transmembrane protein of COPI vesicles, p23, was studied. Interaction of coatomer with this peptide domain results in a conformational change and polymerization of the complex in vitro. This changed conformation also is observed in vivo, i.e., on the surface of authentic, isolated COPI vesicles. An average of four peptides was found associated with one coatomer complex after polymerization. Based on these results, we propose a mechanism by which the induced conformational change of coatomer results in its polymerization, and thus drives formation of the bud on the Golgi membrane during biogenesis of a COPI vesicle.

Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry.

A mass spectrometric method for the quantification of free cholesterol in cells and subcellular membranes is presented. The method is based on a simple one-step chemical derivatization of cholesterol to cholesterol-3-sulfate by a sulfur trioxide-pyridine complex. Quantification is performed by nano-electrospray ionization tandem mass spectrometry (nanoESI-MS/MS) using a stable isotope labeled internal standard. The determination of free cholesterol is demonstrated in about 250 cells of a Chinese hamster ovary (CHO) cell line. With this method a molar ratio of free cholesterol to total phospholipids of 0.34 mol/mol in CHO cells was determined. In a subcellular membrane fraction enriched in Golgi membranes, a molar ratio of free cholesterol to total phospholipids of 0.57 mol/mol was determined. The method should be of value for quantification of other sterols as demonstrated for ergosterol and stigmasterol.

Protein and lipid sorting between the endoplasmic reticulum and the Golgi complex.

Vesicular traffic within the early secretory pathway is mediated by COPI- and COPII-coated vesicles. While COPII-coated vesicles appear to be involved exclusively in the export of secretory proteins and lipids from the endoplasmic reticulum (ER), COPI-coated vesicles seem to function in both anterograde and retrograde transport between the ER-Golgi intermediate compartment (IC) and the Golgi as well as in intra-Golgi transport. Here, we focus on (i) the mechanisms how these transport carriers are formed from a given donor membrane; and (ii) the possible mechanisms involved in sorting of proteins and lipids into such transport vesicles.

A putative heterotrimeric G protein inhibits the fusion of COPI-coated vesicles. Segregation of heterotrimeric G proteins from COPI-coated vesicles.

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.