Uncovering the molecular mechanisms of lignocellulose digestion in shipworms.

Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source in the biosphere. Turnover of woody biomass is a critical component of the global carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil fuels to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon cycling by processing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal's gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics, and microscopy, we show that the digestive proteome of the shipworm Lyrodus pedicellatus is mostly composed of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that functions in the hydrolysis of ß-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass.

The anterior esophageal region of Schistosoma japonicum is a secretory organ.

BACKGROUND: The esophagus of blood-feeding schistosomes has been largely neglected although its posterior portion was designated as a gland decades ago. However, we recently showed it plays a pivotal role in blood processing. It is clearly demarcated into anterior and posterior compartments, both surrounded by a mass of cell bodies. Feeding movies revealed that erythrocytes accumulate in the anterior compartment before entering the posterior, indicating that a distinct process is executed there. We therefore investigated ultrastructural aspects and possible functions of the anterior region. METHODS: The heads of adult Schistosoma japonicum were detached and prepared for both transmission and scanning electron microscopy to define the detailed ultrastructure of the anterior esophagus. Cryosections of heads were also prepared for immunocytochemistry and confocal microscopy to define the pattern of intrinsic host antibody binding in the anterior esophageal lining. RESULTS: The anterior syncytial lining of the esophagus is highly extended by long, thin corrugations of cytoplasm projecting towards the lumen. Strikingly in the male worm, the tips of the corrugations are further expanded by numerous threads of cytoplasm, producing a spaghetti-like appearance in the central lumen. Flattened, pitted cytoplasmic plates are interspersed in the tangled mass of threads. Abundant, morphologically distinct light vesicles of varied size and contents are manufactured in the cell bodies, from where they traffic through cytoplasmic connections to the corrugations and out to the tips. Clusters of vesicles accumulate in expanded tips in males, together with occasional mitochondria whilst females have more mitochondria but fewer vesicles. The membranous contents of light vesicles are secreted mainly from the tips, but also from the sides of the corrugations. They coat the surfaces and then form organised self-adherent membrane figures when shed into the lumen. Host antibody binds strongly in a characteristic pattern to the anterior esophageal lining indicating that the secretions are highly immunogenic. CONCLUSIONS: We suggest that the anterior esophageal region is an independent secretory organ. The contents of light vesicles are released into the esophageal lumen via the tips of corrugation to interact with incoming blood. Our immediate task is to establish their composition and role in blood processing.

The Leishmania major†BBSome subunit BBS1 is essential for parasite virulence in the mammalian host.

Bardet-Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity.

Dopaminergic expression of the Parkinsonian gene LRRK2-G2019S leads to non-autonomous visual neurodegeneration, accelerated by increased neural demands for energy.

Parkinson's disease (PD) is associated with loss of dopaminergic signalling, and affects not just movement, but also vision. As both mammalian and fly visual systems contain dopaminergic neurons, we investigated the effect of LRRK2 mutations (the most common cause of inherited PD) on Drosophila electroretinograms (ERGs). We reveal progressive loss of photoreceptor function in flies expressing LRRK2-G2019S in dopaminergic neurons. The photoreceptors showed elevated autophagy, apoptosis and mitochondrial disorganization. Head sections confirmed extensive neurodegeneration throughout the visual system, including regions not directly innervated by dopaminergic neurons. Other PD-related mutations did not affect photoreceptor function, and no loss of vision was seen with kinase-dead transgenics. Manipulations of the level of Drosophila dLRRK suggest G2019S is acting as a gain-of-function, rather than dominant negative mutation. Increasing activity of the visual system, or of just the dopaminergic neurons, accelerated the G2019S-induced deterioration of vision. The fly visual system provides an excellent, tractable model of a non-autonomous deficit reminiscent of that seen in PD, and suggests that increased energy demand may contribute to the mechanism by which LRRK2-G2019S causes neurodegeneration.

A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei.

The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet-Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.

The orthologue of Sjögren's syndrome nuclear autoantigen 1 (SSNA1) in Trypanosoma brucei is an immunogenic self-assembling molecule.

Primary Sjögren's Syndrome (PSS) is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14) is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13) and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.

Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion.

Proteins of the Leishmania hydrophilic acylated surface protein B (HASPB) family are only expressed in infective parasites (both extra- and intracellular stages) and, together with the peripheral membrane protein SHERP (small hydrophilic endoplasmic reticulum-associated protein), are essential for parasite differentiation (metacyclogenesis) in the sand fly vector. HASPB is a 'non-classically' secreted protein, requiring N-terminal acylation for trafficking to and exposure on the plasma membrane. Here, we use live cell imaging methods to further explore this pathway to the membrane and flagellum. Unlike HASPB trafficking in transfected mammalian cells, we find no evidence for a phosphorylation-regulated recycling pathway in metacyclic parasites. Once at the plasma membrane, HASPB18-GFP (green fluorescent protein) can undergo bidirectional movement within the inner leaflet of the membrane and on the flagellum. Transfer of fluorescent protein between the flagellum and the plasma membrane is compromised, however, suggesting the presence of a diffusion barrier at the base of the Leishmania flagellum. Full-length HASPB is released from the metacyclic parasite surface on to macrophages during phagocytosis but while expression is maintained in intracellular amastigotes, HASPB cannot be detected on the external surface in these cells. Thus HASPB may be a dual function protein that is shed by the infective metacyclic but retained internally once Leishmania are taken up by macrophages.

Flagella and pili are both necessary for efficient attachment of Methanococcus maripaludis to surfaces.

Methanococcus maripaludis has two surface appendages, namely flagella and pili. Flagella have been shown to be required for swimming, but no specific role has been assigned as yet to pili. In this report, wild-type M. maripaludis cells are compared with mutants lacking either pili or flagella or both surface appendages in their ability to attach to a variety of surfaces including nickel, gold and molybdenum grids as well as glass, silicon and mica. Wild-type cells attached to varying degrees to all surfaces tested, except mica, via their flagella as observed by scanning electron microscopy. Large cables of flagella were found to leave the cell and to be unwound on the surface. In addition, such cables were often found to connect cells. In contrast, cells lacking either flagella or pili or both surface appendages were unable to attach efficiently to any surfaces. This indicates a second role for flagella in addition to swimming in M. maripaludis, as well as a first role for pili in this organism, namely in surface attachment.

The small GTPase ARL2 is required for cytokinesis in Trypanosoma brucei.

The Arf-like (Arl) small GTPases have a diverse range of functions in the eukaryotic cell. Metazoan Arl2 acts as a regulator of microtubule biogenesis, binding to the tubulin-specific chaperone cofactor D. Arl2 also has a mitochondrial function through its interactions with BART and ANT-1, the only member of the Ras superfamily to be found in this organelle to date. In the present study, we describe characterization of the Arl2 orthologue in the protozoan parasite Trypanosoma brucei. Modulation of TbARL2 expression in bloodstream form parasites by RNA interference (RNAi) causes inhibition of cleavage furrow formation, resulting in a severe defect in cytokinesis and the accumulation of multinucleated cells. RNAi of TbARL2 also results in loss of acetylated alpha-tubulin but not of total -tubulin from cellular microtubules. While overexpression of TbARL2(myc) also leads to a defect in cytokinesis, an excess of untagged protein has no effect on cell division, demonstrating the importance of the extreme C-terminus in correct function. TbARL2 overexpressing cells (either myc-tagged or untagged) have an increase in acetylated -tubulin. Our data indicate that Arl2 has a fundamentally conserved role in trypanosome microtubule biogenesis that correlates with -tubulin acetylation.

Drosophila indirect flight muscle specific Act88F actin mutants as a model system for studying congenital myopathies of the human ACTA1 skeletal muscle actin gene.

Most human ACTA1 skeletal actin gene mutations cause dominant, congenital myopathies often with severely reduced muscle function and neonatal mortality. High sequence conservation of actin means many mutated ACTA1 residues are identical to those in the DrosophilaAct88F, an indirect flight muscle specific sarcomeric actin. Four known Act88F mutations occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for similar muscle phenotypes. Mutant homozygotes show phenotypes ranging from a lack of myofibrils to almost normal sarcomeres at eclosion. Aberrant Z-disc-like structures and serial Z-disc arrays, 'zebra bodies', are observed in homozygotes and heterozygotes of all four Act88F mutants. These electron-dense structures show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. We conclude that the Drosophila indirect flight muscles provide a good model system for studying ACTA1 mutations.

TbARF1 influences lysosomal function but not endocytosis in procyclic stage Trypanosoma brucei.

The ADP ribosylation factors (Arfs) are a highly conserved subfamily of the Ras small GTPases with crucial roles in vesicle budding and membrane trafficking. Unlike in other eukaryotes, the orthologue of Arf1 in the host bloodstream form of Trypanosoma brucei is essential for the maintenance of endocytosis. In contrast, as shown in this study, knockdown of TbARF1 by RNA interference has no effect on fluid-phase endocytosis in the insect stage of the parasite. The protein remains essential for the viability of these procyclic cells but the major effect of TbARF1-depletion is enlargement of the lysosome. Our data indicate that protein trafficking and lysosomal function are differentially regulated by multiple factors, including TbARF1, during progression through the T. brucei lifecycle.

Aberrant splicing of an alternative exon in the Drosophila troponin-T gene affects flight muscle development.

During myofibrillogenesis, many muscle structural proteins assemble to form the highly ordered contractile sarcomere. Mutations in these proteins can lead to dysfunctional muscle and various myopathies. We have analyzed the Drosophila melanogaster troponin T (TnT) up1 mutant that specifically affects the indirect flight muscles (IFM) to explore troponin function during myofibrillogenesis. The up1 muscles lack normal sarcomeres and contain "zebra bodies," a phenotypic feature of human nemaline myopathies. We show that the up(1) mutation causes defective splicing of a newly identified alternative TnT exon (10a) that encodes part of the TnT C terminus. This exon is used to generate a TnT isoform specific to the IFM and jump muscles, which during IFM development replaces the exon 10b isoform. Functional differences between the 10a and 10b TnT isoforms may be due to different potential phosphorylation sites, none of which correspond to known phosphorylation sites in human cardiac TnT. The absence of TnT mRNA in up1 IFM reduces mRNA levels of an IFM-specific troponin I (TnI) isoform, but not actin, tropomyosin, or troponin C, suggesting a mechanism controlling expression of TnT and TnI genes may exist that must be examined in the context of human myopathies caused by mutations of these thin filament proteins.

Trypanosoma brucei ARF1 plays a central role in endocytosis and golgi-lysosome trafficking.

The ADP ribosylation factor (Arf)1 orthologue in the divergent eukaryote Trypanosoma brucei (Tb) shares characteristics with both Arf1 and Arf6 and has a vital role in intracellular protein trafficking. TbARF1 is Golgi localized in trypanosomes but associates with the plasma membrane when expressed in human cells. Depletion of TbARF1 by RNA interference causes a major decrease in endocytosis, which correlates with Rab5 dissociation from early endosomes. Although the Golgi remains intact, parasites display enlarged flagellar pockets and intracellular flagella. An increase in active GTP-bound TbARF1 in bloodstream parasites is rapidly lethal, correlating with a defect in Golgi-to-lysosome transport. We conclude that the essential Golgi-localizing T. brucei ARF1 has a primary role in the maintenance of both post-Golgi transport and endocytosis and that it is significantly divergent from other characterized ARFs.

Direct measurement of the performance of the Drosophila jump muscle in whole flies.

We have developed a novel apparatus, an ergometer, to simultaneously measure the horizontal and vertical components of the work done during takeoff by the fruitfly, Drosophila. We confirm the anatomical prediction that all the work comes from the middle (mesothoracic) legs. With all six legs on the ergometer platform, displacement is directed roughly 45 degrees forwards or backwards. Both directions are equally likely. This provides for a random, rapid horizontal component to the escape behaviour for flies. When the thoracic stiffness is reduced (due to a mutation in which the indirect flight muscles (IFM) do not form myofibrils), jump output is increased. We conclude that the jump muscle, the tergal depressor of trochanter (TDT), which lacks direct muscle antagonists, performs work during the jump against thoracic stiffness. Both cuticle and IFM contribute to the thoracic stiffness as the TDT still produces repeated contractions in the absence of the IFM. Degeneration of the TDT due to mutants in three sarcomeric proteins results in reduction of the jump output. In one of these, the myosin heavy chain mutant, Mhc5, we show that degeneration occurs with age. The anatomical characteristics of Drosophila mean that we are recording, for the first time in the intact fly, the output of a single muscle that has high homology to vertebrate skeletal muscle. Developing an ergometer for Drosophila offers novel opportunities to assess the functional consequences of mutations in muscle proteins, synaptic physiology, neuromuscular development and aging.

Troponin I is required for myofibrillogenesis and sarcomere formation in Drosophila flight muscle.

Myofibrillar proteins assemble to form the highly ordered repetitive contractile structural unit known as a sarcomere. Studies of myogenesis in vertebrate cell culture and embryonic developmental systems have identified some of the processes involved during sarcomere formation. However, isoform changes during vertebrate muscle development and a lack of mutants have made it difficult to determine how these proteins assemble to form sarcomeres. The indirect flight muscles (IFMs) of Drosophila provide a unique genetic system with which to study myofibrillogenesis in vivo. We show in this paper that neither sarcomeric myosin nor actin are required for myoblast fusion or the subsequent morphogenesis of muscle fibres, i.e. fibre morphogenesis does not depend on myofibrillogenesis. However, fibre formation and myofibrillogenesis are very sensitive to the interactions between the sarcomeric proteins. A troponin I (TnI) mutation, hdp(3), leads to an absence of TnI in the IFMs and tergal depressor of trochanter (TDT) muscles due to a transcript-splicing defect. Sarcomeres do not form and the muscles degenerate. TnI is part of the thin filament troponin complex which regulates muscle contraction. The effects of the hdp(3) mutation are probably caused by unregulated acto-myosin interactions between the thin and thick filaments as they assemble. We have tested this proposal by using a transgenic myosin construct to remove the force-producing myosin heads. The defects in sarcomeric organisation and fibre degeneration in hdp(3) IFMs are suppressed, although not completely, indicating the need for inhibition of muscle contraction during muscle development. We show that mRNA and translated protein products of all the major thin filament proteins are reduced in hdp(3) muscles and discuss how this and previous studies of thin filament protein mutants indicate a common co-ordinated control mechanism that may be the primary cause of the muscle defects.

Microfilament and microtubule organization and dynamics in process extension by central glia-4 oligodendrocytes: evidence for a microtubule organizing center.

Microfilaments in freshly adhering CG-4 cells and differentiated CG-4 oligodendrocytes are concentrated at the tips and edges of rapidly forming processes while microtubules are concentrated in new processes and extend from a concentrated spot of alpha-tubulin staining in the cell body to the cell periphery. In motile bipolar CG-4 cells, microfilaments are heavily concentrated at the flattened end of one process and along the rim of processes and the cell body: microtubules are concentrated along main processes and splay out into process tips and the cell body. In differentiated CG-4 oligodendrocytes, microfilaments are concentrated at the many process tips, in filopodia and in fine processes, but are not obvious in main processes where separate bundles of microtubules, which diverge at process branch points, are concentrated. gamma-tubulin, involved in microtubule nucleation, is concentrated at a small discrete area in the cell body, indicative of a microtubule organizing center. Polymerization of both actin and tubulin is required for initial process elaboration. Depolymerization of microtubules, but not of microfilaments, causes complete retraction of bipolar CG-4 cell processes. This process retraction does not occur if microfilaments are depolymerized first, indicating that process extension/retraction in motile bipolar CG-4 cells may occur by a balance of motor protein-driven forces as suggested for growth cone motility. Cytoskeleton organization in CG-4 cells is very similar to that reported for oligodendrocytes. CG-4 cells are thus a useful model for investigating the signals and mechanisms regulating oligodendrocyte process dynamics.