RESUMO
Prevention of desiccation is a constant challenge for terrestrial organisms. Land insects have an extracellular coat, the cuticle, that plays a major role in protection against exaggerated water loss. Here, we report that the ABC transporter Oskyddad (Osy)-a human ABCA12 paralog-contributes to the waterproof barrier function of the cuticle in the fruit fly Drosophila melanogaster. We show that the reduction or elimination of Osy function provokes rapid desiccation. Osy is also involved in defining the inward barrier against xenobiotics penetration. Consistently, the amounts of cuticular hydrocarbons that are involved in cuticle impermeability decrease markedly when Osy activity is reduced. GFP-tagged Osy localises to membrane nano-protrusions within the cuticle, likely pore canals. This suggests that Osy is mediating the transport of cuticular hydrocarbons (CHC) through the pore canals to the cuticle surface. The envelope, which is the outermost cuticle layer constituting the main barrier, is unaffected in osy mutant larvae. This contrasts with the function of Snu, another ABC transporter needed for the construction of the cuticular inward and outward barriers, that nevertheless is implicated in CHC deposition. Hence, Osy and Snu have overlapping and independent roles to establish cuticular resistance against transpiration and xenobiotic penetration. The osy deficient phenotype parallels the phenotype of Harlequin ichthyosis caused by mutations in the human abca12 gene. Thus, it seems that the cellular and molecular mechanisms of lipid barrier assembly in the skin are conserved during evolution.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Drosophila/genética , Ictiose Lamelar/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Dessecação , Proteínas de Drosophila/metabolismo , Mutação com Perda de FunçãoRESUMO
Parvovirus B19 (B19V) possesses a linear single-stranded DNA genome of either positive or negative polarity. Due to intramolecular sequence homologies, either strand may theoretically be folded in several alternative ways. Viral DNA, when extracted from virions by several procedures, presents as linear single-stranded and/or linear double-stranded molecules, except when one particular commercial kit is used. This protocol yields DNA with an aberrant electrophoretic mobility in addition to linear double-stranded molecules, but never any single-stranded molecules. This peculiar kind of DNA was found in all plasma or serum samples tested and so we decided to analyse its secondary structure. In line with our results for one- and two-dimensional electrophoresis, mobility shift assays, DNA preparation by an in-house extraction method with moderate denaturing conditions, density gradient ultracentrifugation, DNA digestion experiments and competition hybridization assays, we conclude that (i) the unique internal portions of this distinctive single-stranded molecules are folded into tight tangles and (ii) the two terminal redundant regions are associated with each other, yielding non-covalently closed pseudo-circular molecules stabilized by a short (18 nucleotides) intramolecular stem, whereas the extreme 3'- and 5'-ends are folded back on themselves, forming a structure resembling a twin hairpin. The question arises as to whether this fairly unstable structure represents the encapsidated genome structure. The answer to this question remains quite relevant in terms of comprehending the initiation and end of B19V genome replication.
Assuntos
Proteínas do Capsídeo/genética , DNA Viral/genética , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Genoma Viral/genética , Humanos , Conformação de Ácido Nucleico , Replicação Viral/genéticaRESUMO
Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, we find that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu and the extracellular protein Snsl. In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. Our data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Pele/metabolismo , Animais , Drosophila , Matriz Extracelular/metabolismo , Metabolismo dos LipídeosRESUMO
Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is considered to be the main variant surface antigen (VSA) of Plasmodium falciparum and is mainly localized on electron-dense knobs in the membrane of the infected erythrocyte. Switches in PfEMP1 expression provide the basis for antigenic variation and are thought to be critical for parasite persistence during chronic infections. Recently, strain transcending anti-PfEMP1 immunity has been shown to develop early in life, challenging the role of PfEMP1 in antigenic variation during chronic infections. In this work we investigate how P. falciparum achieves persistence during a chronic asymptomatic infection. The infected individual (MOA) was parasitemic for 42 days and multilocus var gene genotyping showed persistence of the same parasite population throughout the infection. Parasites from the beginning of the infection were adapted to tissue culture and cloned by limiting dilution. Flow cytometry using convalescent serum detected a variable surface recognition signal on isogenic clonal parasites. Quantitative real-time PCR with a field isolate specific var gene primer set showed that the surface recognition signal was not correlated with transcription of individual var genes. Strain transcending anti-PfEMP1 immunity of the convalescent serum was demonstrated with CD36 selected and PfEMP1 knock-down NF54 clones. In contrast, knock-down of PfEMP1 did not have an effect on the antibody recognition signal in MOA clones. Trypsinisation of the membrane surface proteins abolished the surface recognition signal and immune electron microscopy revealed that antibodies from the convalescent serum bound to membrane areas without knobs and with knobs. Together the data indicate that PfEMP1 is not the main variable surface antigen during a chronic infection and suggest a role for trypsin sensitive non-PfEMP1 VSAs for parasite persistence in chronic infections.
Assuntos
Antígenos de Protozoários/genética , Malária Falciparum/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Variação Antigênica/genética , Variação Antigênica/imunologia , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Eritrócitos/parasitologia , Citometria de Fluxo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Genótipo , Humanos , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Malária Falciparum/patologia , Plasmodium falciparum/imunologia , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/biossínteseRESUMO
The fusion of founder cells and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila Characteristic events of myoblast fusion include the recognition and adhesion of myoblasts, and the formation of branched F-actin by the Arp2/3 complex at the site of cell-cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette (a homolog of Nap1 and Hem-2, also known as NCKAP1 and NCKAP1L, respectively), a member of the regulatory complex formed by Scar or WAVE proteins (represented by the single protein, Scar, in flies). kette mutants form finger-like protrusions, but the electron-dense plaques are extended. Here, we show that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar complex revealed that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar and WASp in FCMs.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Caderinas/metabolismo , Fusão Celular , Modelos Biológicos , Mutação/genética , Mioblastos/ultraestrutura , Fenótipo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
An early event in Alzheimer's disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-ß peptide (Aß), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aß seeds remain rather elusive. Active Aß seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aß seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aß seeds on mitochondrial or associated membranes efficiently induced Aß aggregation in vitro and seed ß-amyloidosis in vivo. Aß seeds at intracellular membranes may contribute to the spreading of Aß aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aß.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Mitocôndrias/patologia , Membranas Mitocondriais/patologia , Placa Amiloide/patologia , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: Antigenic variation in Plasmodium falciparum is mediated by the multicopy var gene family. Each parasite possesses about 60 var genes, and switching between active var loci results in antigenic variation. In the current study, the effect of mosquito and host passage on in vitro var gene transcription was investigated. METHODS: Thirty malaria-naive individuals were inoculated by intradermal or intravenous injection with cryopreserved, isogenic NF54 P. falciparum sporozoites (PfSPZ) generated from 1 premosquito culture. Microscopic parasitemia developed in 22 individuals, and 21 in vitro cultures were established. The var gene transcript levels were determined in early and late postpatient cultures and in the premosquito culture. RESULTS: At the early time point, all cultures preferentially transcribed 8 subtelomeric var genes. Intradermal infections had higher var gene transcript levels than intravenous infections and a significantly longer intrahost replication time (P = .03). At the late time point, 9 subtelomeric and 8 central var genes were transcribed at the same levels in almost all cultures. Premosquito and late postpatient cultures transcribed the same subtelomeric and central var genes, except for var2csa CONCLUSIONS: The duration of intrahost replication influences in vitro var gene transcript patterns. Differences between premosquito and postpatient cultures decrease with prolonged in vitro growth.
Assuntos
Variação Antigênica , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/biossíntese , Esporozoítos/imunologia , Transcrição Gênica , Adolescente , Adulto , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Adulto JovemRESUMO
Understanding the mechanisms of how bacteria become tolerant toward antibiotics during clinical therapy is a very important object. In a previous study, we showed that increased daptomycin (DAP) tolerance of Staphylococcus aureus was due to a point mutation in pitA (inorganic phosphate transporter) that led to intracellular accumulation of both inorganic phosphate (Pi) and polyphosphate (polyP). DAP tolerance in the pitA6 mutant differs from classical resistance mechanisms since there is no increase in the MIC. In this follow-up study, we demonstrate that DAP tolerance in the pitA6 mutant is not triggered by the accumulation of polyP. Transcriptome analysis revealed that 234 genes were at least 2.0-fold differentially expressed in the mutant. Particularly, genes involved in protein biosynthesis, carbohydrate and lipid metabolism, and replication and maintenance of DNA were downregulated. However, the most important change was the upregulation of the dlt operon, which is induced by the accumulation of intracellular Pi The GraXRS system, known as an activator of the dlt operon (d-alanylation of teichoic acids) and of the mprF gene (multiple peptide resistance factor), is not involved in DAP tolerance of the pitA6 mutant. In conclusion, DAP tolerance of the pitA6 mutant is due to an upregulation of the dlt operon, triggered directly or indirectly by the accumulation of Pi.
Assuntos
Proteínas de Bactérias/metabolismo , Daptomicina/farmacologia , Óperon/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Testes de Sensibilidade Microbiana , Mutação Puntual/genética , Staphylococcus aureus/genética , Ácidos Teicoicos/metabolismo , Transcriptoma/genéticaRESUMO
BACKGROUND: In the visual system of most binocular vertebrates, the axons of retinal ganglion cells (RGCs) diverge at the diencephalic midline and extend to targets on both ipsi- and contralateral sides of the brain. While a molecular mechanism explaining ipsilateral guidance decisions has been characterized, less is known of how RGC axons cross the midline. RESULTS: Here, we took advantage of the zebrafish, in which all RGC axons project contralaterally at the optic chiasm, to characterize Islr2 as an RGC receptor required for complete retinal axon midline crossing. We used a systematic extracellular protein-protein interaction screening assay to identify two Vasorin paralogs, Vasna and Vasnb, as specific Islr2 ligands. Antibodies against Vasna and Vasnb reveal cellular populations surrounding the retinal axon pathway, suggesting the involvement of these proteins in guidance decisions made by axons of the optic nerve. Specifically, Vasnb marks the membranes of a cellular barricade located anteriorly to the optic chiasm, a structure termed the "glial knot" in higher vertebrates. Loss of function mutations in either vasorin paralog, individually or combined, however, do not exhibit an overt retinal axon projection phenotype, suggesting that additional midline factors, acting either independently or redundantly, compensate for their loss. Analysis of Islr2 knockout mice supports a scenario in which Islr2 controls the coherence of RGC axons through the ventral midline and optic tract. CONCLUSIONS: Although stereotypic guidance of RGC axons at the vertebrate optic chiasm is controlled by multiple, redundant mechanisms, and despite the differences in ventral diencephalic tissue architecture, we identify a novel role for the LRR receptor Islr2 in ensuring proper axon navigation at the optic chiasm of both zebrafish and mouse.
Assuntos
Axônios/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Quiasma Óptico/embriologia , Retina/embriologia , Animais , Padronização Corporal/fisiologia , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Vias Visuais/embriologia , Peixe-ZebraRESUMO
In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Interfase , Carioferinas/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.
Assuntos
Adenilil Ciclases/fisiologia , Melanóforos/metabolismo , Poecilia/embriologia , Animais , Padronização Corporal , Diferenciação Celular , Códon de Terminação , Cruzamentos Genéticos , Feminino , Biblioteca Gênica , Genótipo , Masculino , Melanossomas/metabolismo , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutação , Fenótipo , Filogenia , Pigmentação , Reação em Cadeia da Polimerase , Locos de Características Quantitativas , Análise de Sequência de DNA , Diferenciação Sexual , Especificidade da EspécieRESUMO
Excretion of cytoplasmic proteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG-binding protein, which is secreted via the Sec-pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan-binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non-excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.
Assuntos
Membrana Celular/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/metabolismo , Transporte Biológico , Parede Celular/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Fosfopiruvato Hidratase/metabolismo , Ligação ProteicaRESUMO
The contribution of myeloid cells to tumour microenvironments is a decisive factor in cancer progression. Tumour-associated macrophages (TAMs) mediate tumour invasion and angiogenesis through matrix remodelling, immune modulation and release of pro-angiogenic cytokines. Nothing is known about how pathogenic bacteria affect myeloid cells in these processes. Here we show that Bartonella henselae, a bacterial pathogen causing vasculoproliferative diseases (bacillary angiomatosis), reprogrammes human myeloid angiogenic cells (MACs), a pro-angiogenic subset of circulating progenitor cells, towards a TAM-like phenotype with increased pro-angiogenic capacity. B. henselae infection resulted in inhibition of cell death, activation of angiogenic cellular programmes and induction of M2 macrophage polarization. MACs infected with B. henselae incorporated into endothelial sprouts and increased angiogenic growth. Infected MACs developed a vascular mimicry phenotype in vitro, and expression of B. henselae adhesin A was essential in inducing these angiogenic effects. Secretome analysis revealed that increased pro-angiogenic activities were associated with the creation of a tumour-like microenvironment dominated by angiogenic inflammatory cytokines and matrix remodelling compounds. Our results demonstrate that manipulation of myeloid cells by pathogenic bacteria can contribute to microenvironmental regulation of pathological tissue growth and suggest parallels underlying both bacterial infections and cancer.
Assuntos
Bartonella henselae/fisiologia , Interações Hospedeiro-Patógeno , Células Progenitoras Mieloides/fisiologia , Neovascularização Patológica , Diferenciação Celular , Células Endoteliais/microbiologia , Células Endoteliais/fisiologia , Humanos , Macrófagos/microbiologia , Macrófagos/fisiologiaRESUMO
The fitness of male guppies (Poecilia reticulata) highly depends on the size and number of their black, blue, and orange ornaments. Recently, progress has been made regarding the genetic mechanisms underlying male guppy pigment pattern formation, but we still know little about the pigment cell organization within these ornaments. Here, we investigate the pigment cell distribution within the black, blue, and orange trunk spots and selected fin color patterns of guppy males from three genetically divergent strains using transmission electron microscopy. We identified three types of pigment cells and found that at least two of these contribute to each color trait. Further, two pigment cell layers, one in the dermis and the other in the hypodermis, contribute to each trunk spot. The pigment cell organization within the black and orange trunk spots was similar between strains. The presence of iridophores in each of the investigated color traits is consistent with a key role for this pigment cell type in guppy color pattern formation.
Assuntos
Cromatóforos/fisiologia , Cor , Poecilia/fisiologia , Pigmentação da Pele/fisiologia , Nadadeiras de Animais/citologia , Nadadeiras de Animais/fisiologia , Nadadeiras de Animais/ultraestrutura , Animais , Cromatóforos/citologia , Cromatóforos/ultraestrutura , Células Epidérmicas , Epiderme/fisiologia , Epiderme/ultraestrutura , Masculino , Melanóforos/citologia , Melanóforos/fisiologia , Melanóforos/ultraestrutura , Microscopia Eletrônica de Transmissão , Fenótipo , Poecilia/classificação , Poecilia/genéticaRESUMO
BACKGROUND: Chitin produced by membrane-inserted chitin synthases is an important constituent of the arthropod cuticle and midgut peritrophic matrix. Chitin synthesis inhibitors are common insecticides in pest control. As the target of sulfonylurea-derived insecticides such as diflubenzuron, the ABC transporter sulfonylurea receptor (Sur) has been postulated to be an essential cofactor of chitin synthesis. However, direct evidence for this assumption is missing. RESULTS: Here, a study has been made of the phenotype of Drosophila melanogaster larvae suffering completely eliminated Sur function. Taken together, it is found that cuticle architecture is normal and chitin amounts are not diminished in the cuticle of these animals, indicating that Sur is dispensable for chitin synthesis. CONCLUSION: The data obtained suggest that there must exist another sulfonylurea-sensitive ABC transporter that either instead of Sur is the true sulfonylurea-sensitive transporter involved in chitin synthesis or is able to substitute Sur function during cuticle formation. Identification and characterisation of this factor is pivotal for understanding the mode of action of sulfonylurea as insecticide.
Assuntos
Quitina/biossíntese , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Receptores de Sulfonilureias/metabolismo , Animais , Diflubenzuron/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Receptores de Sulfonilureias/genéticaRESUMO
Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous neurodegenerative disorders characterized by spastic weakness of the lower extremities. We have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A. KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP-like pathological changes such as axonal swellings, altered structure and function of synapses, behavioral deficits, and increased mortality.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Cinesinas/genética , Paraplegia Espástica Hereditária/genética , Animais , Transporte Axonal/genética , Transporte Axonal/fisiologia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Camundongos , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Sinapses/genética , Sinapses/patologiaRESUMO
Animals construct a layered skin to prevent dehydration and pathogen entrance. The barrier function of the skin relies on the extensive cross-linking of specialised components. In insects, for instance, epidermal cells produce an apical extracellular cuticle that consists of a network of proteins, chitin and lipids. We have identified mutations in the Drosophila gene coding for the δ-aminolevulinate synthase (Alas) that cause massive water loss. The cuticle of alas mutant larvae detaches from the epidermis and its basal region is frayed suggesting that an Alas dependent pathway is needed to organise the contact between the cuticle and the epidermis and anchor the cuticle to the apical surface of epidermal cells. Concomitantly, reduction of Alas function results in weakening of the extracellular dityrosines network in the cuticle, whereas glutamyl-lysine isopeptide bonds are not affected. The lateral septate junctions of epidermal cells that serve as a paracellular plug are intact, as well. Taken together, we hypothesise that Alas activity, which initiates heme biosynthesis in the mitochondrion, is needed for the formation of a dityrosine-based barrier that confers resistance to the internal hydrostatic pressure protecting both the cuticle from transcellular infiltration of body fluid and the animal from dehydration. We conclude that at least two modules--an apical protein-chitin lattice and the lateral septate junctions, act in parallel to ensure Drosophila skin impermeability.
Assuntos
5-Aminolevulinato Sintetase/genética , Drosophila/genética , Epiderme/metabolismo , Larva/genética , Água/metabolismo , 5-Aminolevulinato Sintetase/metabolismo , Animais , Quitina/metabolismo , Epiderme/fisiologia , Heme/biossíntese , Heme/metabolismo , Larva/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Nuclear pore complexes (NPCs) are large macromolecular assemblies that control all transport across the nuclear envelope. They are formed by about 30 nucleoporins (Nups), which can be roughly categorized into those forming the structural skeleton of the pore and those creating the central channel and thus providing the transport and gating properties of the NPC. Here we show that the conserved nucleoporin Nup93 is essential for NPC assembly and connects both portions of the NPC. Although the C-terminal domain of the protein is necessary and sufficient for the assembly of a minimal structural backbone, full-length Nup93 is required for the additional recruitment of the Nup62 complex and the establishment of transport-competent NPCs.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Células Cultivadas , Glicoproteínas de Membrana/metabolismo , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína , Transporte Proteico , Xenopus , Proteínas de Xenopus/genéticaRESUMO
Conjugation is a major route of horizontal gene transfer, the driving force in the evolution of bacterial genomes. Antibiotic producing soil bacteria of the genus Streptomyces transfer DNA in a unique process involving a single plasmid-encoded protein TraB and a double-stranded DNA molecule. However, the molecular function of TraB in directing DNA transfer from a donor into a recipient cell is unknown. Here, we show that TraB constitutes a novel conjugation system that is clearly distinguished from DNA transfer by a type IV secretion system. We demonstrate that TraB specifically recognizes and binds to repeated 8 bp motifs on the conjugative plasmid. The specific DNA recognition is mediated by helix α3 of the C-terminal winged-helix-turn-helix domain of TraB. We show that TraB assembles to a hexameric ring structure with a central â¼3.1 nm channel and forms pores in lipid bilayers. Structure, sequence similarity and DNA binding characteristics of TraB indicate that TraB is derived from an FtsK-like ancestor protein, suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells.
Assuntos
Proteínas de Bactérias/metabolismo , Conjugação Genética , Transferência Genética Horizontal , Plasmídeos , Streptomyces coelicolor/genética , Sítios de Ligação , Segregação de Cromossomos , Cromossomos Bacterianos/genética , DNA/metabolismo , DNA Bacteriano/metabolismo , Evolução Molecular , Filogenia , Ligação Proteica , Multimerização Proteica , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
In the three-dimensional (3D) structure determination of macromolecules, cryo electron microscopy (cryo-EM) is an important method for obtaining micrographs of unstained specimens for the single-particle reconstruction approach. For cryo-EM, proteins are fixed in a frozen hydrated state by quick-freezing in a thin water layer on a holey carbon film. Cryo-EM of detergent-solubilized membrane proteins is hindered by the fact that detergents reduce the surface tension of water, so that it is difficult to control the ice thickness and the distribution of protein. Amphipols are a new class of amphipathic polymers designed to handle membrane proteins in aqueous solutions under particularly mild conditions. Amphipol A8-35 stabilizes NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa and keeps it water-soluble in the absence of free detergent. Electron microscope images of quick-frozen complex I/A8-35 samples were used for computer-based single-particle averaging and 3D reconstruction, and the reconstruction of unstained frozen-hydrated particles compared with previous detergent-based reconstructions. The potential of amphipols for cryo-EM is discussed.