RESUMO
The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.
Assuntos
Leishmania mexicana , Leishmania , Parasitos , Psychodidae , Animais , Leishmania mexicana/genética , Ciclo Celular , Divisão Celular , Psychodidae/parasitologia , MamíferosRESUMO
The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm.
Assuntos
Eimeria tenella , Parasitos , Animais , Eimeria tenella/metabolismo , Eimeria tenella/ultraestrutura , Tomografia com Microscopia Eletrônica , Organelas/metabolismo , Parasitos/metabolismo , Proteínas de Protozoários/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1-G2 tether and S-M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement-loss of orthogonal orientation and ability to separate and move apart-are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells.
Assuntos
Corpos Basais/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/citologia , Citoesqueleto/metabolismo , DNA de Cinetoplasto/genética , Flagelos/metabolismo , Proteínas de Protozoários/genética , Interferência de RNA , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismoRESUMO
The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.
Assuntos
Citoesqueleto/metabolismo , Flagelos/fisiologia , Interações Hospedeiro-Parasita , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Morfogênese , Psychodidae/parasitologia , Animais , Membrana Celular , Citocinese , Feminino , Flagelos/ultraestrutura , Leishmania/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Leishmania kinetoplastid parasites infect millions of people worldwide and have a distinct cellular architecture depending on location in the host or vector and specific pathogenicity functions. An invagination of the cell body membrane at the base of the flagellum, the flagellar pocket (FP), is an iconic kinetoplastid feature, and is central to processes that are critical for Leishmania pathogenicity. The Leishmania FP has a bulbous region posterior to the FP collar and a distal neck region where the FP membrane surrounds the flagellum more closely. The flagellum is attached to one side of the FP neck by the short flagellum attachment zone (FAZ). We addressed whether targeting the FAZ affects FP shape and its function as a platform for host-parasite interactions. Deletion of the FAZ protein, FAZ5, clearly altered FP architecture and had a modest effect in endocytosis but did not compromise cell proliferation in culture. However, FAZ5 deletion had a dramatic impact in vivo: Mutants were unable to develop late-stage infections in sand flies, and parasite burdens in mice were reduced by >97%. Our work demonstrates the importance of the FAZ for FP function and architecture. Moreover, we show that deletion of a single FAZ protein can have a large impact on parasite development and pathogenicity.
Assuntos
Cílios/fisiologia , Flagelos/fisiologia , Leishmania/fisiologia , Leishmania/patogenicidade , Psychodidae/parasitologia , Animais , Membrana Celular/metabolismo , Cílios/genética , Cílios/ultraestrutura , Endocitose , Flagelos/genética , Flagelos/ultraestrutura , Deleção de Genes , Interações Hospedeiro-Parasita , Junções Intercelulares , Leishmania/genética , Leishmania/ultraestrutura , Camundongos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Virulência/genéticaRESUMO
The 9 + 2 axoneme structure of the motile flagellum/cilium is an iconic, apparently symmetrical cellular structure. Recently, asymmetries along the length of motile flagella have been identified in a number of organisms, typically in the inner and outer dynein arms. Flagellum-beat waveforms are adapted for different functions. They may start either near the flagellar tip or near its base and may be symmetrical or asymmetrical. We hypothesized that proximal/distal asymmetry in the molecular composition of the axoneme may control the site of waveform initiation and the direction of waveform propagation. The unicellular eukaryotic pathogens Trypanosoma brucei and Leishmania mexicana often switch between tip-to-base and base-to-tip waveforms, making them ideal for analysis of this phenomenon. We show here that the proximal and distal portions of the flagellum contain distinct outer dynein arm docking-complex heterodimers. This proximal/distal asymmetry is produced and maintained through growth by a concentration gradient of the proximal docking complex, generated by intraflagellar transport. Furthermore, this asymmetry is involved in regulating whether a tip-to-base or base-to-tip beat occurs, which is linked to a calcium-dependent switch. Our data show that the mechanism for generating proximal/distal flagellar asymmetry can control waveform initiation and propagation direction.
Assuntos
Dineínas/química , Flagelos/fisiologia , Axonema/química , Flagelos/química , Multimerização ProteicaRESUMO
The distal end of the eukaryotic flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.
Assuntos
Axonema/metabolismo , Flagelos/metabolismo , Cinesinas/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Axonema/química , Flagelos/química , Cinesinas/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/químicaRESUMO
The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.
Assuntos
Cílios/metabolismo , Ciliopatias/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/metabolismo , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Corpos Basais/metabolismo , Corpos Basais/ultraestrutura , Compartimento Celular , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Ciliopatias/genética , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Encefalocele/genética , Encefalocele/metabolismo , Flagelos/genética , Flagelos/metabolismo , Flagelos/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mutação , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismo , Proteoma/genética , Proteínas de Protozoários/genética , Interferência de RNA , Retinose Pigmentar , Trypanosoma/genética , Trypanosoma/ultraestruturaRESUMO
Leishmania species, members of the kinetoplastid parasites, cause leishmaniasis, a neglected tropical disease, in millions of people worldwide. Leishmania has a complex life cycle with multiple developmental forms, as it cycles between a sand fly vector and a mammalian host; understanding their life cycle is critical to understanding disease spread. One of the key life cycle stages is the haptomonad form, which attaches to insect tissues through its flagellum. This adhesion, conserved across kinetoplastid parasites, is implicated in having an important function within their life cycles and hence in disease transmission. Here, we discover the kinetoplastid-insect adhesion proteins (KIAPs), which localise in the attached Leishmania flagellum. Deletion of these KIAPs impairs cell adhesion in vitro and prevents Leishmania from colonising the stomodeal valve in the sand fly, without affecting cell growth. Additionally, loss of parasite adhesion in the sand fly results in reduced physiological changes to the fly, with no observable damage of the stomodeal valve and reduced midgut swelling. These results provide important insights into a comprehensive understanding of the Leishmania life cycle, which will be critical for developing transmission-blocking strategies.
Assuntos
Flagelos , Leishmania , Psychodidae , Animais , Leishmania/fisiologia , Leishmania/genética , Leishmania/metabolismo , Psychodidae/parasitologia , Flagelos/metabolismo , Adesão Celular , Insetos Vetores/parasitologia , Interações Hospedeiro-Parasita , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Estágios do Ciclo de Vida , Leishmaniose/parasitologia , Leishmaniose/transmissão , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , FemininoRESUMO
Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian hosts. To better understand the 3D organisation of male gametogenesis, we used serial block face scanning electron microscopy (SBF-SEM) and serial-section cellular electron tomography (ssET) of P. berghei microgametocytes to examine key structures during male gamete formation. Our data reveals an elaborate organisation of axonemes coiling around the nucleus in opposite directions forming a central axonemal band in microgametocytes. Furthermore, we discover the nucleus of microgametes to be tightly coiled around the axoneme in a complex structure whose formation starts before microgamete emergence during exflagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlight some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite.
Assuntos
Anopheles , Plasmodium berghei , Masculino , Animais , Plasmodium berghei/genética , Haploidia , Células Germinativas , Anopheles/parasitologia , Flagelos/genética , MamíferosRESUMO
Attachment to a substrate to maintain position in a specific ecological niche is a common strategy across biology, especially for eukaryotic parasites. During development in the sand fly vector, the eukaryotic parasite Leishmania adheres to the stomodeal valve, as the specialised haptomonad form. Dissection of haptomonad adhesion is a critical step for understanding the complete life cycle of Leishmania. Nevertheless, haptomonad studies are limited, as this is a technically challenging life cycle form to investigate. Here, we have combined three-dimensional electron microscopy approaches, including serial block face scanning electron microscopy (SBFSEM) and serial tomography to dissect the organisation and architecture of haptomonads in the sand fly. We showed that the attachment plaque contains distinct structural elements. Using time-lapse light microscopy of in vitro haptomonad-like cells, we identified five stages of haptomonad-like cell differentiation, and showed that calcium is necessary for Leishmania adhesion to the surface in vitro. This study provides the structural and regulatory foundations of Leishmania adhesion, which are critical for a holistic understanding of the Leishmania life cycle.
Assuntos
Leishmania , Psychodidae , Animais , Microscopia EletrônicaRESUMO
Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown functions. Here, using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localization of 89% of the T. brucei proteome, a resource we call TrypTag. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultraconserved cellular architecture of eukaryotes, including the first comprehensive 'cartographic' analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.
Assuntos
Parasitos , Trypanosoma brucei brucei , Animais , Humanos , Trypanosoma brucei brucei/fisiologia , Parasitos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteoma/análise , GenomaRESUMO
Most motile flagella have an axoneme that contains nine outer microtubule doublets and a central pair (CP) of microtubules. The CP coordinates the flagellar beat and defects in CP projections are associated with motility defects and human disease. The CP nucleate near a 'basal plate' at the distal end of the transition zone (TZ). Here, we show that the trypanosome TZ protein 'basalin' is essential for building the basal plate, and its loss is associated with CP nucleation defects, inefficient recruitment of CP assembly factors to the TZ, and flagellum paralysis. Guided by synteny, we identified a highly divergent basalin ortholog in the related Leishmania species. Basalins are predicted to be highly unstructured, suggesting they may act as 'hubs' facilitating many protein-protein interactions. This raises the general concept that proteins involved in cytoskeletal functions and appearing organism-specific, may have highly divergent and cryptic orthologs in other species.
Assuntos
Flagelos/fisiologia , Locomoção , Proteínas de Protozoários/metabolismo , Trypanosoma/fisiologia , Leishmania/genética , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia de Sequência , Sintenia , Trypanosoma/genéticaRESUMO
Flagella have multiple functions that are associated with different axonemal structures. Motile flagella typically have a 9 + 2 arrangement of microtubules, whereas sensory flagella normally have a 9 + 0 arrangement. Leishmania exhibits both of these flagellum forms and differentiation between these two flagellum forms is associated with cytoskeletal and cell shape changes. We disrupted flagellum elongation in Leishmania by deleting the intraflagellar transport (IFT) protein IFT140 and examined the effects on cell morphogenesis. Δift140 cells have no external flagellum, having only a very short flagellum within the flagellar pocket. This short flagellum had a collapsed 9 + 0 (9v) axoneme configuration reminiscent of that in the amastigote and was not attached to the pocket membrane. Although amastigote-like changes occurred in the flagellar cytoskeleton, the cytoskeletal structures of Δift140 cells retained their promastigote configurations, as examined by fluorescence microscopy of tagged proteins and serial electron tomography. Thus, Leishmania promastigote cell morphogenesis does not depend on the formation of a long flagellum attached at the neck. Furthermore, our data show that disruption of the IFT system is sufficient to produce a switch from the 9 + 2 to the collapsed 9 + 0 (9v) axonemal structure, echoing the process that occurs during the promastigote to amastigote differentiation.
Assuntos
Axonema/metabolismo , Proteínas de Transporte/metabolismo , Flagelos/metabolismo , Leishmania mexicana/metabolismo , Proteínas de Protozoários/metabolismo , Axonema/genética , Proteínas de Transporte/genética , Flagelos/genética , Leishmania mexicana/citologia , Leishmania mexicana/genética , Proteínas de Protozoários/genéticaRESUMO
Trypanosomes and Leishmanias are important human parasites whose cellular architecture is centred on the single flagellum. In trypanosomes, this flagellum is attached to the cell along a complex flagellum attachment zone (FAZ), comprising flagellar and cytoplasmic components, the integrity of which is required for correct cell morphogenesis and division. The cytoplasmic FAZ cytoskeleton is conspicuously associated with a sheet of endoplasmic reticulum termed the 'FAZ ER'. In the present work, 3D electron tomography of bloodstream form trypanosomes was used to clarify the nature of the FAZ ER. We also identified TbVAP, a T. brucei protein whose knockdown by RNAi in procyclic form cells leads to a dramatic reduction in the FAZ ER, and in the ER associated with the flagellar pocket. TbVAP is an orthologue of VAMP-associated proteins (VAPs), integral ER membrane proteins whose mutation in humans has been linked to familial motor neuron disease. The localisation of tagged TbVAP and the phenotype of TbVAP RNAi in procyclic form trypanosomes are consistent with a function for TbVAP in the maintenance of sub-populations of the ER associated with the FAZ and the flagellar pocket. Nevertheless, depletion of TbVAP did not affect cell viability or cell cycle progression.
Assuntos
Flagelos/fisiologia , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/ultraestrutura , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Flagelos/ultraestrutura , Técnicas de Silenciamento de Genes , Humanos , Imageamento Tridimensional , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Interferência de RNA , Trypanosoma brucei brucei/ultraestruturaRESUMO
INTRODUCTION: Non-small cell lung cancer (NSCLC) with certain activating mutations in the epidermal growth factor receptor (EGFR) is sensitive to the small molecule EGFR tyrosine kinase inhibitors gefitinib and erlotinib, although acquired resistance eventually develops. Resistance is often mediated by acquisition of the T790M mutation in the activated EGFR allele. The aim of this study was to investigate in an EGFR tyrosine kinase inhibitor sensitive NSCLC cell line model, the impact of induced EGFR T790M expression on the cell biology and sensitivity to novel therapeutic strategies. METHODS: Doxycycline inducible EGFR T790M-mediated drug resistance was generated in the clinically relevant HCC827 NSCLC cell line. Cell fate, the activities of EGFR and downstream signaling molecules, and the sensitivity to downstream inhibition of EGFR signaling networks were examined in the presence or absence of induced EGFR T790M expression. RESULTS: Inducible EGFR T790M expression generated acquired resistance to EGFR inhibitors in HCC827 cells as expected. However, induced EGFR T790M expression did not affect activity of EGFR downstream signaling pathways or cell proliferation under the conditions tested. Moreover, sensitivity to inhibition of signaling molecules downstream of EGFR was unaffected by induced EGFR T790M. Importantly, HCC827 cells remained sensitive to class I phosphatidyl-inositol-3-kinase and mammalian target of rapamycin inhibition, which provoked pronounced autophagy, without significant apoptosis. CONCLUSIONS: Phosphatidyl-inositol-3-kinase /mammalian target of rapamycin inhibition is a potentially effective therapeutic strategy against NSCLC with acquired resistance to EGFR inhibition. However, the implications of drug-induced autophagy in NSCLC need further exploration.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/genética , Furanos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gefitinibe , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Serina-Treonina Quinases TORRESUMO
Fanconi anemia (FA) is an inherited disease with congenital abnormalities and an extreme risk of acute myeloid leukemia (AML). Genetic events occurring during malignant transformation in FA and the biology of FA-associated AML are poorly understood, but are often preceded by the development of chromosomal aberrations involving 3q26-29 in bone marrow of FA patients. We report here the molecular cytogenetic characterization of FA-derived AML cell lines SB1685CB and SB1690CB by conventional and array comparative genomic hybridization, fluorescence in situ hybridization, and SKY. We identified gains of a 3.7 MB chromosomal region on 3q26.2-26.31, which preceded transformation to overt leukemia. This region harbors the oncogenic transcription factor EVI1. A third FA-derived cell line, FA-AML1, carried a translocation with ectopic localization of 3q26 including EVI1. Rearrangements of 3q, which are rare in childhood AML, commonly result in overexpression of EVI1, which determines specific gene expression patterns and confers poor prognosis. We detected overexpression of EVI1 in all three FA-derived AML. Our results suggest a link between the FA defect, chromosomal aberrations involving 3q and overexpression of EVI1. We hypothesize that constitutional or acquired FA defects might be a common factor for the development of 3q abnormalities in AML. In addition, cryptic imbalances as detected here might account for overexpression of EVI1 in AML without overt 3q26 rearrangements.
Assuntos
Proteína BRCA2/genética , Cromossomos Humanos Par 3/genética , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Amplificação de Genes , Leucemia Mieloide/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Translocação Genética , Doença Aguda , Linhagem Celular , Criança , Proteínas de Ligação a DNA/biossíntese , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Fatores de Transcrição/biossínteseRESUMO
Throughout its elongation, the new flagellum of the procyclic form of the African trypanosome Trypanosoma brucei is tethered at its tip to the lateral aspect of the old flagellum. This phenomenon provides a cytotactic mechanism for influencing inheritance of cellular pattern. Here, we show that this tethering is produced via a discrete, mobile transmembrane junction - the flagella connector. Light and electron microscopy reveal that the flagella connector links the extending microtubules at the tip of the new flagellum to the lateral aspect of three of the doublet microtubules in the old flagellar axoneme. Two sets of filaments connect the microtubules to three plates on the inner faces of the old and new flagellar membranes. Three differentiated areas of old and new flagellar membranes are then juxtaposed and connected by a central interstitial core of electron-dense material. The flagella connector is formed early in flagellum extension and is removed at the end of cytokinesis, but the exact timing of the latter event is slightly variable. The flagella connector represents a novel form of cellular junction that is both dynamic and mobile.