RESUMO
Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.
Assuntos
Acetilcolina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/farmacocinética , Proteínas de Membrana Transportadoras , Neurônios/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Vesículas Revestidas por Clatrina/metabolismo , Endocitose/fisiologia , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde , Indicadores e Reagentes/farmacocinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/farmacocinética , Dados de Sequência Molecular , Mutagênese/fisiologia , Neurônios/citologia , Transmissão Sináptica/fisiologia , Transfecção , Células Tumorais Cultivadas , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
Pathogenic trypanosomatids cause a plethora of diseases marked by the lack of efficient vaccines and therapies. As a consequence, studies are being conducted that are geared towards the understanding of basic mechanisms and various biological aspects of these parasites that might be used as targets for new developments in these areas. One such aspect is the understanding of specific cellular trafficking mechanisms that might be attacked with the intention of disease control. In this paper, we give an overview of our current knowledge of cellular targeting mechanisms in trypanosomatids, with special emphasis on our data related to lysosomal targeting of cysteine proteinases in Leishmania.
Assuntos
Antiprotozoários/farmacologia , Leishmania/metabolismo , Trypanosoma/metabolismo , Animais , Antiprotozoários/farmacocinética , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmaniose/tratamento farmacológico , Proteínas Luminescentes/genética , Proteínas Luminescentes/farmacocinética , Lisossomos/enzimologia , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/farmacocinética , Trypanosoma/efeitos dos fármacos , Trypanosoma/genética , Tripanossomíase Africana/tratamento farmacológicoRESUMO
Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be approximately equal to 10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5-10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Starburst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions.