Exosomy, jejich biogeneze, složení a role

Exosomy jsou podtyp extracelulárních váčků, oproti ostatním extracelulárním váčkům se liší svým endozomálním původem a svým typickým tvarem. Tvorba váčků začíná při maturaci časných endozomů a invaginaci váčků dovnitř lumen. Při invaginaci se pomocí proteinových komplexů ESCRT třídí ubiquitinované proteiny dovnitř váčků. Do těchto váčků je také uzavřeno malé množství cytosolu. Po vytvoření se tyto váčky nazývají intraluminální váčky a celé těleso se nazývá multivezikulární tělísko. Multivezikulární tělísko splývá s plazmatickou membránou a intraluminální váčky jsou sekretovány jako exosomy. Exosomy jsou přítomné v mnoha tělesných tekutinách a jsou sekretovány mnoha druhy buněk. V organismu prezentací antigenu na svém povrchu aktivují imunitní systém nebo slouží v buněčné komunikaci přenosem malých RNA molekul.Exosomes are a subtype of extracellular vesicles. Exosomes are distinguishable from other extracellular vesicles by their endosomal origin and their typical cup-shaped morphology. The biogenesis of exosomes begins in the early endosomes by inward budding. The endosomal sorting complex required for transport sorts ubiquitinylated proteins into the vesicles. The small volume of cytosol is also encapsulated during budding. These vesicles are called intraluminal vesicles and the whole body is called multivesicular body. Multivesicular body fuses with the plasma membrane and vesicles are released as exosomes into the extracellular space. Exosomes are present in all bodily fluids and are secreted by a high number of cells. Exosomes present antigens on their surface to trigger immunity or serve in the cellular communication by the transfer of small RNAs.Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc

Exosomes, their biogenesis, composition and role

Exosomes are a subtype of extracellular vesicles. Exosomes are distinguishable from other extracellular vesicles by their endosomal origin and their typical cup-shaped morphology. The biogenesis of exosomes begins in the early endosomes by inward budding. The endosomal sorting complex required for transport sorts ubiquitinylated proteins into the vesicles. The small volume of cytosol is also encapsulated during budding. These vesicles are called intraluminal vesicles and the whole body is called multivesicular body. Multivesicular body fuses with the plasma membrane and vesicles are released as exosomes into the extracellular space. Exosomes are present in all bodily fluids and are secreted by a high number of cells. Exosomes present antigens on their surface to trigger immunity or serve in the cellular communication by the transfer of small RNAs

Study of exosomes in polyomavirus infection

Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells..

Studium exosomů při polyomavirové infekci

Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells...Exosomy jsou extracelulární váčky endosomálního původu. Původně se myslelo, že exosomy slouží pouze k vylučování buněčného odpadu, avšak se ukázalo, že slouží i ke komunikaci mezi buňkami a hrají roli i ve virových infekcích. Exosomy jsou využívány viry např. k přenosu virového proteinu či jejich RNA/DNA. Jedním z virů, u kterého není role exosomů při infekci objasněna je myší polyomavirus. Myší polyomavirus patří do čeledi Polyomaviridae, do které spadá i řada lidských virů, jako je např. JC virus či virus Merkelova karcinomu. Myší polyomavirus kóduje malý, velký a střední T antigen a tři kapsidové proteiny. O středním T antigenu je známo, že se váže na buněčné membrány. Vzhledem k tomu, že exosomy jsou membránově odvozené struktury, zaměřili jsme se možný přenos středního T antigenu. K tomuto cíli bylo potřeba nejdříve zvládnout metody izolace exosomů a jejich charakterizace. Exosomy byly izolovány pomocí ultracentrifugace a dále purifikovány na hustotním gradientu OptiPrep. Charakterizovány byly pomocí elektronové mikroskopie, přístroje NanoSight a proteinových exosomových markerů. Mezi tyto markery patří například Alix, či flotillin-1. Dále byly buňky transfekovány, aby exprimovali střední T antigen. Bylo ukázáno, že exosomy izolované z těchto buněk skutečně obsahovali střední T antigen....Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult