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Monitoring of protein network dynamics: the role of FcRL proteins during sperm-egg membrane interaction
Bašus, Kryštof ; Komrsková, Kateřina (advisor) ; Šušor, Andrej (referee)
Sperm--egg membrane interaction and fusion is mediated by various molecules of the different protein network that are located on both egg and sperm membrane. So far, many proteins have been selected to be fusion candidates, some of them (Izumo1, CD9, Juno) were proven to be essential, whereas others were discovered to play an unsuspected new active role (CD46, tetraspanins). After the adhesion of sperm to an egg, Juno located on the oolema associates with monomeric Izumo1 on sperm membrane, which is results in Izumo1 dimerization following quick removal of Juno from the egg surface as described in mouse. It implies that additional receptor on the egg membrane is required to play a role in sperm--egg fusion. To find a human fusogenic receptor for IZUMO1 protein we used one--bead--one--compound (OBOC) assay, a random screening approach. A bead, fulfilling all the requirements when interacting with the human sperm, carried a peptide sequence showing homology with the conserved Ig domain of the human specific Fc receptor--like protein 3 (FcRL3). In general, the ...
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Monitoring of protein network dynamics: the role of FcRL proteins during sperm-egg membrane interaction
Bašus, Kryštof ; Komrsková, Kateřina (advisor) ; Šušor, Andrej (referee)
Sperm--egg membrane interaction and fusion is mediated by various molecules of the different protein network that are located on both egg and sperm membrane. So far, many proteins have been selected to be fusion candidates, some of them (Izumo1, CD9, Juno) were proven to be essential, whereas others were discovered to play an unsuspected new active role (CD46, tetraspanins). After the adhesion of sperm to an egg, Juno located on the oolema associates with monomeric Izumo1 on sperm membrane, which is results in Izumo1 dimerization following quick removal of Juno from the egg surface as described in mouse. It implies that additional receptor on the egg membrane is required to play a role in sperm--egg fusion. To find a human fusogenic receptor for IZUMO1 protein we used one--bead--one--compound (OBOC) assay, a random screening approach. A bead, fulfilling all the requirements when interacting with the human sperm, carried a peptide sequence showing homology with the conserved Ig domain of the human specific Fc receptor--like protein 3 (FcRL3). In general, the ...
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Visualization of RNA and its translation in mammalian oocyte
Bašus, Kryštof ; Tětková, Anna (advisor) ; Macůrková, Marie (referee)
Translation is one of the fundamental biological processes. It is assumed that the locali- zation of mRNA and its translation in a certain time and at specific locations plays a role in many cellular processes. The mammalian oocyte becomes transcriptionally inactive when it reaches its full size and utilizes only RNA that has been synthesized and stored in the early stages of development. Thus the regulation of protein synthesis at the translational level is cri- tical for the correct completion of meiotic maturation of oocytes and early development of embryos. To monitor cellular physiology, it is necessary to be able to visualize and monitor spe- cific molecules and processes at single cell level. This is enabled by the development of light microscopy and fluorescence probes that specifically bind to certain organelles, cellular struc- tures, proteins or other molecules. In this thesis I describe selected methods of visualization of RNA, global translation as well as translation of specific transcripts, and proteins. The methods that this thesis describes are RNA FISH, visualization of translation using methionin analogs, FUNCAT, SUnSET, FlAsH, ReAsH, TRICK, SINAPS, FUNCAT-PLA, PURO-PLA. Key words: Translation, RNA, Protein, Visualization, Oocyte
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