Cel
The secretory pathway of eukaryotic cells has been studied mostly in mammalian and yeast cells. The disadvantage of these systems are the complicity of the secretor pathway in mammalian cells and morphological peculiarities of yeasts, especially the presence of a thick cell wall and dense cytoplasm, which make the morphological analysis rather difficult. To analyse molecular mechanisms, experimental models with simple organization of secretor traffic are urgently demanded. Only very few of primitive unicellular eukaryotic systems have been studied in the respect of presence of minimal Golgi.
Microsporidians (MS) are intracellular obligate parasites with a wide host range from Protozoa to Primates. MS are especially abundant in Arthropods and fishes and are increasingly recognized as human pathogens especially in individuals with HIV. They lack the Golgi stacked cisternae through the whole life cycle; though their sporogenic phase possesses a compartment with atypical structure, identified biochemically as Trans - Golgi. Practically nothing has been known so far about the organization of exocytotic pathway in MS, yet through the whole life cycle (that occurs totally inside one host cell), MS display secretory activities on the proliferative phase, connected with genesis of membranes for proliferation and with releasing substrates inside the host cell, and on the sporogenic phase - with the spore morphogenesis. The presumable absence of Golgi complex makes the proliferative phase of MS development an attractive model to study minimal secretory machinery. The peculiarities of microsporidian spore morphogenesis - the presence of conglomerates of a polar tube protein inside Golgi containers), give an opportunity to find out the precursors of the secretory compartment in earlier stages of parasite development and to study the mechanisms of cargo maturation, thus providing one more important advantage of the system. We believe that MS represent a promising model for analysis the minimal machinery for protein secretion in eukaryotes.
The following objectives are determined.
(1) To visualize the entire bio-secretory pathway in various phases of microsporidian ontogenesis: (i) in proliferative stages, (ii) in sporoblasts and (iii) in spores, using three-dimensional serial reconstruction combined with more powerful methods of cell fixation, such as rapid-freezing-cryosubstitution or glutaraldehyde, dissolved in highly buffered solution combined with the application of reduced osmium;
(2) To identify the transformation of different bio-secretory compartments during microsporidian life cycle, using immuno electron microscope labeling for the proteins involved into the constitutive secretion and being highly conserved during the evolution, such as COPII, ARFs, COPI, clathrin, p24;
(3) To investigate the characteristics of intracellular transport in microsporidians in different phases of their development and under the action of specific drugs affecting ARF/COPI machinery and microtubules, namely brefeldin A and nocadosol. The following experimental systems (already established), will be used in the experiments: (a) parasite-host system: Nosema grylli - Gryllus bimaculatus; (b) primary short-term culture of the infected tissue; (c) N. algeri - insect cell lines; (d) N. algeri - mammalian cell line; (e) Encephalitozoon cuniculi - mammalian cell line.
In this project the particular interests of four teams in intracellular morphology, membrane-traffic pathways and the biology of protozoan parasites meet together. The advantage of the project is the previous collaboration of all involved teams, which will facilitate the organization of work. The research design is based on the achieved results (already raised antibodies against MS proteins, established methods of MS cultivation, isolation of developmental stages) and a strong expertise in modern methodology of cell biology. The planned research will bring new information about MS physiology, indicate new targets for suppressive therapy and contribute to the defining the minimal machinery for protein secretion in eukaryotes.
Temat(-y)
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66030 S.Maria Imbaro Chieti
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