Studies On Ciliature Microtubular Organelles And Tubulins In The Hypotrich Ciliate Stylonychia Pustulata

Microtubular cytoskeleton of ciliated protozoa is composed of ciliature microtubular organelles and non-ciliated region microtubules.The former locate in the outer surface of cell which named cortex.Ciliature microtubular organelles organize into ciliary units.These units,which are the most complicated microtubular systems,array in different models,and construct into oral ciliature apparatus,somatic ciliature apparatus and ciliature base-associated microtubules.As the structural unit of cytoskeleton,microtubules are closely coupled with lots of biological activities happening in ciliates,and also are studied largely by researchers from different fields. At the same time,researches about ciliature microtubular organelles are always important contents in protozoology,cellular and molecular biology.At present,the structures of microtubules in ciliates,whose kinetosomes array dispersedly,have been well studied,while related researches on hypotrich ciliates are restricted mainly to several species due to complicatedness of the composition and construction of their cytoskeletons.In addition,ciliatures of hypotrich ciliates are all composed by cilia.A problem is why the arrangement and localization models of ciliatures in different species are also different,but is identical in the same species?So the resolution of this problem is of great significance for further elucidation of the heredity of ciliatures in ciliates and the formation mechanism of eukaryotic cell ciliary pattern.In this dissertation,by the studies on ciliature microtubules,ciliature base-associated microtubules and tubulins,the constructive characters of cortical microtubular organelles in Stylonychia pustulata will be clearly illustrated by using TEM,SEM, FLUTAX direct-fluorescent labeling,immunofluorescent labeling,immunoeletron microscopy labeling,biochemistry extraction and western blotting.Expect to provide basic informations for the researches about the formation and regulation of cellular structures in ciliates.Results are as follows: 1 Ciliature MicrotubulesCortical ciliature microtubular orgenelles of S.pustulata is composed of ciliature microtubules and ciliature base-associated microtubules.These ciliature microtubules consist of adoral zone of membranelles(AZM),undulating membranes(UM), frontal-ventral-transverse cirri(FVTC),caudal cirri(CC),left and right marginal cirri (L-and RMC)and dorsal kineties(DK).In the front of cell,the AZM possesses 50-60 adoral membranelles,each of the membranelle consisted of 3 rows of cilia;UM possesses 2 rows of cilia;FVTC distribute into 8 frontal,5 ventral and 5 transverse cirri in the pattern of Oxytricha-Stylonychia;1 marginal row of cirri on each side do not connect at the caudal part,and there are 25 left and 35 right marginal cirri;6 DK rows,and the kinetosomen unit in the dorsal kineties consists of one shafted-kinetosome and another condycondylocilium;3 CC.In addition,the TEM showed that ciliary shafts possess"9+2"doublet microtubules;basal bodies possess"9+0"triplet microtubules,and basal bodies of the cirri are also linked by different kinds of connectives.Nonetheless,compared with other species of hypotrich ciliates, the arrangemental models of ciliatures and the numbers of cilia are also different. Especially,in higher evolutionary ciliates,its ciliatures congregate,so the functions of these ciliatures will also be centralized in some ventral ciliatures.During morphogenesis,the ciliature microtubules experience a wholly new process of assembly and disassembly,with the ciliatures duplicating at the previous models.It is worth noting that the old AZMs will be completely retained for the proter in division,whereas the AZM of the opisthe,FVTC primordia and L-and RMC primordia of proter and opisthe would generate from the same site where the old ciliatures stayed.So the old cirri,besides playing important parts in the morphogenesis and orientation of the new cirri,they also harmonize the morphogenetic process.However,at the site of old cirri,the morphogenetic characteristics of 5 rows of FVTC primordia are especially unique in Oxytrichid ciliates.And all these results suggested that difference exists in the morphogenesis of the ciliature microtubular organelles in the ventral cortex of Hypotrich ciliates,even among ciliates with close relation in genetics. 2 Ciliature Base-Associated MicrotubulesThe microtubular cytoskeleton of AZM comprises membranelle brackets, post-membranelle brackets microtubules,membranelle brackets microtubules and the converse V-shaped microtubules between membranelle brackets;and that the base-associated microtubules of UM form the microtubule cytoskeletal net;and the fibrillar cirral basket(FCB)locate at the base of FVTC,the anterior longitudinal microtubules(ALM),posterior longitudinal microtubules(PLM)and transverse microtubules(TM)of FVTC,L-and RMC send forth from FCB,while the base-associated microtubules at different locations differ from each other in developmental degrees.The ALM and PLM of FV cirri are thick,but the TM are invisible;5 ALM of TC extend forwards and converge together,TM extend to the right side of cell,PLM are thick;TM of L-and RMC both extend to the left side of cortex,and displayed a non-mirror symmetrical image,ALM are different from PLM in developmental degrees;Nonetheless,the ALM and PLM of 6 rows of DKs send forth from the base of ciliatures and extend to anterior and posterior cortex respectively,forming the microtubule net below the surface of the dorsal cortex.From the above results,we then infer that the three types of base-associated microtubules (ALM,PLM and TM)are the essential components in the base of microtubular organelles of Hypotrich ciliates.And in comparison with other hypotrich ciliates, there exists significant difference in orientation of the TM of L-and RMC,while the constructive characters of the base-associated microtubules of DKs share striking similarity with Stylonychia mytilus and Allotricha curdsi.Thus it could be concluded that the constructive characters of the TM of L-and RMC are conservative within the same species,while the characters of the base-associated microtubules only enjoy similarity on the level of family.In addition,TEM showed that the cortical non-ciliated regions consist mainly of monolayer and multilayer microtubules.The latter,distribute to dorsal cortex,is highly developed.We thus conclude that the multilayer microtubules and the ciliature microtubular organelles of dorsal cortex might cooperate in the reinforcement of dorsal cortex. 3 Localization and Function of Tubulinsα-andβ-tubulin could be localized to all microtubular organelles,including the ciliature microtubules,ciliature base-associated microtubules and cortical non-ciliated regions microtubules,whileδ-andγ-tubulin are colocalized to basal bodies.Besides, immunogold decoration showed that the localization ofγ-tubulin to the proximal part of basal bodies is also coupled with that ofδ-tubulin,which reveals thatγ-tubulin andδ-tubulin might coexist to function together or interact with each other.Interestingly, once the morphogenetic process of AZM of the ciliate S.pustulata finishes,the disassembly and renewal of basal bodies microtubules of oral apparatus stop simultaneously,so we infer that during early morphogenesis in AZM of S.pustulata,γ-tubulin,though closely associated with microtubular assembly,only acts in maintenance of the oral ciliature base-associated microtubules after the morphogenetic process.The fact thatδ-tubulin also presents in the mid-connections between basal bodies but with sporadic distribution in the lumen and distal part of basal bodies,we thus conclude thatδ-tubulin is a basal body-associated protein required for extension or stability of the triplet microtubules of basal bodies or connections.To characterizeα-,γ-andδ-tubulins in S.pustulata and compare their distributions in soluble and insoluble fractions,proteins were extracted from harvested cells and their profiles are exhibited as follows by ways of SDS-PAGE and Coomassie blue staining.The whole cytoskeletal and soluble fractions displayed a highly complex protein pattern,abundant with tubulins as its component,and the contents are 31.6%and 21.7%respectively,whereas the protein content in the insoluble fraction decreases significantly.In addition to confirm the molecular weights ofα-,γ-,andδ-tubulin to be 50 kD,48 kDa,and 55 kDa respectively, immunoblotting also showed that the expression levels of the three tubulins differ dramatically in the three above fractions extracted,which further demonstrates their different functions as a results of their characteristics.It is then could be concluded that the solubleα-tubulin in living cells possesses a higher potential of assembling and disassembling in a temporary"oligomeric"state,while the presence ofγ-tubulin in both soluble and insoluble fractions with a greater proportion in the later than in the former showed that it may be demand for duplication of basal bodies, and also required for stability of the microtubules of basal bodies or ciliary shafts.On the other hand,the solubleα-,γ-andδ-tubulin might interact cooperatively in the reassembly and disassembly of microtubules in S.pustulata.4 ConclusionsThe results acquired above showed that ciliature microtubular orgnelles are important components of cortical microtubular cytoskeletons in Hypotrich ciliates,but the constructive model of ciliature and the developmental degrees and constructive models of their ciliature base-associated microtubules are quite different.During morphogenesis,the ciliature microtubular organelles in the ventral and dorsal cortex regenerate orderly,and compared with other species,the construct and morphogenetic models are also quite different.Further,it is also showed thatα-andβ-tubulin are the important components in cortical microtubular cytoskeletons,whileγ-andδ-tubulin co-exist in basal bodies though,finer localizing also revealed minute difference in their distributions.Moreover,according to the different biochemical characters,we concluded that the functions of the three tubulins are also different,yet they interact closely together or on their own in the formation of the complicated cortical microtubular cytoskeletons.To sum up,the findings in this research are of important scientific significance for further elucidation of the structural complexity and diversity of cortical ciliature microtubular organelles,the assembling mechanism of cortical microtubular cytoskeleton,the heredity and regulation of ciliatures in ciliates as well as the formation mechanism of eukaryotic cell ciliary pattern...