Studies On Microtubular Cytoskeleton And γ-Tubulin In The Hypotrich Ciliate Euplotes Eurystomus

There are abundant of microtubule cytoskeletons in the protozoa ciliate, which composed of the ciliature microtubular and non-ciliated microtubular system. The former contains the adoral ciliature microtubules, somatic ciliature microtubules and their base-associated microtubules; the later contains the microtubules not connect with ciliatures directorly, such as membrane microtubule cytoskeletons, subpellicular microtubule layers, microtubules in the deep cytoplasm and that connect with other organelles. It is noteworthy that ciliates in different group characterized with different cilia modals have microtubule cytoskeleton differentiation inordinately, and it is important for revealing the formation of eykaryocyte structure and its regulatory mechanisim to study the ciliature microtubules and the construction and heredity of microtubule organelles in this kind of cells. Researches on the formation of cilia modals and its regulation in the ciliates became a hot topic since the 80's of last century. In the resent 15 years, researches on the ciliature microtubules deep into the levels of microtubule units and the tubulins, and it is focused on the genesis of microtubule units during the cell cycle, the assembly of the microtubules into the cilia units and the cortical ciliatures, and the regulation during the genesis and assembly of ciliture microtubules. Since it is discovered that theγ-tubulin is an important part of microtubule organizing centers (MTOCs) in the ciliate Paramecium and Tetrahymena, it was found taken part in the regulation of the assembly of the tubulin heterdimers during the cell cycles and maintains the stability of the MTOCs. Researches on the locations of the functional proteins in the ciliatures and exploration of the microtubule assembly and regulation become an important issue. But correlational studies is just limited in the cilites which have a scattered arranged sometic ciliature, and the organization of ciliature microtubules and its relationship in the Hypotrich Ciliates which have specialized recombined ciliatures is not clear. In the present study, the Hypotrich Ciliate Euplotes eurystomus was used. On the cell level, the organization characterizes and the morphogenesis of the ciliature microtubules, the ciliature associated microtubules and the cortical microtubules in the non-ciliated regions were studied in the micro-and ultra-levels with the help of the fluorescent labeling and the transmission electrion microscopy, to get more datas for the microtubular ciliature construction, the differentiation and regulation of microtubules during the morphogenesis in this kind of ciliates. On protein levels, the RNAi approach by feeding was using to interfere the y-tubulin gene expression and it function on the ciliature microtubules and the cortical microtubules in the non-ciliated regions was explored, to provide some molecular biological and morphological evidences for the mechanism of this protein in the microtubule assembles.1. The composition of cortical microtubule cytoskeletons.The director and immune fluorescence labeling displayed that the cortical microtubular organelles of Euplotes eurystomus were composed of ciliature microtubules, ciliature base-associated microtubules and cortical non-ciliated regions microtubules. In the ventral cortex, the adoral ciliature and its base-associated microtubules consisted of adoral zone of membranelles(AZM), membranelle brackets microtubules and the short microtubules inner side the adoral cortex; undulating membranes(UM) and its base-associated microtubule nets; the sometic ciliature and its base-associated microtubule consisted of frontal-ventral-transverse-caudal cirri (FVTCC), left marginal cirri (LMC) and the anterior longitudinal microtubules(ALM), posterior longitudinal microtubules(PLM),transverse microtubules(TM), which contain a developed ALM of TC extended into the right side of the first FC. The dorsal kineties (DK) and its base-associated microtubules is the rosette-like skeletal structure. The cortical non-ciliated regions microtubules composed leptos microtubule nets in the dorsal and ventral cortex; the oblique microtubules in the dorsal cortex; the longitudinal microtubules. The results show that the ciliate Euplotes are characteristic of the enormous AZM, the mixed type FVCs, the developed anterior longitudinal microtubules of TC and the simplified marginal cirri. Furthermore, they have a developed microtubular system in the non-ciliated regions formed into microtubule nets and microtubule layers. We have shown the two kinds of microtubule organelles system perfectly in this kind of ciliates, and provided more dates for the understanding deeply of diversity of the cytoskeleton and the complexity of the cytoarchitecture in the cell and organelle levels in ciliates.2 The ultrastructure of the cortical microtubule cytoskeleton.The ultrastructures show that in the ciliature microtubules the basal-bodies of the cilium is composed of the a symmetrical array of nine triplet microtubules with a 9+0 arrangement like a cartwheel and are connected by the longitudinal and transverse fibers, all of them are enveloped in the basal body bracket; under the basal-bodies, there are cilium scaffolds contain dense matters; in the basal of basal-bodies, there are single microtubules associated with the basal-bodies and the microrubule bundles associated with the cilium. The dorsal kineties locate in the concavity of the dorsal crista, and its base-associated microtubules extend into the cytoplasm like a root. The cortical non-ciliated regions microtubular cytoskeleton is a complex organelle composed with single or poly microtubules, and located in the specific regions as a form of column, bands or network. In the ventral subpellicular microtubule layers, single microtubules arrange in a line; in the dorsal subpellicular microtubule layers, a microtubule unit consisted with three microtubules in a form of microtubule triplets arrange in a line; in the subpellicular granular layer, the microtubules arrange parallel or angled to the membrane, and cross into network; in the cytoplasm, microtubules gathered into bands and extend along the cytoplasm in different direction; in the cortical preiection-layers, there also some microtubules arrange parallel. The result indicate that the cortical ciliature is consist of not only cilium and basal bodies but also their linking-fibers, the basal body bracket, the associated microtubules and the fibrillar cirral basket; and the cortical non-ciliated regions microtubular cytoskeleton is consist of the subpellicular microtubule layers, the microtubules in the subpellicular granular layer, the cytoplasmic microtubules and the microtubules in the cortical preiection-layers. We have displayed the two kinds of microtubule cytoskeleton system comprehensively in the cell level, and provide some dates for the formulization of the formation of ciliature and non-ciliated microtubules in this kand of ciliates.3 The morphogenesis of the cortical microtubular organelles.During the process of the morphogenesis of the ciliatures, AZM of proter were generated from the old AZM, the primordium of the opisthe AZM and UM genesis from the left basal side of the old AZM. Then, they developed and differentiate into the opisthe AZM and UM. The primordiums of the FVTCC originated between the old FVTCCs cortex. During the differentiation of the primordiums of FVTCCs, the microtubule buds originated from the base of these primordiums. The anterior longitudinal microtubules primordiums (ALMPs) of TC occurred earlier than the posterior longitudinal microtubules primordiums (PLMPs) of the FVC. The former extend to the anterior end of the cytoplasm when the TC migrates to the posterior part of the cytoplasm. On the contrary, the later extend to the reverse direction. It is noteworthy that the old ciliature base-associated microtubules degenerated as the FVTCC of proter and opisthe morphogenesis and differentiate and the new ciliature base-associated microtubules were observed. We have displayed the formation, differentiation and the location of the ciliature microtubule primordiums during the morphogenesis of ciliates Euplotes, and the process of the genesis, growth and location of the ciliature base-associated microtubules was also observed, which has not been reported before. And it provided more dates for the understanding of the morphogenesis of ciliature base-associated microtubules.4 The location and function of theγ-tubulin.The y-tubulin mainly location in the basal-bodies of the interphase Euplotes cells, include the membranelle brackets of AZM, the basal-bodies of FVTCC and DKs. In addition, the microtubules between the base of the TC, the pores of the contractile vacuole and the macronucleus membranes were also stained by the fluorescein. During the morphogenesis, this protein also located in basal-bodies of the primordiums of the FVTCCs.The y-tubulin of Euplotes eurystomus was inserted in to the double-T7-promoter of expression vector L4440, and the recombinant plasmid L4440-TGU was transformed into the RNaseⅢdeficient E. coli strain HT115. The IPTG was used to induce the expression of dsRNA in the E. coli strain HT115, which was then fed to the ciliated Euplotes eurystomus to interfere its y-tubulin gene expression. Ciliates interfered by RNAi seemed to be hard to ingest, and the undigested food congregated to the post-oral cytoplasm area. No cells could survive as the cell could not divide. The ultrastructure show that most of microtubule triplets were structurally incomplete, that is to say, A-tubule and B-tubule existed while C-tubule disappeared, and B-tubule disintegrated in some microtubule triplets. In the dorsal subpellicular microtubule layer some of the inner-located microtubules seen in the three microtubule-containing units in the normal cells are disappeare, and some microtubules are disappeared from the microtubule row in the ventral subpellicular microtubule layer. As a result, the interference of y-tubulin gene can not only affect the expression of the correlative protein, but also result in the morphological changes, and it is lethal for the cells. In addition, y-tubulin is important for maintenance of stability of the nine microtubule triplets in basal body, and is necessary for the integrity of microtubule structure in the dorsal and ventral subpellicular microtubular layers.