| Protozoa ciliates are the most complex a single cell due to the highly specialized cell structure.A variety of organelles in cytoplasm of ciliates accomplishes many life activities through synergistic action.Ciliates' extrusome possesses many important functions such as defense,predation,intercellular communication and moreover.At the same time,it's important to protozoan genealogy and taxonomy.Therefore,ciliates' extrusome has always been the study emphasis of domestic and overseas scholars.At present,the studies on extrusome of Tetrahymena and Paramecium--model organism are explored in depth,but only on the morphology level.Hypotrichous ciliate is less studied and the research on its ingredients and functions is still a blank field.With differential interference contrast microspy,scanning electron microscopy and transmission electron microscopy,the structure of Oxytricha longigranulosa extrusome is studied in this paper.With staining with methyl green-pyronin,alcian blue staining,scanning electron microscopy and high performance liquid chromatography tandem mass spectrometry,the protein composition of Oxytricha granulifera extrusome is discussed profoundly.The main results are listed as follows:1 The morphology and extrusomes of O.longigranulosaO.longigranulosa cells presented the form of long and thin strip,which wre dorsoventrally compressed,with the length ranged from 80-110 ?m and width ranged from 30-50 ?m.It was the Oxytrichidea with typical "8-5-5" ciliata mode,with one column for each left right marginal cirri.The colorless circular granules under ellipse were visible by light microscope,with the diameter around 0.5 ?m.Dorsalobservation showed that the spheroidal particles were 3-5 columns between adjacent two columns of dorsal kinety,bending along with the body bent;Ventral observation showed that few spheroidal particles distributed surrounding ventral cirri,and most of them were distributed in the head and tail of cells,as well as nearby right marginal cirri.Scanning electron microscope observation showed that the extrusomes of O.longigranulosa were elongated ellipse,and distributed in long or short column.They were slightly gathered around dorsal kineties where the differences was distinctive with ventral.Most of extrusomes were gathered near adoral zone of membranelles in the ventral side,while below transverse cirri,3 to 4 rows of extrusome were arrayed with "arc".The extrusomes were 2-3 rows which distributed surrounding left of right marginal cirri passed through the whole body;2-3 rows of extrusomes distributed surrounding right side of left marginal cirri,which started from the middle of ventral side and extended to the tail.The extrusomes which were secreted in vitro were distributed on the body surface vertically or diagonally,presenting "shuttle shape".The length was 1 ?m,and the diameter of top was 0.5 ?m.It can be seen clearly the mature extrusomes elongated the ellipse vertically when splited the cells.The transmission electron microscope observated that mature extrusomes were ellipsoidal structure with a single membrane.The length was 1 ?m and width was 0.5 ?m,which was consistent with the result observed under scanning electron microscope.It was almost no electron density at cavity on the anterior,while the tail with high electron density.Colorless cortical granules of O.longigranulosa had high consistence with extrusomes in the aspects such as organelles distribution and morphological structure.It can be secreted in vitro when being affected by stimuli,and therefore,it was considered that its colorless cortical granules were a kind of extrusomes.This kind of extrusomes had a great differences with mucocysts and toxicysts in structure andemitting mechanism,while it had a high consistence with O.granulifera and Architricha indica in position and morphology.Therefore,the author considered this extrusomes may be the same type with O.granulifera and A.indica,and all above it were "secreting type" extrusomes.By comparing other species of Oxytrichidea who had colorless cortical granules,we can speculate that all colorless cortical granules of Oxytrichidea was the "secreting type" extrusomes.2 Analysis on protein components of O.granulifera extrusomesThe tests showed that extrusomes of O.granulifera will form a layer of"capsule" structure on the cell surface after Methyl Green-Pyronin solution stimulates.And then O.granulifera was divorced from "capsule" through constant contraction motion.Concentrated and collected the "capsule" by centrifugation.We identified extrusome-related proteins from the specific band gained from SDS-PAGE with shotgun LC-MS/MS by checking Oxytricha trifallax protein database,respectively,in this report.The result showed that:a total of 25 proteins were detected in the extrusomes,4 of these proteins were high credible proteins and 21 of these proteins were credible proteins;while 8 of these proteins were characterized proteins and 17 of these proteins were uncharacterized proteins.8 known proteins were listed as follows:Ribosomal protein S5,Tubulin beta chain,Amidinotransferase,Protein kinase domain containing protein,PAS domain S-box family protein,Thymidine kinase,PP2C,calmodulin,and Calcium-dependent protein kinase.In addition,many high credible proteins were uncharacterized proteins,and we can speculate that these proteins had close relationship with ejection mechanism and formation of extrusomes.Compared with other common extrusomes,the proteins' composition of Oxytrichidea extrusome in this report was difference.Compared with the Tetrahymena mucocysts,its main composition was mucopolysaccharide,aspartic protease,Cth4p and others,and these proteins were not reported in O.granulifera.There were no mucocyst related proteins were discovered in O.granulifera.Compared with toxicysts and trichocysts,the protein related with toxin analogues were also not discovered in this study.So author considered that the reported proteins were the specific composition of Oxytrichidea extrusomes,at the same time,the ejection mechanism and function were different with lower groups of ciliates.Different proteins plaied different roles in extrusomes.The calmodulin-dependent protein kinase may interact with calmodulin and Ca2+,which was beneficial for extrusomes to be secreted when being affected by stimuli;S5 ribosomal protein may have important role for promoting that the lower temperature stimulates extrusomes to be secreted;Amidinotransferase may promote the synthesis of ATP through the action on creatine and phosphocreatine,which can provide power support for extrusomes.Besides,the many uncharacterized proteins were high credibile,and the function of these proteins needs to be further studied.In this paper,the morphology and distribution of O.longigranulosa extrusomes and protein composition of O.granulifera extrusomes were described in detail,and the dynamic relationship between the extrusomes and subpellicular microtubules were deeply analyzed,which enriched data of the studies of ciliate extrusomes.The method separating extrusomes from ciliate cells was reported in detail for the first time,and the protein composition of extrusome was revealed.This study might be able to provide new idea to recognize the composition of extrusome,and improve the understanding of the structure,formation,evolution and regulation of protozoa cells. |
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