| Dense core vesicles existed widely in higher eukaryotic cells,and contained abundant contents.They were named so because its opaque dense core observed under the electron microscope.Contents in dense core vesicles could regulated and maintained a variety of different physiological functions.The dense core vesicles also existed in ciliate protozoa,called extrusomes,which were usually located under the pellicle of the cell,and the substances were rapidly ejected when stimulated.Extrusomes played an important role in the communication between protozoa with the outside world,which made cells respond quickly when stimulated,and realize various physiological functions by releasing the stored contents.The structure of dense core vesicles and their exocytosis behaviors in protozoa cells were more complex compared with that in higher eukaryotes,so the study of extrusomes of protozoa was of great reference significance for understanding of the evolution of eukaryotes.In this study,protrichocysts of Pseudourostyla cristata,which was a unique type of extrusome in subclass Hypotrichia,was taken as the research object.Chemical treatments were used to explore a stable scheme for inducing of protrichocysts,and differential interference phase contrast microscope and electron microscope were used to observe the specific discharging process in vivo.Proteins of protrichocysts was studied by staining of Alcian blue,specific labeling of FLUTAX-2,polyacrylamide gel electrophoresis and high performance liquid chromatography-tandem mass spectrometry.Study on the methods of inducing and ejection process of protrichocysts in vivoIn this paper,different final concentrations of methyl green-pyronin solution and calcium chloride solution were used to the normal vegetative cells of Pseudourostyla cristata,and the discharging states of protrichocysts were observed in a short time.Results showed that the cell was best induced when the final concentration of methyl green-pyronin solution was at 0.010%.In an instant,filiform projectiles were observed all over the cell when discharge was induced,and it could finally fall off from the cells’ surface after a period because of the activities of the cell;Discharge of protrichocysts could also be induced by calcium chloride solution treatment.Particles could be observed on the cells’ surface,and the morphology was consistent at high magnification with that induced by methyl green-pyronin solution.In this paper protrichocysts of Pseudourostyla cristata could be both induced by the treatments of two solutions.And though a large number of discharge of protrichocysts could be induced by calcium chloride solution,it was difficult to be observed because of the colorless of the solution.Moreover,the proportion of induced cells of total number was unstable when treated with calcium chloride solution,which made the experiment uneasy to control.However,the proportion above was stable when treated by methyl chloride-pyronin solution,and almost every cells could have been induced by different concentrations of treatment of methyl chloride-pyronin solution,and protrichocysts would be dyed in obvious red,which made the observation easier.Cells remained active after clear water was added to terminated the inducing by methyl chloride-pyronin solution.Due to the protrichocysts in the cell was sharply decrease after treated with methyl chloride-pyronin solution,the protrichocysts-deficient cells could be collected after a period of induced.Therefore,we take methyl green-pyronin solution as a basis inducing method for subsequent experiment,e.g.separation,collection,further component exploration and predation of protrichocysts.2 Discharging process of protrichocystsMature protrichocyst,2 μm in length,were rod-shaped and located below the pellicle of the cell.The organelle could be divided into four different parts:the central shaft,the cap,the tip and the body.In this paper,method of adhesion by polylysine was used to the cell,combined with observation of electron microscope and light microscope,results were same under two treatments of inducing,and the discharging process was summarized to three stages:(1)The microtubule-like structure of the cap gradually loosened and dissolved,and the less dense posterior part of the body extended,stretched at 2-3 times to the original one.(2)The dense anterior part of the body extended and the central shaft gradually been pushed out,while the less dense posterior part of the body continued to extend.At this time,the length of the protrichocyst would reach at 20-30 μm;(3)The dense anterior part of the body extended and the central shaft was pushed out finally.With the disintegration of the less dense posterior of the body,the central shaft was completely exposed,and finally,the tip at the front end finally fell off from the central shaft.Compared the discharging process of protrichocysts with the known type of extrusomes,trichocysts.We found that they were similar but slightly different:both of them extended quickly and ejected some sharp structures,and an annular particulate was observed at the front end of the trichocyst which was similar to that in the protrichocysts,and might be also related to the function of membrane fusion in the discharging process.But protrichocysts was more complicated than trichocysts in the discharging process:the cap disintegrated,then the body extended in two steps,and finally ejected the central shaft.It further confirmed that the protrichocysts were indeed different from the trichocysts.Compared with toxicysts which exist in the classes Haptorida and Prostomatea,although the extension mechanism of protrichocyst was completely different from that in toxicyst,the final push-out of the central shaft was similar to the process that toxicyst finally ejected the toxins inside the structure.Substances in the packaged structure of both protrichocysts and toxicysts were eventually exposed,so it was not excluded that the central shaft also contains some toxic components.Comparing with the other two types of extrusomes,we considered that the protrichocyst may have a defensive function,whether from chemical or physical point of view.3 Study on the components of protrichocystsAfter dyed with 0.0025%Alcian blue solution,the cap of protrichocyst was blue,but the body was not.Acidic mucus can be specifically marked by Alcian blue solution,it indicated that the cap may contained acidic mucus but the body did not.Tubulin protein can be specifically fluorescence labeled by FLUTAX-2.And after the specific fluorescence labeling of FLUTAX-2,the cap of protrichocyst could be labeled but the body part did not.The results of electron microscope and specific fluorescence labeling of FLUTAX-2 indicated that the cap of protrichocyst contained tubulin protein,but the body did not.Samples of untreated cells,protrichocysts,and protrichocysts-deficient cells were analyzed by polyacrylamide gel electrophoresis and high performance liquid chromatography.Bands of proteins and peaks of chromatographic of different samples were compared.Bands of proteins at 110 kDa,95kDa,50kDa were considered as the main components of protrichocysts;The peaks of chromatographic at 20.54-20.56,15.44-15.53 and 15.89-16.00 min were considered as the characteristic peaks of the main components of protrichocysts proteins.In addition,the specific peaks at 11.35,19.33,19.96 and 27.40 min of protrichocysts might corresponded to some active substances related to the discharging process.Results of secondary mass spectrometry of three samples indicated that,most of the specific proteins of protrichocysts,e.g.small guanine protein of Rab family,guanine nucleotide regulatory protein,transitional endoplasmic reticulum protein,cation transporter protein,and two kinds of proton pumps(V-type proton pump,vesicle proton transfer inorganic pyrophosphatase)were related to membrane fusion.And these proteins have strong homology in eukaryotes.We speculated that signals of membrane fusion might be transferred to the cell by guanine nucleotide regulatory protein to produce signal Then through the combination of small guanine protein of Rab family and its effect factors,membrane fusion was prepared to occur.Transitional endoplasmic reticulum protein and cation transporter played a regulatory role in this process by regulating proteins and polypeptides related to it and transporting cations,respectively.Two kinds of proton pumps might promote membrane fusion by transporting hydrogen ions and participated in the formation and discharge of protrichocysts.In addition,vesicle proton transfer inorganic pyrophosphatase could use energy more efficiently.The vesicle proton transfer inorganic pyrophosphatase has not been found in the dense core vesicles of higher animals.The findings of this study confirmed the previous view that the evolution in extrusomes of protozoa and dense core vesicles in higher animals were belong to convergent evolution.A stable scheme of discharge inducing and collecting of protrichocysts was established for the first time in this paper.The discharging process of protrichocysts and its characteristic were summarized in detail.The composition of this complex organelle was identified for the first time,and proteins related to the discharging process were compared with other dense core vesicles in eukaryotes.This study provided deeper understandings of the discharging mechanism and its function of the protrichocysts from different perspectives.The results also provided new data for us to understand the complexity structure in ciliate,as well as the evolution of dense core vesicles in ciliates and eukaryotes. |
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