Autophagy regulates the formation of primary cilia, which in turn affects autophagy. The relationship between autophagy and cilia is known to be bidirectional although the specific mechanisms involved have yet to be elucidated. In this study, we found for the first time that ATG8 protein localizes in the basal body of the dorsal kineties and the base of the ventral cirri in Euplotes amieti. ATG8 protein maintains the structural integrity of cilia and plays a role in the construction of the cortical ciliature and microtubule cytoskeleton associated with cilia. ATG8 gene interference leads to the degradation of IFT88, the transport protein in cilia, thus inhibiting the generation of cilia, and affecting the swing of cilia. This influences the swimming speed and cilia pattern, leading to death in Euplotes amieti.

FOP is a centriole satellite protein involved in ciliogenesis. Although centriole satellites are involved in centrosome and ciliumrelated protein trafficking, their functions related to ciliary assembly and maintenance of ciliary microtubule stability remain unclear. In this study, the function of the FOP gene in Euplotes amieti was investigated by interfering with its expression using RNAi. As a result, expression levels of the ciliary assembly-related proteins BBS8 and IFT88 were down-regulated. Swimming speeds also decreased and the Euplotes were only able to spin in circles, which suggested that the FOP protein is an important protein involved in ciliary motion. Further observations of Euplotes amieti microstructure and ultrastructure via immunofluorescence and transmission electron microscopy revealed that FOP not only participated in the formation of the ventral ciliary basal body but also played an important role in the maintenance of cortical microtubules, which is fundamental for the morphological structure of Euplotes amieti.

CEP43 is a centriole satellite protein involved in ciliogenesis. Although centriole satellites are involved in centrosome and cilium-related protein trafficking, their functions related to ciliary assembly and maintenance of ciliary microtubule stability remain unclear. In this study, the function of the CEP43 gene in Euplotes amieti was investigated by interfering with its expression using RNAi. As a result, expression levels of the ciliary assembly-related proteins BBS8 and IFT88 were down-regulated. Swimming speeds also decreased and the Euplotes were only able to spin in circles, which suggested that the CEP43 protein is an important protein involved in ciliary motion. Further observations of Euplotes amieti microstructure and ultrastructure via immunofluorescence and transmission electron microscopy revealed that CEP43 not only participated in the formation of the ventral ciliary basal body, but also played an important role in the maintenance of cortical microtubules, which is fundamental for the morphological structure of Euplotes amieti.

艾美游仆虫(Euplotes amieti)含有几乎所有已知的纤毛病基因,但绝大多数基因及其表达产物的细胞定位和功能未知.为明确中心粒蛋白43(CEP43)和卷曲螺旋域蛋白13(CCDC13)在艾美游仆虫中的细胞以及亚细胞定位,本研究采用免疫荧光和免疫电镜技术对其进行显微与超微结构观察.免疫荧光结果显示,CEP43主要定位于艾美游仆虫的细胞核、腹面纤毛器(口围带、尾棘毛、额腹横棘毛)的基体及其附属微管,CCDC13主要定位于腹面纤毛器杆部和基体以及银线系统,附属微管及大核未见其定位.CEP43与γ-微管蛋白定位相同,CCDC13与γ-微管蛋白仅在腹面定位相同.2种蛋白在免疫电镜下显示与荧光标记定位相同,而且CCDC13在额腹棘毛基部的胶体金数量远多于CEP43.结合已有研究推测,纤毛形成后多余的CEP43受γ-微管蛋白复合体调控且被募集于细胞核,CCDC13参与形成银线系统,但为增加生长期艾美游仆虫的微管再生能力,附属微管结构不需要CCDC13的参与.本结果为进一步研究上述蛋白在腹毛类纤毛虫中调节和维持皮层微管类细胞骨架的装配和稳定性中的作用和机制提供资料.

The pattern of blood fluid shear stress (FSS) is considered the main factor that affects ciliogenesis in human umbilical vein endothelial cells (hUVECs), the underlying mechanism is unclear. Microfluidic chamber experiments were carried out to load hUVECs with low fluid shear stress (LSS, 0.1 dynes/cm2) or high fluid shear stress (HSS, 15 dynes/cm2). Van Gogh2 (Vangl2), a core protein in the planar cell polarity (PCP) pathway, was silenced and overexpressed in hUVECs. Immunofluorescence analysis showed that primary cilia assemble under LSS while disassembling under HSS. Vangl2 expression was consistent with cilia assembly, and its localization showed a polar distribution under LSS. Furthermore, the average number of ciliated cells and primary cilia length were increased in the Vangl2 overexpressing cell lines (the OE group) but decreased in the Vangl2 silenced cell lines (the SH group). When these cells were loaded with different FSS, more ciliated cells with longest primary cilia were observed in the LSS loaded OE group compared with those in the other groups. Immunoprecipitation showed that the interaction between Bardet-Biedl syndrome 8 (BBS8) and Vangl2 was enhanced following LSS loading compared to that under HSS. However, the interactions between phosphorylated dishevelled segment polarity protein 2 (pDvl2), kinesin family member 2a (Kif2a), and polo-like kinase 1 (Plk1) and Vangl2 were restrained following LSS loading. Overall, the results indicated that Vangl2 played a significant role during LSS-induced primary cilia assembly by recruiting BBS to promote the apical docking of basal bodies and by restraining Dvl2 phosphorylation from reducing primary cilia disassembly.

为获得艾美游仆虫(Euplotes amieti)大核基因组结构特征、分析基因功能及其表达调控方式,研究采用高通量测序技术对艾美游仆虫进行了大核基因组与转录组测序,结果显示基因组测序最终得到原始reads数据为10.92 Gb,过滤后得到50287条Contigs.GC含量较低,为31％;其中两端同时具有端粒的微染色体数量为27542条,占54.76％,只含有一端端粒的基因数量为6118条.Contigs进行基因结构分析,96.5％的基因能够被预测出功能,最终得到27650条基因,平均外显子长度为311.69 bp;内含子较短,平均长度为150 bp.转录组测序结果为76219898条,拼接后获得38588条转录组Unigenes,其平均长度为1189 bp.将转录组的38588条Unigenes比对发现有2％—3％基因发生了编程性移码,其中,绝大多数为+1PRF;除此之外,艾美游仆虫的终止密码子还存在重配现象,其终止密码子为UAA和UAG,而UGA编码半胱氨酸或硒代半胱氨酸.这与游仆虫属的编程性移码及终止密码子重配的特点一致.将27650条基因组Contigs与38588条转录组Unigenes成功获得注释.基因功能分析显示转录本显著富集于细胞生长与死亡、膜转运、运输与细胞学过程等.将基因组与转录组随机各抽取50个基因进行PCR验证.95％的基因均验证成功.结果 表明艾美游仆虫除了具有游仆虫特有的微染色体、密码子重新分配和程序性移码等基因组特征外,还具有大量"联合微染色体",编码大量与感受外界环境变化、细胞周期与蛋白表达调控等相关的特殊蛋白质,并通过一定量的miRNA与锌指类转录因子对基因进行表达调控.

Objective To study how does reduction of blood flow perfusion regulates cytoskeleton rearrangement and its relationship with inflammation during atherosclerosis (AS). Methods The carotid artery stenosis model was established by carotid cannula in New Zealand rabbits. The normal animals were set as control group, and animals in high-fat diet were set as high-fat group. The localization and expression of vascular inflammation-related proteins MCP-1, planar cell polarity signaling pathway-related proteins DVL2 and VANGL2, and cytoskeletal proteins BBS8 and γ-tubulin were determined by immunohistochemistry staining,qPCR and Western blot. Results The cannula group and high-fat group but not control group showed intimal hyperplasia.The localizations of DVL2 and VANGL2 were found mainly at the endovascular hyperplasia in the cannula group, while the positive localizations of them were hardly found in the other two groups. Moreover, the expressions of the seproteins have been significantly up-regulated in the cannula group as compared to that in the control or high-fat group. Conclusions Reduction of blood flow perfusion is involved in the early inflammation of AS by increasing expression of MCP-1, and regulates the BBS8 and γ-tubulin during the rearrangement of microtubule cytoskeleton.

动脉粥样硬化是心血管疾病的主要发病原因.大量研究显示,动脉粥样硬化的形成与内皮细胞的初级纤毛相关.探讨内皮细胞中的初级纤毛装配和解聚的机制,对于揭示动脉粥样硬化的发病机制具有重要的理论和实践意义.目前,作为平面细胞极化(PCP)信号通路的特有组分VANGL平面细胞极性蛋白2(VANGL2)在内皮细胞中与初级纤毛相关的研究甚少.因此,本研究构建VANGL2的慢病毒稳定干扰和过表达人脐静脉内皮细胞(hUVEC)系.从形态学上对初级纤毛分布的情况进行标记,同时用免疫荧光对VANGL2可能相互作用的蛋白质进行共定位检测,并利用免疫印迹进一步对其下游蛋白质蓬乱蛋白(DVL2)、初级纤毛装配相关蛋白质巴特-比德尔综合征8(BBS8)和纤毛内转运系统88(IFT88)、初级纤毛解聚相关蛋白质运动蛋白家族成员2A(KIF2A)和143位点处丝氨酸磷酸化的蓬乱蛋白(pDVL2)进行检测,用免疫共沉淀检测与VANGL2、下游蛋白质DVL2相互作用的蛋白质.结果 表明,免疫荧光标记初级纤毛的荧光图显示,过表达VANGL2后初级纤毛数量显著增多(P＜0.05),其长度也较其他组更长,其定位方向更倾向于VANGL2富集的位置,并位于初级纤毛的基部.免疫荧光共定位显示,BBS8、IFT88均与VANGL2共定位,在过表达VANGL2组中均呈极性分布,PLK1也与VANGL2存在共定位情况,但整体上PLK1的表达量较低.免疫印迹结果显示,过表达VANGL2时,其下游蛋白质DVL2上调(P＜0.01).同时,初级纤毛解聚相关蛋白质KIF2A也上调(P＜0.01).此外,与初级纤毛解聚途径激活有关的pDVL2下调(P＜0.05),初级纤毛装配相关的蛋白质BBS8、IFT88发生上调(P＜0.01).免疫共沉淀结果显示,VANGL2与DVL2存在相互作用关系,且VANGL2和DVL2均与KIF2A相互作用,但DVL2与KIF2A相互作用的程度较强.综上结果表明,VANGL2可能通过上调DVL2并增加DVL2与KIF2A的相互作用而促进初级纤毛的解聚过程.同时,通过下调pDVL2、上调初级纤毛装配相关蛋白质BBS8和IFT88而增加初级纤毛的装配过程.但在过表达VANGL2组中,初级纤毛装配的过程比解聚占优势,两者最终的平衡决定初级纤毛的发生情况,为进一步研究VANGL2在内皮细胞中初级纤毛装配和解聚的机制研究提供见解.

This study aims to explore the mechanism of fluid shear stress in regulating the primary cilia assembly or disassembly in human umbilical vein endothelial cells (hUVECs) using microfluidic chamber experiments. Immunofluorescence analysis showed that primary cilia assembled under disturbed fluid shear stress (DF) of 1 dyne/cm2, while disassembled under unidirectional shear stress (USS) of 15 dynes/cm2. Disheveled (Dvl2) in Wnt signaling pathway was effectively co-immunoprecipitated with Bardet–Biedl syndrome proteins 8 (Bbs8) and γ-tubulin. Compared with those in the control group, the percentages of ciliated cells with Dvl2 overexpression were found to be 67% and 59.667%, respectively, under USS and DF (an increment of 21–38.7%); while, those with Dvl2 silencing were 16% and 32.667%, respectively, under USS and DF (a decrement of 23–30%). Further, the expression of Bbs8 and γ-tubulin was decreased by RNA interference of Dvl2 but increased with Dvl2 overexpression. The results indicated that Dvl2 played a pivotal role during DF-induced primary cilia assembly, and was important for apical docking of basal bodies through Bbs8 and γ-tubulin.

Wnt信号路通是一类以分泌型Wnt蛋白为配体激活的信号通路,包括经典Wnt信号通路和非经典Wnt/PCP信号通路和Wnt/Ca2+信号通路三类.近年来,大量研究显示Wnt信号通路参与了血管系统的多种生理和病理过程,其中在血管新生和血管钙化的研究最为深入,现就该信号通路在此过程中的作用及其潜在的心血管病诊治价值进行综述.

目的:通过转录组测序分析获得不同切应力作用下人脐静脉内皮细胞的基因的表达谱,为进一步探索切应力影响内皮细胞形态和功能的机制提供依据.方法:以人脐静脉内皮细胞为材料,通过Streamer系统建立6通道可调控切应力的流体动力学细胞模型,以层流切应力(15 dynes/cm2)为对照,以低切应力(0.1 dynes/cm2)为实验组,分别加载细胞18h.提取总RNA逆转录合成cDNA,建立文库,以二代测序平台Illumina HiSeq中进行扩增和测序.结果:序列比对结果显示,有19986个基因比对上,新转录本分析显示各组新转录本数约占总转录本数的50％.基因表达差异分析显示,较对照组,低切应力组表达上调基因983个,表达下调基因701个.GO分析显示,有18499个基因得到了归类注释,绝大多数基因富集到生物学过程.KEGG分析显示,富集Top20的信号通路与细胞周期、DNA复制和细胞分裂、细胞应激和凋亡等生物学过程相关.结论:低切应力作用不仅仅激活内皮细胞中细胞的增殖相关基因,同时也涉及到DNA损伤修复和凋亡相关基因.

目的 获得蓬乱蛋白2(DVL2)基因干扰和过表达慢病毒表达系统,并在人脐静脉内皮细胞中(hUVECs)稳定表达.方法 ①聚合酶链反应扩增目的基因,设计合成shRNA,以慢病毒表达质粒为基础构建DVL2干扰和过表达载体,酶切电泳、实时荧光定量聚合酶链反应(qRT-PCR)和测序技术鉴定载体构建是否成功;②以3质粒系统在HEK293T细胞中包装病毒,通过荧光显微镜观察计数并计算病毒滴度;以嘌呤霉素筛选稳定转染的hUVECs,通过荧光显微镜观察计数获得转染效率.将感染好的细胞分为BC组(hUVECs空白对照)、NC组(HBLV-GFP-PURO阴性对照)、干扰组(pHB-shRNA-HDVL2)及过表达组(pHBLV-HDVL2).③通过qRT-PCR与Western blotting检测分别从mRNA和蛋白表达水平验证目的基因的干扰和过表达水平.结果 ①插入慢病毒表达载体的基因片段与目的基因的碱基序列完全一致.干扰序列峰形图为单峰,无突变.②病毒包装后,NC组、干扰组、过表达组滴度分别为2×108、2×108和1×108 TU/ml,感染人脐静脉内皮细胞的感染效率达98％.③干扰组DVL2的表达较BC组降低,差异有统计学意义(P<0.05),过表达组较BC组升高,差异有统计学意义(P<0.05),其中干扰效率为61％,蛋白质过表达水平为BC组的2.7倍.结论 DVL2基因干扰和过表达慢病毒载体构建成功,并能够在原代hUVECs中稳定表达.

The primary cilia,as an independent cellular component,are widely distributed on the surface of various types of non-proliferating cells.Their life cycle begins at rest and ends before mitosis.Although the structure can only be observed by electron microscopy,it has the powerful functions of sensing mechanical,fluid,light,and chemical signals. When its structure and function are disturbed,it will lead to a series of cilia-related diseases,involving bone formation, kidney,heart and other organs and fields.During the heart development,the normal cardiovascular system rhythm mainte-nance and the pathologic process,the primary cilia play an important role by regulating a series of signaling pathways,and it is the key receptor for the cardiovascular system to adapt to stress and maintain homeostasis.In the future,the study of primary cilia function and regulation mechanism will provide a theoretical basis for the prevention and treatment of cilia diseases.

Fluid shear stress (FSS) may alter ciliary structures and ciliogenesis, and it has been reported that the Wnt signaling pathway may regulate cilia assembly and disassembly. The present study aimed to investigate the effects of FSS on primary cilia, the Wnt/beta-catenin and Wnt/PCP signaling pathways, and the association among them. In the present study, human umbilical vein endothelial cells were subjected to FSS of differing velocities for various periods of time using a shear stress device. Subsequently, immunofluorescence and quantitative polymerase chain reaction were used to detect the expression and localization of the following core proteins: P-catenin in the Wnt/beta-catenin signaling pathway; and dishevelled segment polarity protein 2 (Dv12), fuzzy planar cell polarity protein (Fuz) and VANGL planar cell polarity protein 2 (Vang12) in the Wnt/planar cell polarity (PCP) signaling pathway. Furthermore, the colocalization of Dv12 with the basal body was analyzed under low FSS and laminar FSS. The results demonstrated that low FSS promoted the expression of Dv12 and its colocalization with the basal body. Although Fuz expression was decreased with increasing duration of FSS, no visible alterations were detected in its localization, it was ubiquitously localized in the ciliated region. Conversely, the expression of Vang12 was increased by laminar FSS, and beta-catenin was translocated into the nucleus at the early stage of low FSS. These findings suggested that Dv12 may participate in low FSS-induced ciliogenesis and beta-catenin may participate at the early stage, whereas Vang12 may be associated with laminar FSS-induced cilia disassembly.

齐墩果酸(oleanolic acid,OA)是一种广泛存在于自然界中的五环三萜类天然化合物,已有研究表明OA具有抗肺癌活性,但是具体机制尚未完全阐释清楚.本研究旨在证实OA是否能够诱导非小细胞肺癌A549细胞凋亡,并探索该效应是否与线粒体凋亡通路相关.我们将不同浓度的OA作用于细胞后,使用MTT法检测A549细胞的生长情况.采用AV/PI法进行染色后,经流式细胞仪检测其凋亡率,同时使用Hoechst 33342染色并使用荧光显微镜观察并拍照;Westen-blotting检测OA对A549细胞的线粒体凋亡通路相关蛋白相对表达水平的影响.MTT结果显示OA能够抑制A549细胞的生长,并呈时间剂量依赖性.AV/PI法染色结果显示OA能够诱导A549细胞凋亡,亦呈时间剂量依赖性.Hoechst 33342法染色结果相似.Westen-blotting结果表明OA能够上调A549细胞线粒体凋亡通路相关蛋白Bax,tBid,Cleaved caspase 3的相对表达水平,同时下调该通路Bcl-xl,Bcl 2的相对表达水平.以上所有的结果表明OA能够显著抑制肺癌A549细胞的生长并通过调节线粒体凋亡通路相关蛋白的表达水平而诱导其凋亡.

OBJECTIVE:The present study aimed to investigate the ability of hesperidin to suppress the migration and invasion of A549 cells, and to investigate the role of the SDF-1/CXCR-4 cascade in this suppression.METHODS:We performed a Transwell migration assay to measure the migratory capability of A549 cells treated with 0.5% DMSO, SDF-1α, AMD3100 or hesperidin. The SDF-1 level in the culture medium was determined by an enzyme-linked immunosorbent assay (ELISA) to detect whether different concentrations of hesperidin affected SDF-1 secretion. A wound-healing assay was performed to determine the effects of different concentrations of hesperidin on the migration inhibition of A549, H460 and H1975 cells. Additionally, the effect of various hesperidin concentrations on the rate of A549 cell invasion and migration was examined with and without Matrigel in Transwell assays, respectively. Western blot analysis was used to evaluate the protein levels of CXCR-4, MMP-9, CK-19, Vimentin, p65, p-p65, p-IκB, IκB, p-Akt and Akt. RT-qPCR was used to detect the mRNA levels of CXCR-4, MMP-9, CK-19, Vimentin, p65, IκB, SDF-1 and Akt.RESULTS:The Transwell migration assay indicated that SDF-1α promoted A549 cell migration, while AMD3100 and hesperidin significantly inhibited the migratory capability. The wound-healing assay demonstrated that hesperidin treatment significantly reduced the rate of wound closure compared with the control group in a dose-dependent manner. Similarly, the migration and invasive abilities of A549 cells, H460 and H1975 cells treated with hesperidin were significantly decreased compared with the control group. The ELISA data suggested that hesperidin attenuated the secretion of SDF-1 from A549 cells in a dose-dependent manner. Furthermore, western blot analysis indicated that SDF-1α treatment significantly increased the levels of CXCR-4, p-p65, p-IκB and p-Akt in A549 cells. In contrast, AMD3100 or hesperidin reversed the effect induced by SDF-1α through decreasing the expression of CXCR-4. Subsequent RT-qPCR and western blot analyses also confirmed that hesperidin had a significant effect on the expression of EMT-related proteins, including MMP-9, CK-19 and Vimentin, in A549 cells.CONCLUSION:In summary, we demonstrated that hesperidin inhibited the migratory and invasive capabilities of A549 human non-small cell lung cancer cells by the mediation of the SDF-1/CXCR-4 signaling cascade, thus providing the foundation for the development of hesperidin as a safer and more effective anticancer drug for non-small cell lung cancer.

Lung cancer has high incidence and mortality rates worldwide. In the present study, the mechanisms by which hesperidin decreases the viability and induces the apoptosis of human non-small cell lung cancer (NSCLC) A549 cells were investigated. Initially, MTT and flow cytometric assays were performed to evaluate the effects of hesperidin on the viability and apoptosis of A549 cells and human normal lung epithelial BEAS-2B cells. The results revealed that hesperidin has no negative effects on the human normal lung epithelial BEAS-2B cells and the viability of cells treated with various concentrations of hesperidin was inhibited in a time- and dose-dependent manner compared with the control groups. Subsequently, the expression levels of proteins involved in the mitochondria-associated apoptotic pathway were studied by western blot analysis. Hesperidin was identified to induce A549 cell apoptosis by downregulating the levels of B-cell lymphoma-2 (Bcl-2) and Bcl extra large protein and simultaneously upregulating the levels of Bcl-2-associated X protein, BH3 interacting-domain death agonist (Bid), tBid, cleaved caspase-9, cleaved caspase-3 and cleaved poly(adenosine diphosphate ribose)polymerase. The effect of hesperidin on the cell cycle was assessed by flow cytometry. Hesperidin was observed to cause G0/G1 arrest of A549 cells by decreasing the expression of cyclin D1 and increasing the expression of p21 and p53. In summary, it was demonstrated that hesperidin induced apoptosis through the mitochondrial apoptotic pathway and induced G0/G1 arrest in human NSCLC A549 cells. Therefore, hesperidin may be developed as a potential therapeutic drug for the treatment of NSCLC.

Objective To investigate the effects of rabbit carotid artery cannula on atherosclerosis formation and β-catenin expression.Methods Fifteen 2-month-old New Zealand rabbits weighing(2.0±0.2)kg were randomly divided into three groups,high-fat diet group,left common carotid artery cannula group and left common carotid artery cannula plus high-fat diet group,5 cases in each group,and taking the right blood vessels in the left common carotid artery cannula group served as the control group.The animals were sacrificed after 9-week feeding,and the total rabbit carotid artery in each group was taken;the real-time PCR and immunohistochemistry were used to detect β-catenin mRNA and protein expression targeting in rabbit common carotid artery tissue.Results The real-time PCR results showed that the β-catenin mRNA expression in the left common carotid artery cannula group was higher than that in the control group,high fat diet group and the left common carotid artery cannula + high-fat diet group.The immunohistochemistry results showed that,except for the control group,various groups had the β-catenin protein location in the cytoplasma,moreover which in the left carotid artery cannula group and left common carotid artery cannula +high-fat diet group mainly located in the area of intimal hyperplasia.Conclusion β-catenin is highly expressed in the atherosclerotic vessel wall caused by rabbit carotid artery cannula.

生物化学课程是医学本科生的专业必修课,主要讲述生命体细胞中的化学进程.其内容抽象、复杂,学生普遍反映较难理解.因此,如何提高学生的学习兴趣,帮助其更加深入地理解生物化学课程的众多知识点是该课程教学改革的重要内容.选用生物化学中物质代谢章节内容,设计代谢与疾病发生的专题式文献,探讨其在辅助学生理解物质代谢相关知识点的作用,取得了较好的实践结果.

This study aimed to investigate the assembly characteristics of ciliature and cortical microtubules and the localization of tubulins in different depths and regions of the cortex. The hypotrichous ciliates have closely arranged cilia and a highly complex microtubular system. Direct fluorescence and immunofluorescence labeling were used to observe ciliary organelles and cortical microtubular cytoskeleton in Euplotes eurystomus. An immunofluorescence analysis demonstrated that alpha-tubulin localized to the ventral and dorsal ciliary organelles and their associated microtubules, while gamma-tubulin localized to the basal bodies of ciliary organelles, macronuclear membrane, and excretory pore of a contractile vacuole in the interphase. A direct fluorescence analysis showed that the ciliature and cortical microtubules in the deep cortex were more clearly marked by fluorescent taxoid (FLUTAX). Interestingly, alpha- and gamma-tubulins also colocalized to the ringlike ciliary base-associated microtubules of dorsal kineties. The short microtubular bundles between the bases of transverse cirri could be marked by FLUTAX and gamma-tubulin rather than alpha-tubulin, suggesting that tubulins in cortical microtubules in E. eurystomus varied, and the differentiation of cortical microtubules in the hypotrichous ciliate tended to be versatile. Also, during morphogenesis, gamma-tubulin also localized to the base of ciliary primordium, where new basal bodies were formed, suggesting that the endocellular position of gamma-tubulin in ciliates was related to basal bodies and regulated by the cell cycle. This study might help understand the assembly characteristics and tubulin composition of microtubules in different depths and regions of the cortex in hypotrichous ciliate E. eurystomus.