【作者】 韩轶；

【导师】 侯连生；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 在变形虫盘基网柄菌(Dictyostelium discoideum)中,细胞自主机制和细胞内信号的共同作用控制着细胞类型的分化、空间模式和形态发生。约10~5的细胞趋化性群聚,形成了一个多细胞有机体,沿着有机体的中轴,细胞分化成不同的类型,最后,形成柄细胞和孢子细胞组成的一个成熟的子实体。在发育期间,细胞类型的分化、空间模式的形成和形态发生的完成与细胞表面的蛋白分子有密切的关系。gp150分子是一种跨膜糖蛋白,由lagC基因编码,通过异嗜性粘着作用来调节细胞间的粘着。为探索gp150分子的功能,我们以野生型KAX-3细胞和突变型AK127细胞(gp150缺失突变型)为材料,比较了他们在形态发生,膜蛋白组成和蛋白激酶活性的不同。通过此次实验,得到了以下几个结果: 1) KAX-3细胞可以顺利的完成多细胞发育,并最终形成子实体;AK127细胞的多细胞发育只能聚集成疏松细胞团,无法跨越紧密细胞丘阶段。疏松细胞团又解聚,最终死亡。 2) gp150蛋白的缺失明显改变了许多膜蛋白的表达情况,其中68kD,65kD,60kD,50kD,45kD,37kD,34kD,31kD,17kD等膜蛋白的表化较明显。最值得注意的是17kD蛋白。当野生型细胞发育进行到细胞丘阶段,17kD膜蛋白的含量大大增加,提示了该蛋白与细胞发育有密切关系。17kD为一种富含组蛋白的膜周边蛋白,可促进肌动蛋白的聚合。它的结构与白细胞介素1β(IL1β)和成纤维细胞生长因子的结构相似。提示了gp150除了粘附功能外,对其他膜蛋白的表达有一定影响。 3) 在盘基网柄菌多细胞的发育的聚集阶段,蛋白激酶A(PKA)的活性在两种细胞中的变化情况基本一致,这说明gp150并不影响发育早期PKA的活性。在蛞蝓体时期,PKA的活性在KAX-3细胞细胞中表现为振荡波,并且在此期的最后阶段,有一个大的振幅,这可能是为了最终的发育做准备。而AK127细胞在蛞蝓体的初期也表现为震荡信号,然后PKA的活性到达的水平与其在聚集时期相当。这表明随着发育进程gp150蛋白调控PKA的活性于适当的范围。这些研究表明,gp150不仅仅作为粘附分子发挥作用,它还作为一种信号分子,对不同的信号途径有调控作用。 4) 在盘基网柄菌多细胞的发育的聚集阶段,蛋白激酶C(PKC)的活性在两种细胞中的变化情况基本一致,这说明gp150并不影响发育早期PKC的活性。在细胞丘和蛞蝓体时期,PKC活性在两种细胞中都表现为一种震荡波。不同的是,PKC活性在AK127细胞中的振动波幅大于KAX-3细胞。这暗示着gp150蛋白本身,或者与其他信号分子一起,调控PKC活性在适当的范围内发挥作用。如更多还原

【Abstract】 In Dictyostelium amoebae, cell-type differentiation, spatial patterning, and morphogenesis are controlled by a combination of cell- autonomous mechanisms and intercellular signaling. A chemotactic aggregation of ~ 10~5 cells leads to the formation of a multicellular organism. Cell-type differentiation and sorting result in a small number of defined cell types organized along an anteroposterior axis. Finally a mature fruiting body is created by the terminal differentiation of stalk and spore cells. During development, membrane proteins play an important role in cell-type differentiation, including spatial patterning, and morphogenesis. The membrane glycoprotein gpl50 is encoded by the lagC gene and mediates cell-cell adhesion by heterophilic binding during Dictyostelium discoideum development. In experiment, we take KAX-3(wild type) cell and AK127(lagC-null) as experiment material. To study the function of gpl50, we have examined the effect of gpl50 on morphogenesis, membrane proteins and protein kinases during Dictyostelium discoideum development1) The KAX-3 cells can complete development, and form a mature fruiting body finally. The AK127 cells fail to establish tight mound and the loose mound disaggregates before the cells eventually die.2) The absence of gpl50 change the expression of many membrane proteins.The membrane proteins which change apparently include 68kD, 65kD, 60kD, 50kD, 45kD, 37kD, 34kD, 31kD, 17kD. One of the most interesting changes is about protein of 17kD. In mound stage, The content of 17kD increases distinctly, and it suggests that the protein of 17kD is important during Dictyostlium multicellular development. The protein of 17kD is an histidine rich actin binding protein from Dictyostelium discoideum. The structure of the protein is similar to interleukin and the fibroblast growth factor. It indicates that gpl50 may function in other signal transduction pathways and may affect on the expression of the membrane proteins.3) At aggregation stage during Dictyostlium multicellular development, the activity of PKA changes alike in both KAX-3 cells and AK127 cells. It suggest gpl50 does not influence the activity of PKA at aggregation stage. At slug stage, the activity of PKA in KAX-3 cells functions in the form of oscillatory waves, and has a large amplitude at the end of this stage, which could prepare for terminal differentiation. While AK127 cells have the same oscillatory waves at the beginning of slug stage, then the activity of PKA reach the level of the aggregation stage. It suggests that thegpl50 plays a role to keep the activity of PKA in appropriate range as well as time. 4) At aggregation stage during Dictyostlium multicellular development, the activity of PKC changes alike in both KAX-3 cells and AK127 cells. It suggest gpl50 does not influence the activity of PKC at aggregation stage. In mound and slug stage, the activity of PKC functions in the form of oscillatory waves in both KAX-3 cells and AK127 cells, but mutant cells have oscillatory waves with large amplitude. It indicates that gpl50 and other signal molecules keep the activity of PKC in appropriate range. The regulation of PKC activity was not accurately mediated, if no gpl50 protein.These studies indicate that gpl50 functions not only as cell adhesion molecule, but as signal molecule mediating signal transduction pathways.更多还原