Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.
Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization.
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DOI：
10.1083/jcb.136.6.1271
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发表时间：
1997-03-24
期刊：
影响因子：
--
通讯作者：
Cardelli J
中科院分区：
文献类型：
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作者：
Buczynski G;Grove B;Nomura A;Kleve M;Bush J;Firtel RA;Cardelli J
磷脂酰肌醇3-激酶（PI 3-激酶）参与控制细胞增殖、肌动蛋白细胞骨架组织和调节胞内细胞器之间的囊泡运输。在盘基网柄藻中至少有三个基因，DdPIK 1、DdPIK 2和DdPIK 3，编码与哺乳动物110-kD PI-3激酶在激酶结构域内的氨基酸序列最密切相关的蛋白质。DdPIK 1和DdPIK 2的突变体（Δ ddpik 1/ddpik 2）在液体培养基中生长缓慢。使用FITC-葡聚糖（FD）作为液相标记物，我们确定突变株在胞饮作用中受损，但在珠粒或细菌的吞噬作用中正常。显微镜和生物化学方法表明，从酸性溶酶体到非酸性溶酶体后空泡的液相运输速率在突变细胞中降低，导致液相流出减少。通过透射电镜测定，突变细胞也几乎完全没有大的溶酶体后空泡。而Δ ddpik 1/ddpik 2细胞在其他膜运输的调节中功能正常。例如，放射性标记脉冲追踪实验表明，在突变株中，溶酶体酶α-甘露糖苷酶沿着分泌途径的转运速率和分选效率是正常的。此外，膜的收缩空泡网络（可能通过膜运输连接到内体途径）在突变细胞中功能和形态正常。光学显微镜观察发现Δ ddpik 1/ddpik 2细胞比野生型细胞更小，形状更不规则; 1-3%的突变细胞也通过薄的胞质桥连接。扫描电镜表明，突变体细胞含有大量的丝状伪足突出横向和垂直从细胞表面，和荧光显微镜表明，这些丝状伪足富含F-肌动蛋白积累在皮质模式在控制细胞。最后，Δ ddpik 1/ddpik 2细胞响应并更快地向cAMP移动。总之，这些结果表明，DdPIK 1和DdPIK 2基因产物调节内体途径中的多个步骤，并可能通过肌动蛋白组织的变化在细胞形状和运动的调节中起作用。
Phosphatidylinositide 3-kinases (PI 3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization, and the regulation of vesicle trafficking between intracellular organelles. There are at least three genes in Dictyostelium discoideum, DdPIK1, DdPIK2, and DdPIK3, encoding proteins most closely related to the mammalian 110-kD PI-3 kinase in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (Δddpik1/ddpik2) grows slowly in liquid medium. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic postlysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large postlysosomal vacuoles as determined by transmission EM. However, Δddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radiolabel pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme α-mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologically normal in mutant cells. Light microscopy revealed that Δddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1–3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a cortical pattern in control cells. Finally, Δddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization.