Two distinct functions for PI3-kinases in macropinocytosis
Two distinct functions for PI3-kinases in macropinocytosis
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DOI：
10.1242/jcs.134015
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发表时间：
2013-09-15
影响因子：
4
通讯作者：
Kay, Robert R.
中科院分区：
文献类型：
--
作者：
Hoeller, Oliver;Bolourani, Parvin;Kay, Robert R.
类-1 PI 3激酶是肌动蛋白细胞骨架的主要调节剂，其对趋化性、吞噬作用和巨胞饮作用的精确贡献尚未得到解决。我们使用系统的遗传切除来研究这个问题，在不断增长的网骨藻细胞。质谱显示，缺乏1类PI 3-激酶的整个基因组互补的五重突变体仅保留野生型PtdIns（3，4，5）P-3水平的10%。对叶酸的趋化性和细菌的吞噬作用在五倍突变体中正常进行，但巨胞饮作用被废除。在这种情况下，PI 3-激酶显示出专门的功能，其中只有一个直接与总PtdIns（3，4，5）P-3水平相关：大胞饮体起源于PtdIns（3，4，5）P-3的斑块，具有相关的富含F-肌动蛋白的皱褶，两者都依赖于PI 3-激酶1/2（PI 3 K1/2）而不是PI 3 K4，而皱褶转化为囊泡需要PI 3 K4。一个生物传感器来自Ras结合域的PI 3 K1表明，Ras激活整个囊泡形成。结合试验表明，RasG和RasS与PI 3 K1/2和PI 3 K4的相互作用最强，并且任一Ras的单个突变体具有严重的巨胞饮缺陷。因此，PI 3-激酶在生长的网骨藻细胞中的基本功能是巨胞饮，其中它们具有两种不同的功能，由至少两种单独的Ras蛋白支持。
Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P-3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P-3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P-3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.