Karjalainen Mikko, Kakkonen Elina, Upla Paula, Paloranta Heli, Kankaanpää Pasi, Liberali Prisca, Renkema G Herma, Hyypiä Timo, Heino Jyrki, Marjomäki Varpu
Department of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, FI-40351 Jyväskylä, Finland.
Mol Biol Cell. 2008 Jul;19(7):2857-69. doi: 10.1091/mbc.e07-10-1094. Epub 2008 Apr 30.
We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-alpha2beta1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that alpha2beta1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.
我们之前已经表明，人微小核糖核酸病毒埃可病毒1型（EV1）在2小时内与其受体α2β1整合素一起被转运至小窝小体。在此，我们表明大多数早期摄取并非通过小窝进行。由抗体或EV1结合聚集的α2β1整合素最初从脂筏内化至微管泡状结构中。这些囊泡积累液相标记物，但最初并不与小窝蛋白-1或内化的猴病毒40（SV40）共定位。此外，内化的内体不包含糖基磷脂酰肌醇（GPI）锚定蛋白或小窝蛋白1，这表明聚集的α2β1整合素分别不进入富含GPI锚定蛋白的内体区室或小窝蛋白途径。内体在15分钟至2小时之间进一步成熟为更大的多囊泡体，并同时在内部募集小窝蛋白-1或SV40。在SAOS-α2β1细胞中，细胞进入受p21激活激酶（Pak）1、Rac1、磷脂酰肌醇3激酶、磷脂酶C和肌动蛋白调节，但不受发动蛋白2调节。一种氨氯地平类似物5-（N-乙基-N-异丙基）氨氯地平可阻断感染，导致整合素在早期微管泡状结构中积累，并阻止其结构成熟为多囊泡结构。我们的结果共同表明，α2β1整合素聚集定义了其自身的进入途径，该途径依赖于Pak1，但不依赖网格蛋白和小窝蛋白，并且能够将货物分选至小窝小体。
