title: A Raft-derived, Pak1-regulated entry participates in alpha2beta1 integrin-dependent sorting to caveosomes.
abstract: 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.
authors.affiliation: Department of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, FI-40351 Jyväskylä, Finland.
authors.forename: Mikko
authors.initials: M
authors.lastname: Karjalainen
authors.forename: Elina
authors.initials: E
authors.lastname: Kakkonen
authors.forename: Paula
authors.initials: P
authors.lastname: Upla
authors.forename: Heli
authors.initials: H
authors.lastname: Paloranta
authors.forename: Pasi
authors.initials: P
authors.lastname: Kankaanpää
authors.forename: Prisca
authors.initials: P
authors.lastname: Liberali
authors.forename: G Herma
authors.initials: GH
authors.lastname: Renkema
authors.forename: Timo
authors.initials: T
authors.lastname: Hyypiä
authors.forename: Jyrki
authors.initials: J
authors.lastname: Heino
authors.forename: Varpu
authors.initials: V
authors.lastname: Marjomäki
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism*; D022461:Caveolins / Q000737:chemistry; D045744:Cell Line, Tumor; D002966:Clathrin / Q000378:metabolism; D029822:Enterovirus B, Human / Q000378:metabolism; D006801:Humans; D038982:Integrin alpha2beta1 / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D054462:p21-Activated Kinases / Q000378:metabolism*
chemical_list: D000952:Antigens, Polyomavirus Transforming; D022461:Caveolins; D002966:Clathrin; D038982:Integrin alpha2beta1; D000584:Amiloride; C515903:PAK1 protein, human; D054462:p21-Activated Kinases; D010738:Type C Phospholipases