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Vol. 19, Issue 7, 2857-2869, July 2008

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A Raft-derived, Pak1-regulated Entry Participates in 2β1 Integrin-dependent Sorting to Caveosomes

Mikko Karjalainen^*,, Elina Kakkonen^*,, Paula Upla^*, Heli Paloranta^*, Pasi Kankaanpää, Prisca Liberali, G. Herma Renkema^||, Timo Hyypiä^¶, Jyrki Heino, and Varpu Marjomäki^*

*Department of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, FI-40351 Jyväskylä, Finland; Department of Biochemistry, University of Turku, FI-20014 Turku, Finland; Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro (Chieti), Italy; ^||Institute of Medical Technology, University of Tampere and Tampere University Hospital, FI-22014 Tampere, Finland; and ^¶Department of Virology, University of Turku, FI-20520 Turku, Finland

Submitted October 31, 2007; Revised April 15, 2008; Accepted April 22, 2008
Monitoring Editor: Jean Gruenberg

We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor 2β1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. 2β1 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 2β1 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-2β1 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 2β1 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.

These authors contributed equally to this work.

Address correspondence to: Varpu Marjomäki (vmarjoma{at}cc.jyu.fi)

Abbreviations used: CTxB, cholera toxin B; EV1, echovirus 1; GPI-AP, glycosyl phosphatidylinositol-anchored protein; SV40, simian virus 40; Pak, p21-activated kinase; HRP, horseradish peroxidase; PFA, paraformaldehyde.