title: Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan cross-linkage activates Rac1 for internalization.
abstract: The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction.
authors.affiliation: Department of Emerging Infectious Diseases, Graduate School of Medicine, Tohoku University, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Imamura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Gonda
authors.forename: Chandra Nath
authors.initials: CN
authors.lastname: Roy
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Gatanaga
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ohuchi
authors.forename: Hideo
authors.initials: H
authors.lastname: Higuchi
mesh_terms: D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D019812:Heparan Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D010873:Pinocytosis / Q000235:genetics; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D019812:Heparan Sulfate Proteoglycans; C107953:RAC1 protein, human; D054322:tat Gene Products, Human Immunodeficiency Virus; D020830:rac1 GTP-Binding Protein