title: Single quantum dot tracking reveals that an individual multivalent HIV-1 Tat protein transduction domain can activate machinery for lateral transport and endocytosis.
abstract: The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.
authors.affiliation: Department of Emerging Infectious Diseases, Graduate School of Medicine, Tohoku University, Sendai, Japan. suzukiy39@med.tohoku.ac.jp
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Chandra Nath
authors.initials: CN
authors.lastname: Roy
authors.forename: Warunya
authors.initials: W
authors.lastname: Promjunyakul
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Hatakeyama
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Gonda
authors.forename: Junji
authors.initials: J
authors.lastname: Imamura
authors.forename: Biju
authors.initials: B
authors.lastname: Vasudevanpillai
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ohuchi
authors.forename: Makoto
authors.initials: M
authors.lastname: Kanzaki
authors.forename: Hideo
authors.initials: H
authors.lastname: Higuchi
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Kaku
mesh_terms: D000199:Actins / Q000378:metabolism; D004705:Endocytosis; D015658:HIV Infections / Q000378:metabolism / Q000821:virology*; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism / Q000821:virology; D018613:Microscopy, Confocal / Q000379:methods; D008870:Microtubules / Q000378:metabolism / Q000821:virology; D057054:Molecular Imaging / Q000379:methods; D011554:Pseudopodia / Q000378:metabolism / Q000821:virology; D045663:Quantum Dots; D053586:Virus Internalization; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000032:analysis* / Q000378:metabolism*
chemical_list: D000199:Actins; D054322:tat Gene Products, Human Immunodeficiency Virus