The transactivator protein of human immunodeficiency virus type 1 Tat has the unique property of mediating the delivery of large protein cargoes into the cells when present in the extracellular milieu. Here we show that Tat fusion proteins are internalized by the cells through a temperature-dependent endocytic pathway that originates from cell membrane lipid rafts and follows caveolar endocytosis. These conclusions are supported by the study of the slow kinetics of the internalization of Tat endosomes, by their resistance to nonionic detergents, the colocalization of internalized Tat with markers of caveolar endocytosis, and the impairment of the internalization process by drugs that disrupt lipid rafts or disturb caveolar trafficking. These results are of interest for all those who exploit Tat as a vehicle for transcellular protein delivery.

The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse cell membranes. This ability has generated great interest due to potential therapeutic applications. However, the relevant cellular pathway and its dynamics have not been elucidated yet. Here we unravel the intracellular fate of exogenously added Tat fused with green fluorescent protein (GFP) in live HeLa and CHO cells, from the early interaction with the plasma membrane up to the long-term accumulation in the perinuclear region. We demonstrate that the internalization process of full-length Tat and of heterologous proteins fused to the transduction domain of Tat exploits a caveolar-mediated pathway and is inhibited at 4 degrees C. Remarkably, a slow linear movement toward the nucleus of individual GFP-tagged Tat-filled caveolae with an average velocity of 3 micro m/h was observed. No fluorescence was observed in the nucleus, possibly suggesting that Tat fusion protein unfolding is required for nuclear translocation. In addition, early sensitivity to cytochalasin-D treatment indicates the essential role of the actin cytoskeleton in the displacement of Tat vesicles toward the nucleus. Our results imply that HIV-1 Tat mediates the internalization of protein cargos in a slow and temperature-dependent manner by exploiting the caveolar pathway.

We describe the screened Korringa-Kohn-Rostoker (KKR) method and the thirdgeneration linear muffin-tin orbital (LMTO) method for solving the single-particle Schrödinger equation for a MT potential. In the screened KKR method, the eigenvectors c RL,i are given as the non-zero solutions, and the energies ε i as those for which such solutions can be found, of the linear homogeneous equations: RL K a R ′ L ′ ,RL (ε i ) c RL,i = 0, where K a (ε) is the screened KKR matrix. The screening is specified by the boundary condition that, when a screened spherical wave ψ a RL (ε, r R ) is expanded in spherical harmonics Y R ′ L ′ (r R ′ ) about its neighboring sites R ′ , then each component either vanishes at a radius, r R ′ =a R ′ L ′ , or is a regular solution at that site. When the corresponding "hard" spheres are chosen to be nearly touching, then the KKR matrix is usually short ranged and its energy dependence smooth over a range of order 1 Ry around the centre of the valence band. The KKR matrix, K (ε ν ) , at a fixed, arbitrary energy turns out to be the negative of the Hamiltonian, and its first energy derivative,K (ε ν ) , to be the overlap matrix in a basis of kinked partial waves, Φ RL (ε ν , r R ) , each of which is a partial wave inside the MT-sphere, tailed with a screened spherical wave in the interstitial, or taking the other point of view, a screened spherical wave in the interstitial, augmented by a partial wave inside the sphere. When of short range, K (ε) has the two-centre tight-binding (TB) form and can be generated in real space, simply by inversion of a positive definite matrix for a cluster. The LMTOs, χ RL (ε ν ) , are smooth orbitals constructed from Φ RL (ε ν , r R ) andΦ RL (ε ν , r R ) , and the Hamiltonian and overlap matrices in the basis of LMTOs are expressed solely in terms of K (ε ν ) and its first three energy derivatives. The errors of the single-particle energies ε i obtained from the Hamiltonian and overlap matrices in the Φ (ε ν )-and χ (ε ν ) bases are respectively of second and fourth order in ε i − ε ν . Third-generation LMTO sets give wave functions which are correct to order ε i − ε ν , not only inside the MT spheres, but also in the interstitial region. As a consequence, the simple and popular formalism which previously resulted from the atomic-spheres approximation (ASA) now holds in general, that is, it includes downfolding and the combined correction. Downfolding to few-orbital, possibly short-ranged, low-energy, and possibly orthonormal Hamiltonians now works exceedingly well, as is demonstrated for a high-temperature superconductor. First-principles sp 3 and sp 3 d 5 TB Hamiltonians for the valence and lowest conduction bands of silicon are derived. Finally, we prove that the new method treats overlap of the potential wells correctly to leading order and we demonstrate how this can be exploited to get rid of the empty spheres in the diamond structure.

We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).

The reversible photoinduced structural changes of a green fluorescent protein (GFP) mutant and their optical control are reported. A photoreversible optically inactive configuration is demonstrated with the absorption peak at 365 nm, which is consistent with a photoisomerization pathway associated with hydrogen-bond breaking in the chromophore environment. We show that this state is involved in the switching dynamics recently discovered in these molecules and we determine the transition rates of the reversible photoconversion processes. These experiments combine to provide the framework for the implementation and optimization of efficient room-temperature GFP-based all-optical memories that use the fluorescent properties of these proteins.