[Three-dimensional motion analysis for GLUT4 vesicles in TIRF microscopy].

In this paper, GLUT4 vesicles are observed in real-time under TIRF microscopy and a new three-dimensional single particle tracking algorithm according to the unique features of TIRF is put forward. Firstly a fluorescence correction procedure was processed to solve the problem of fluorescence bleaching over time and mobile vesicles were segmented by an adaptive background subtraction method. Kalman filtering was then introduced to track the granules so as to reduce the searching range and to avoid the disturbance of background noise and false targets. In the experiments the algorithm was applied in analyzing the long-distance movement of GLUT4 vesicles. The experimental results indicate that the algorithm has achieved robust tracking of the vesicles in the imaging plane and has effectively calculated the position in the direction orthogonal to the imaging plane.

Bi-directional transport of GLUT4 vesicles near the plasma membrane of primary rat adipocytes.

Insulin stimulates glucose uptake into adipocytes by mobilizing intracellular membrane vesicles containing GLUT4 proteins to the plasma membrane. Here we applied time-lapse total internal reflection fluorescence microscopy to study moving parameters and characters of exogenously expressed GLUT4 vesicles in basal, insulin and nocodazole treated primary rat adipocytes. Our results showed that microtubules were essential for long-range transport of GLUT4 vesicles but not obligatory for GLUT4 distribution in rat adipocytes. Insulin reduced the mobility of the vesicles, made them tethered/docked to the PM and finally had constitutive exocytosis. Moreover, long-range bi-directional movements of GLUT4 vesicles were visualized for the first time by TIRFM. It is likely that there are interactions between insulin signaling and microtubules, to regulating GLUT4 translocation in rat adipocytes.

Anti-proliferative effects of recombinant iron superoxide dismutase on HepG2 cells via a redox-dependent PI3k/Akt pathway.

The coding sequence for an iron superoxide dismutase (fe-sod) was amplified from the Nostoc commune genome. Recombinant Fe-SOD was overexpressed in Escherichia coli, accounting for approximately 76% of total bacterial protein. Fe-SOD was purified from bacterial lysate by Ni-NTA column chromatography and used to generate an anti-SOD antibody. The purified Fe-SOD was encapsulated in liposomes and delivered to HepG2 liver tumor cells to eliminate cellular superoxide anions. The SOD-loaded cells exhibited lower reactive oxygen species (ROS) levels and higher reduced glutathione (GSH) levels. In Fe-SOD-treated cells, the cell cycle was delayed in the G(1) phase, and HepG2 cell growth slowed in association with dephosphorylation of the serine-threonine kinase Akt. Low-dose H(2)O(2) stimulated Akt phosphorylation, implying that Akt activation in HepG2 cells is redox-sensitive. Akt phosphorylation was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors, suggesting that PI3K is an upstream mediator of Akt activation in HepG2 cells. This study provides insight into recombinant Fe-SOD-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Fe-SOD as an antitumor agent.