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For the purpose of gaining molecular information such as molecular distances and molecular motion under the fluorescence microscope, we have developed a time-resolved microfluorimetric method, a time-resolved fluorescence spectroscopic technique combined with the optical microscopy: the excitedstate lifetime of fluorescent molecules (about 1-20 nsec) in a small spot in a single living cell is measured under the microscope using a focussed pulsed laser beam as an excitation light source (0.5 micron spot diameter, (lambda)=365 nm, FWHH= 14 psec, 4 MHz repetition rate) and a synchroscan streak camera as a detector with time-resolution. A signal-to-noise ratio better than 100 was obtained for fluorescein labelled band 3 (1 label/band 3) in an area of 2.5 micron-diameter (containing 2x105 band 3) in a single erythrocyte ghost after signal accumulation for 50 sec. The time-resolved microfluorimetry has been applied to the studies of(l) the assembly mechanism ofcell adhesion protein (E-cadherin, a calcium-dependent cell adhesion receptor protein) at the site of cellcell contact in keratinocytes in culture and (2) endosome-endosome fusion in fibroblasts in culture. (1) Observation by video microscopy indicated that the calcium-induced assembly ofE-cadherin at the sites of cell contact takes place via lateral migration of cadherin in the plasma membrane of keratinocytes. Association of cadherin molecules at the molecular level has been assessed by observing the resonance energy transfer (RET) from fluorescein (donor) to reactive red 8 (acceptor), both attached to monoclonal IgG antibody specific to E-cadherin (Occurrence of RET would decrease the lifetime of the donor). The fluorescence lifetime decay data of fluorescein at various locations in cells indicated the occurrence ofRET only at the boundary region ofkeratinocytes in the high-calcium medium, suggesting that cadherin molecules form aggregates at the sites of cell contact, where they function as a cell adhesion molecule between two cells. (2) Quantitative observation of fusion of endosomes in single cultured cells (NIHI3T3) has been made. Our method is based on the observation of internal content mixing of endosomes by detecting RET between 7-nitrobenz-2-oxa- 1 ,3-diazol-4-yl (NBD)-labelled a2-macroglobulin (energy donor) and water-soluble sulforhodamine (energy acceptor), which are sequentially endocytosed by a cell. One of the most important characteristics of the fusion assay reported here is that it allows fast quantitative measurement (measuring time, 10-30 sec) of fusion of each endocytotic vesicle discernible under the fluorescence microscope. The excited-state lifetime of the donor in each endosome was measured (1.6 micron-diameter spot) in various parts of the cell. Acceleration of the fluorescence decay of the donor (occurence of RET) was detected, the extent of which increased with an increase of the incubation time with the acceptor. This result indicates the mixing of the energy donor with the acceptor in endosomes, showing that fusion between successively formed endosomes can take place in cells. Blocking of endosome acidification with NH4C1 failed to supress endosome fusion. The cells treated with cytochalasin D at concentrations sufficient to destroy the actin filaments did not show any indications of endosome fusion, suggesting that actin filaments are required for enconter and/or fusion processes of endosomes.
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