Satoshi Kagiwada

Specificity of amphiphilic anionic peptides for fusion of phospholipid vesicles.

We have synthesized five amphiphilic anionic peptides derived from E5 peptide [Murata, M., Takahashi, S., Kagiwada, S., Suzuki, A., Ohnishi, S. 1992. Biochemistry 31:1986-1992. E5NN and E5CC are duplications of the N-terminal and the C-terminal halves of E5, respectively, and E5CN is an inversion of the N- and the C-terminal halves. E5P contains a Pro residue in the center of E5 and E8 has 8 Glu residues and 9 Leu residues. We studied fusion of dioleoylphosphatidylcholine (DOPC) large unilamellar vesicles assayed by fluorescent probes. The peptides formed alpha-helical structure with different degrees; E5NN, E5CN, and E8 with high helical content and E5CC and E5P with low helical content. These peptides bound to DOPC vesicles at acidic pH in proportion to the helical content of peptide. The peptides caused leakage of DOPC vesicles which increased with decreasing pH. The leakage was also proportional to the helicity of peptide. Highly helical peptides E5NN, E5CN, and E8 caused hemolysis at acidic pH but not at neutral pH. The fusion activity was also dependent on the helicity of peptides. In fusion induced by an equimolar mixture of E5 analogues and K5 at neutral pH, E8, E5NN, and E5CN were most active but E5CC did not cause fusion. In fusion induced by E5-analogue peptides alone, E5CN was active at acidic pH but not at neutral pH. Other peptides did not cause fusion. Amphiphilic peptides also appear to require other factors to cause fusion.

Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity.

The amphiphilic anionic peptides E5 and E5L can mimic the fusogenic activity of influenza hemagglutinin(HA). These peptides induced fusion of egg yolk phosphatidylcholine small or large unilamellar vesicles only at acidic pH in a similar manner to viral HA. Acetylation or acetimidylation of the N-terminus of the peptides drastically reduced the fusion activity of the intact peptides, while C-terminal amidation left the activity unchanged. The binding assay suggested that the interaction of the modified peptides with lipid membranes was almost unchanged in comparison with those of the parent peptides, and the CD spectra showed that these peptides were alpha-helical. The results showed the importance of the N-terminus of the peptides on the membrane fusion activity, although why the N-terminal modifications affect the activity is still unclear.

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol‐stabilized bovine‐brain microtubules without microtubule‐associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential‐interference‐contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine‐labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N‐ethylmaleimide, brefeldin A, or GTP‐γ‐S. The association is mediated by trypsin‐sensitive peripheral protein(s) on the Golgi membranes.

Fusion of dioleoylphosphatidylcholine vesicles induced by an amphiphilic cationic peptide and oligophosphates at neutral pH.

Peptide E5 is an analogue of the fusion peptide of influenza virus hemagglutinin and K5 is a cationic peptide which has an arrangement of electric charges complementary to that of E5. We reported that a stoichiometric mixture of E5 and K5 caused fusion of large unilamellar vesicles (LUV) of neutral phospholipids (Murata, M., Kagiwada, S., Takahashi, S. and Ohnishi, S. (1991) J. Biol. Chem. 266, 14353-14358). K5 caused fusion of LUV composed of dioleoylphosphatidylcholine (DOPC) at pH > 10, but not at neutral pH. In the presence of oligophosphates, such as 1 mM ATP, GTP, or polyphosphate, K5 caused rapid and efficient fusion of DOPC LUV at neutral pH without hydrolysis of oligophosphate groups, but another anions such as citrate, acetate, AMP, phosphate, or EDTA were ineffective. The peptide/oligophosphate-induced fusion behaviors have been investigated by a fluorescence resonance energy transfer assay for lipid mixing of LUV and negative staining electron microscopy. At higher ionic strengths ( > 0.3 M KCl) or in the presence of 5.0 mM MgCl2, the fusion was inhibited. Even at the inhibitory conditions, the association of K5 with lipid vesicles at neutral pH was directly confirmed by the Ficoll gradient assay method and by blue shifts of the tryptophan fluorescence of the peptide. A nonhydrolyzable GTP analogue, GTP gamma S, also induced fusion. These observations suggested that the electrostatic interactions between the positive and negative charges of K5 and oligophosphate, respectively, induced complex formation, triggering membrane fusion.

Development of time-resolved microfluorimetry and its application to studies of cellular membranes

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.