Inner-arm dynein-f of Chlamydomonas flagella is a heterodimeric dynein. We performed conventional in vitro motility assays showing that dynein-f translocates microtubules at the comparatively low velocity of approximately 1.2 microm/s. From the dependence of velocity upon the surface density of dynein-f, we estimate its duty ratio to be 0.6-0.7. The relation between microtubule landing rate and surface density of dynein-f are well fitted by the first-power dependence, as expected for a processive motor. At low dynein densities, progressing microtubules rotate erratically about a fixed point on the surface, at which a single dynein-f molecule is presumably located. We conclude that dynein-f has high processivity. In an axoneme, however, slow and processive dynein-f could impede microtubule sliding driven by other fast dyneins (e.g., dynein-c). To obtain insight into the in vivo roles of dynein-f, we measured the sliding velocity of microtubules driven by a mixture of dyneins -c and -f at various mixing ratios. The velocity is modulated as a function of the ratio of dynein-f in the mixture. This modulation suggests that dynein-f acts as a load in the axoneme, but force pushing dynein-f molecules forward seems to accelerate their dissociation from microtubules.

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Polyethylene glycol (PEG) plays an important role as an inhibitor of the electrodeposition. In this study, we investigated the potential dependence of PEG (molecular weight: 3000) adsorption and desorption processes on the gold single crystal substrate. High-speed atomic force microscopy (HS-AFM) was applied for observing the dynamic behaviors of the nucleation and the growth of PEG adsorption. The in-situ observation was conducted at the several potentials where the cyclic voltammogram measurement showed the broad peak of the reduction current. As the applied potential became more cathodic, the adsorption morphology changed from film-like, through the sphere, to the large irregular cluster. When the potential was switched to anodic potential, HS-AFM could show the dissolution process of PEG. Finally we demonstrated the effect of the tip force by modifying the feed-back circuit of AFM.

SummaryThe cytoplasmic tail (CT) of the influenza virus hemagglutinin (HA) consists of an amino acid sequence that is highly conserved among various subtypes of the influenza A virus. To study the effect of CT size on HA-mediated membrane fusion, we constructed a cDNA encoding a chimeric protein in which the size of CT of influenza virus HA (from A/Hong Kong/1/68; subtype H3) was enlarged by tagging with enhanced green fluorescent protein (EGFP). The EGFP-tagged HA (HA-EGFP) or wild-type HA was individually expressed in HeLa cells, and cell membrane fusion activities were examined using dual-labeled chicken red blood cells. The red blood cells were labeled with both lipidic and aqueous dyes to verify the fusion activity of the HA-EGFP at different steps of the fusion process: 1) lipid-mixing, 2) content-mixing and 3) fusion pore formation. Our data showed that the binding activity of HA towards red blood cells was not affected by enlargement of the CT of HA with EGFP. However, its lipid-mixing, subsequent content-mixing, and fusion pore formation activities were drastically impaired by the enlargement. Therefore, we conclude that CT size is crucial for progression from the binding step to the lipid-mixing step in the process of HAmediated membrane fusion.