Two-Track Control of Cellular Machinery for Photomovement in Spirogyra varians (Streptophyta, Zygnematales).

Plants and freshwater algae devoid of flagella evolved various photomovements to optimize their photosynthetic efficiency. The filaments of Spirogyra varians exhibit complex swaying and undulating movement and form a compact mat which enables them to adjust their light exposure. Photomovement of filament fragments (1-10 cells) was analyzed using various photoreceptor and cytoskeleton inhibitors under monochromatic light. Different patterns of movement were observed under red and blue light. The filaments showed positive phototropism under blue light. Under red light, the filaments bent to undulating shape, but rapidly became unbent by a short exposure to far-red light suggesting the involvement of phytochrome in this movement. The mechanical effector for the red-light response was microtubules; the movement was inhibited effectively by the microtubule inhibitor, oryzalin. The blue-light movement was partially inhibited by the single treatment of either cytochalasin D or oryzalin, but was completely blocked when both chemicals were applied together. Phototropin-signaling inhibitors, wortmannin and LY294002, reversibly inhibited the blue-light movement. Caffeine treatment reversibly stopped the blue-light movement, while the red-light movement was not affected by calcium inhibitors. Our results suggest that the complex photomovement of S. varians is the result of a two-track control of microtubules and microfilaments signaled by the combination of phytochrome and phototropin-like receptors.

A promising new wavelength region for three-photon fluorescence microscopy of live cells.

We report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.

Studies on conjugation of Spirogyra using monoclonal culture.

We succeeded in inducing conjugation of Spirogyra castanacea by incubating algal filaments on agar plate. Conjugation could be induced using clone culture. The scalariform conjugation was generally observed, while lateral conjugation was rarely. When two filaments formed scalariform conjugation, all cells of one filament behaved as male and those of other filament did as female. Very rarely, however, zygospores were formed in both of pair filaments. The surface of conjugation tube was stained with fluorescently labeled-lectins, such as Bandeiraea (Griffonia) simplicifolia lectin (BSL-I) and jacalin. BSL-I strongly stained the conjugation tubes, while weakly did the cell surface of female gamete first and then that of male gamete. Jacalin stained mainly the conjugation tubes. Addition of jacalin inhibited the formation of papilla, suggesting some important role of jacalin-binding material at the initial step of formation of the conjugation tubes.