Despite being a major phytoplankton group in high energy environment, dinoflagellates are especially sensitive to turbulence. Under laboratory conditions, agitations are known to reduce cell proliferation. The mechanism for the phenomena was suggested to be disruption of the "cell division apparatus" implying the activation of the spindle assembly checkpoint. To investigate spindle checkpoint in a dinoflagellate, an anti-microtubular drug, nocodozole, was used. We demonstrated that nocodazole reversibly prolonged the G₂+M phase of the dinoflagellate cell cycle.

Spindle checkpoint induced cell cycle arrest in C. cohnii, however, was short when compared with that of other eukaryotes. We also observed that mechanically-induced transient cell cycle arrest at G₁ phase, in both the heterotro...[ Read more ]

Despite being a major phytoplankton group in high energy environment, dinoflagellates are especially sensitive to turbulence. Under laboratory conditions, agitations are known to reduce cell proliferation. The mechanism for the phenomena was suggested to be disruption of the "cell division apparatus" implying the activation of the spindle assembly checkpoint. To investigate spindle checkpoint in a dinoflagellate, an anti-microtubular drug, nocodozole, was used. We demonstrated that nocodazole reversibly prolonged the G₂+M phase of the dinoflagellate cell cycle.

Spindle checkpoint induced cell cycle arrest in C. cohnii, however, was short when compared with that of other eukaryotes. We also observed that mechanically-induced transient cell cycle arrest at G₁ phase, in both the heterotrophic species Crypthecodinium cohnii and the photosynthetic species Heterocapsa triquetra. This suggested that activation of spindle checkpoint, which involves G₂/M arrest, is not essential to the mechanically-induced cell cycle arrest in dinoflagellates.

We further investigated the signaling pathway involved in mechanically-induced G₁ arrest in dinoflagellate. Calcium is the second messenger in many cytological processes, including transduction pathways in many mechano-transduction. We demonstrate here that mechanical shaking and caffeine, the ryanodine receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate C. cohnii. Dantrolene, an ryanodine receptor antagonist, but not the IP₃ receptor antagonist 2-APB, dose-dependently inhibited shaking-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently induced cell cycle arrest in dinoflagellates without arresting cellular growth. Prolonged shaking can substantially abolish caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores. Fluorescence-conjugated ryanodine gave positive labellings, which can be blocked by ryanodine, in the cortice of C. cohnii cells. Both caffeine and shaking abolished the fluorescence of chlorotetracycline-labelled cortical calcium stores. Calcium ionophores did not induce cell cycle arrest, and calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. This implicated that calcium depletion of the internal stores, but not calcium elevation per se, is involved.

Using fluorescence-conjugated diphydropyridine (DHP), positive labellings were observed in the cortice of C. cohnii cells, which can be blocked by DHP antagonist, nifedipine. DHP agonist, Bay K, induced calcium mobilization of the CTC-stained calcium stores. Using membrane potential sensitive dye, DiSC3(5), membrane potential changes were recorded when C. cohnii cells subjected to mechanical stimulation and potassium ion. Pharmacological studies also suggested that the DHPR-like protein acted upstream of the RyR-like protein in the dinoflagellate. Accumulated data, therefore, is consistent with a skeletal muscle type excitation-contraction coupling-like mechanism involved in the mechanically-induced cell cycle arrest in a unicellular organism.