Eiji Kamitsubo

Cytoplasmic streaming in internodal cells of Nitella under centrifugal acceleration: a study done with a newly constructed centrifuge microscope

SummaryWe constructed a new centrifuge microscope of the stroboscopic type, with which the cytoplasmic streaming inNitella internodal cells under centrifugal acceleration was studied. Under moderate centrifugal acceleration (ca. 50–100×g), the direction of cytoplasmic streaming in an internodal cell ofNitella is parallel to the direction of the subcortical fibrils. The speed of endoplasm flowing contiguous to the subcortical fibrils is neither accelerated nor retarded by moderate centrifugal acceleration. The endoplasmic flow, however, stops suddenly following an electrical stimulus. The endoplasm contiguous to the subcortical fibrils is immobilized transiently at the time of streaming cessation induced by an electrical stimulus under centrifugal acceleration at 50–100×g, even at 900×g. It is suggested that transitory cross bridges between the immobilized endoplasm and the subcortical fibrils are formed at the time of streaming cessation. The bulk endoplasm flows as a whole in the direction parallel to that of the subcortical fibrils and stops promptly upon electrical stimulation. Soon after the stoppage the bulk endoplasm starts to flow passively in the direction parallel to that of the centrifugal acceleration as a result of the centrifugal force.

Effect of supraoptimal temperatures on the function of the subcortical fibrils and an endoplasmic factor inNitella internodes

SummaryThe effect of supraoptimal temperatures onNitella cells was studied with special reference to the function of subcortical fibrils and an endoplasmic factor. Local heat-treatment (50 °C for 1 minute) of an internodal cell ofNitella disclosed that 1. the subcortical fibrils in the treated area remain normal, not affected by the treatment, 2. the subcortical fibrils alone produce no cytoplasmic streaming, 3. the endoplasm contains an extremely heat-labile factor which is indispensable for streaming, and 4. the stagnant endoplasm in the heat-treated area is neither coagulated nor gelated by heat.

CENTRIFUGE MICROSCOPE AS A TOOL IN THE STUDY OF CELL MOTILITY

The centrifuge microscope (CM) is composed of a centrifuge and a microscope optical system designed to observe minute objects, especially living cells, during the application of centrifugal acceleration. Structures and characteristics of various types of CM designed and constructed up to the present and studies done with the CM on cell biology, especially cell motility, are reviewed. These studies include observations of the behavior of cells and cell components in a centrifugal field, determination of the mechanical properties of the cell surface and cytoplasm, microsurgical operations on cells with centrifugal force, and determination of the magnitude and the site of generation of motive force for cell motility.

Light-Induced Changes in the Behavior of Chloroplasts under Centrifugation in Vallisneria Epidermal Cells

Summary Using a centrifuge microscope of the stroboscopic type, effects of light irradiation and chemical applicationon the behavior of chloroplasts in epidermal cells of Vallisneria gigantea were examined. Movements of chloroplasts in the cytoplasmic layer that faces the outer periclinal wall (P side) were exclusively observed. Irradiation with blue light (451.0nm) at a high-fluence rate (24.9 μmol m-2 · sec-1) accelerated the passive gliding of chloroplasts produced by the centrifugal force of 25-40 × g. A similar acceleration was caused by application of EGTA to the dark-adapted cells in the presence of cytochalasin B. In cells preirradiated with red light (650.0nm) at a low-fluence rate (2.2 μmol m-2 · sec-1) for 3 - 4h, the minimal centrifugal force required to induce the passive gliding of chloroplasts increased to over 50 × g. The application of EGTA to these cells brought about the movement of chloroplasts in the central area of the P side, which exhibited a close resemblance to the movement induced by irradiation with blue light in the cells preirradiated with red light. Light irradiation seems to affect some mechanical properties of the cytoplasmic matrix. The significance of these findings in the light of orientation movements of chloroplasts in these cells is discussed.

Apparent Viscosity of the Endoplasm of Characean Internodal Cells Measured by the Centrifuge Method

The apparent viscosity of Nitella endoplasm was determined in vivo by measuring the movement of oil droplets through the endoplasm under various centrifugal accelerations (100–500 × g). It was shown that the endoplasm is highly viscous when a shearing force is absent and is very fluidic at fairly high rates of shear.

The mechanical structure of the cytoplasm of the echinoderm egg determined by gold particle method' using a centrifuge microscope

A method was developed to investigate the mechanical structure of the cytoplasm based on the movement of an intracellular gold particle subjected to centrifugal acceleration (the gold particle method). The movement of the particle in the cell was observed and recorded with a new centrifuge microscope of stroboscopic type (13). In eggs and oocytes of the echinoderms, Clypeaster japonicus, Asterias amurensis, and Asterina pectinifera, the particle moved in the cytoplasm by an applied centrifugal acceleration in the centrifugal direction, but the course was not exactly straight and the velocity fluctuated during the movement, suggesting the existence of a network structure in the cytoplasm. In fertilized eggs, the movement of the particle by the centrifugal acceleration was impeded by the structures of the sperm aster and the cleavage diaster. The apparent viscosity of the cytoplasm in fertilized eggs changed in parallel to the development of the sperm aster and the mitotic diaster in the cell. These results indicate that the asters are really rigid structures in the cell as previously shown by the magnetic particle method (8).

Immobilization of endoplasm flowing contiguous to the actin cables upon electrical stimulus inNitella internodes

SummaryUsing a stroboscopic centrifuge microscope, we demonstrated that, when actin cables (=bundles of F-actin) had been previously removed locally from the cell cortex ofNitella internodes, the passively flowing endoplasm found there under centrifugal force did not stop at all upon electrical stimulus, while the actively flowing endoplasm contiguous to the actin cables at the normal cell cortex promptly stopped following the stimulus and was immobilized for several seconds. The results present evidence that, upon electrical stimulus, the presence of actin cables is required to immobilize the endoplasm flowing contiguous to the actin cables in a state that resists displacement by centrifugal force.

Visualization of a rapid, red/far-red light-dependent reaction by centrifuge microscopy

SummaryUsing a centrifuge microscope with stroboscopic illumination, we examined the effects of light irradiation on the passive movement of chloroplasts in dark-adapted mesophyll cells ofVallisneria gigantea. While irradiation with red light accelerates the passive gliding of chloroplasts produced by centrifugal force, irradiation with far-red light negates this effect. Irradiation with blue light does not accelerate the passive gliding, while red light is completely effective even in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthesis. An apparently active movement of chloroplasts can be induced by irradiation with red or blue light only in the presence of the far-red light-absorbing form of phytochrome. The significance of the reaction in the light with respect to the regulation of cytoplasmic streaming is discussed.

Measurement of motive force for cytoplasmic streaming in characean internodes

SummaryWith an attempt to measure the motive force responsible for cytoplasmic streaming in characean internodal cells, the difference between densities of cytoplasm and vacuolar sap was heightened by about 10 times (density of vacuolar sap was made larger than that of cytoplasm) by replacing the natural vacuolar sap ofChara corallina with an artificial one of higher density. Endoplasmic flow contiguous to the peripheral actin cables (peripheral flow of endoplasm) in the centrifugal direction was not influenced at all by the application of centrifugal acceleration up to 1400 g. We thus concluded that the motive force for the peripheral flow should be much larger than 12dyn/cm2, a figure more than 10 times larger than that for bulk endop lasmic flow so far reported.

Bidirectional flow of endoplasm inPhysarum plasmodium under centrifugal acceleration

SummaryThe behavior of cytoplasmic streaming in plasmodial strand ofPhysarum polycephalum was studied under centrifugal acceleration using a centrifuge microscope of the stroboscopic type. Cytoplasmic streaming in the plasmodium was greatly affected by changes in the acceleration. The endoplasmic flow in the centrifugal direction was accelerated, while that in the centripetal was retarded, by centrifugal acceleration. The centrifugal acceleration required to stop the endoplasmic flow in the centripetal direction did not cause total cessation of streaming but always induced a bidirectional flow of endoplasm in one and the same strand. Each profile of velocity distribution of the bidirectional flow was both parabola with flattened apex. One possible cause of the bidirectional flow is discussed.