Protoplasmic streaming of an internodal cell of Nitella flexilis; its correlation with electric stimulus

The sudden cessation or sudden decrease in velocity of the protoplasmic streaming of Nitella flexilis is observed whenever an action potential is elicited. The action potential can be generated by an electric stimulus after its refractory period, whether the flow is at a complete standstill or on the way to recovery. The membrane potential is generally decreased more or less when the rate of flow is decreased on application of salts or other agents. There is, however, no parallelism between these two. The membrane potential decreases proportionally with applied voltage of subthreshold intensity, while the rate of flow does not change appreciably. Only on application of a superthreshold voltage does the flow stop suddenly. In one case the rate of flow decreased to half without appreciable decrease in membrane potential. In another case it continued flowing at about one-half rate, although the membrane potential was almost zero. The Q₁₀ of the rate of flow is about 2, while it is 1.1 to 1.5 for the membrane potential. The sudden cessation of the protoplasmic streaming is supposed to be caused by the temporary formation of certain interlinkages among contractile protein networks in the endoplasm during excitation at the cathodal half of Nitella.     

Rhythmicity in the protoplasmic streaming of a slime mold,Physarum polycephalum. I. A statistical analysis of the electric potential rhythm

The electric potential difference (1 to 15 mv.) between two loci of the slime mold connected with a strand of protoplasm changes rhythmically with the same period (60 to 180 seconds) as that of the back and forth protoplasmic streaming along the strand. Generally some phase difference is observed between them. Periods of the electric potential rhythm show a Gaussian distribution. Amplitudes give a somewhat different distribution curve. Wave forms are not always simple harmonic ones, but are distorted more or less. However, auto-correlation analysis proves that there is a dominant rhythm of a nearly constant period which coincides with the mean period of the Gaussian distribution curve. Calculations made on an assumption that the electric potential rhythm is the result of many elementary rhythms (i.e., same periodicity, arbitrary phase angles) distributed throughout the plasmodium, give a satisfactory coincidence with the observed distribution for the amplitude. The predominance of a rhythm of a nearly constant periodicity suggests the existence of well organized interactions among components of a contractile protein network, the rhythmic deformation of which is supposed to be responsible for the protoplasmic streaming and for the electric potential rhythm.     

DISTRIBUTION OF MATURATION-PROMOTING FACTOR IN STARFISH OOCYTE STRATIFIED BY CENTRIFUGATION

In starfish, cytoplasm taken from maturing oocytes is capable of inducing breakdown of the germinal vesicle and subsequent maturation when injected into immature oocytes. The cytoplasmic factor has been designated as "maturation-promoting factor" (MPF). Ooplasm was stratified by centrifugation of maturing oocytes in density-graded Ficoll-seawater, without disrupting the cell. Three strata were distinguished beginning with the centripetal side: oil cap, hyaline layer and yellow layer. MPF activity was shown to be localized in the hyaline layer. Electron microscopic observation revealed that the hyaline layer contains Golgi complexes, many lucent vesicles and multi-vesicular bodies as distinct organelles, but seldom contains such inclusions as the lipid droplets forming the oil cap, mitochondria, yolk and pigment granules contained in the yellow layer. Based on these observations, a possible cytoplasmic component with MPF activity is discussed.     

IONIC COMPOSITION OF THE CYTOPLASM OF NITELLA FLEXILIS

The K, Na and Cl concentrations of the chloroplast layer andthe flowing cytoplasm of Nitella flexilis have been determinedby applying an internal perfusion technique, which enabled usto avoid contamination of ions from the cell sap. K, Na andCl concentrations of the chloroplast layer are 110, 26 and 136mM and those of the flowing cytoplasm are 125, 5 and 36 mM respectively.The cell sap contains 80 mM K, 28 mM Na and 136 mM Cl. Althoughthere are some variations in these values among samples, theflowing cytoplasm is rich in K and poor in Cl and especiallyin Na. The exchange of K and Na across the tonoplasl occursfairly easily (half-time, a few hours), while that of Cl occursextremely slowly (half-time, a few days). ¹This work was supported by Research Grants from the Ministryof Education of Japan     

Oocyte Competence to Maturation-inducing Hormone. I. Breakdown of Germinal Vesicles of Small Oocytes in Starfish,Asterina pectinifera*

1-Methyladenine (1-MeAde) is the endogenous maturation-inducing substance (MIS) in starfish. However, small oocytes have no competence to 1-MeAde even at the concentration of 10^?5M. Furthermore, when they were injected with cytoplasm of fully-grown (large) and maturing (1-MeAde-treated) oocytes, known to contain maturation-promoting factor (MPF), they did not undergo germinal vesicle breakdown (GVBD). On the other hand, germinal vesicles (GV) of the small oocytes underwent nuclear breakdown when the small oocytes were fused with the large maturing oocytes. Therefore it is concluded that the GV of the small oocytes are capable of undergoing nuclear breakdown in the presence of the sufficient MPF, but that the small oocytes can not amplify the injected MPF. Fused cells displayed particular shape changes during the course of nuclear breakdown of both the large and the small oocytes.     

INDUCTION OF OOCYTE MATURATION BY DISULFIDE-REDUCING AGENT IN THE SEA CUCUMBER, STICHOPUS JAPONICUS

1-Methyladenine (1-MeAde) is known to be a natural inducer of starfish oocyte maturation. Disulfide-reducing agents such as dithiothreitol (DTT) and 2, 3-dimercapto-1-propanol (BAL) are known to mimic the action of 1-MeAde in inducing starfish oocyte maturation. Although 1-MeAde failed to induce oocyte maturation in sea cucumbers, breakdown of germinal vesicles and subsequent meiotic behaviour of chromosomes were induced by the treatment with DTT in the pronase-treated oocytes of the sea cucumber, Stichopus japonicus. These findings suggest that reduction of disulfide bonds plays an important role in triggering oocyte maturation in some marine forms such as echinoderms.     

STUDIES ON NITELLA HAVING ARTIFICIAL CELL SAP II. RATE OF CYCLOSIS AND ELECTRIC POTENTIAL

The rate of cyclosis, the value of resting potential and theexcitability were studied on the Nitella internode whose cellsap was replaced with artificial solutions. K⁺ to Ca⁺⁺ ratioin the cell sap should be within 2 to 50 in order to maintainthe normal rate of cyclosis and the resting and action potentialsat least for several days. Replacement of the cell sap witha solution containing Li⁺, Na⁺ or Rb⁺ in place of K⁺ had noappreciable effect on the activity of the internode for severaldays, while gradual but marked decrease in the rate of cyclosisand the resting potential was observed with Cs⁺. There was noappreciable difference between the effects of Cl^– andNO₃^– on cyclosis, resting and action potentials, whileSO₄^–– decreased them to some extent. The rate ofcyclosis was not affected appreciably by changes in osmoticconcentration between 0.17 and 0.30 M. (Received September 6, 1963; )     

FORMATION OF THE FERTILIZATION MEMBRANE BY INSEMINATION OF IMMATURE STARFISH OOCYTES PRETREATED WITH CALCIUM-FREE SEAWATER

When immature oocytes of the starfish, Asterina pectinifera , were treated with calcium-free seawater for 1 hr and then inseminated in normal seawater, they formed several blisters, indicative of polyspermy, and raised fertilization membranes. These oocytes continued to have intact germinal vesicles. Electron microscopic study revealed that the egg surface remained virtually unchanged after the treatment with calcium-free seawater. Upon insemination, however, the cortical granules broke down and the fertilization membrane was formed. These immature oocytes with ferilization membranes underwent maturation (germinal vesicle breakdown) after treatment with 1-methyladenine.
The treatment with calcium-free seawater seems to bring about some physiological change on the surface of immature oocyte, which bestows some attributes of maturation but is insufficient to mature the oocytes completely.