MICRURGICAL STUDIES ON PROTOPLASM1

Summary 1. Relation between nucleus and cytoplasm. Evidence is reviewed of interrelations between the interkinetic nucleus and the cytoplasm, together with recent findings concerning such interrelations between the female nucleus, the male pronucleus, and the karyocytoplasm of the fertilized starfish egg. 2. Structural components of the protoplasm. Relatively stable and unstable structures (sol-gel transformations) can be distinguished. Illustrations are given of cell nuclei, some of which are liquid (spinning glands) so that the progressive deposition of viscid material in the cytoplasm distorts the nucleus into a highly multilobulated shape. Other nuclei (dipteran salivary glands) react as solids from being packed with chromosomes. The boundaries of these chromosomes can be detected by injecting carbon particles which collect in narrow spaces between them. 3. Miscibility of protoplasm with water. Cells can be injected with water which spreads through the cytoplasm. More is tolerated if the water contains monovalent salts, particularly potassium chloride. 4. Internal osmotic pressure. The capillarity of spaces within a living cell, e.g. muscle fibre, complicates the evaluation of osmotic pressures when determining the freezing-point of the living cell interior. The question also arises whether the freezing-point depression of cellular extracts is valid for determining the osmotic pressure of the living cell. Fundulus eggs, considered impermeable to water and to electrolytes, can be shown to be permeable to water by the fact that they develop high internal pressures when immersed in hypotonic solutions. The lack of swelling is due to the inelastic envelopes of the individual eggs. 5. An electrolytic solution compatible with cytoplasm. The preparation of a solution approaching that of the interior of a myxomycete has been done by micro-injecting solutions of varying constituents and ascertaining those exhibiting least toxicity. 6. Extraneous coats. A study of the physical properties of the protoplasmic surface film is complicated by the presence of extraneous coats. The most generalized extraneous coat serves as an intercellular cement, the consistency of which is conditioned by the pH and the proportion of calcium and sodium in the medium. The permeability of multicellular membranes is affected by the condition of this cement. 7. Hyaluronidase. A hyaluronidase-like substance from sea-urchin sperm dissolves the jelly coats of the eggs, but has no effect on the intercellular cement. In this way it resembles the hyaluronidase of mammals which dissolves the mucinous collagen but not the intercellular cement. 8. The ‘plasma membrane’. Removal of extraneous coats leaves behind a bounding film on the protoplasm. This film has flowing characteristics and is liquid in the presence of calcium in the environment. 9. The hydrogen-ion concentration of the cell interior. The determination is valid for the continuous aqueous phase of protoplasm. The pH virage of this is slightly, if at all, affected by the presence of the living protein which is relatively inert. Intracellular vacuoles have their own pH values which are independent of the pH of the protoplasmic aqueous phase. Neutral red, a chloride of a coloured base, accumulates in acidic vacuoles and stains fatty globules, while phenol red, a sodium or potassium salt of coloured acid, colours the cytoplasm diffusely and only later tends to accumulate in alkaline vacuoles and in the nucleus which is alkaline. Therefore, in a consideration of the pH of protoplasm it is essential to take into account various factors. Lack of such consideration and the fact that certain regions in the cell are affected by variations in external pH have given rise to confusion in the literature. The only stable region which is well buffered and which, as long as the cell is alive, maintains a constant pH value, is the continuous aqueous phase of protoplasm, that of the cytoplasm being in the close neighbourhood of pH 6–8 and that of the cell nucleus, 7-6-7-8. 10. The action of salts on the interior of protoplasm. Reactions to the micro-injection of salts indicate that the proteins in protoplasm are predominantly on the alkaline side of their isoelectric points. Calcium induces coagulation, while sodium and potassium have a dispersive action. In some cases, cell inclusions exist which are agglutinated by sodium chloride. This feature may lead to erroneous conclusions in centrifugation experiments in which the degree of sedimentation of visible granules is used as a criterion for viscosity. 11. Living and dying protoplasm. Living proteins are relatively inert to chemical interaction. Upon the initiation of cytolysis the interactions become prominent accompanied by denaturation of the proteins. The acid of injury may be explained by the increase of ionizable carboxyl groups resulting from the conversion of protein macromolecules to low molecular weight proteins. 12. Relation of structure to ‘one-way’ permeability. The cells of the renal proximal tubule in tissue culture lose their ‘one-way’ permeability to phenol red when they grow out as flattened sheets. They regain it on becoming oriented about incipient centres of secretion.