General Physiology: An Outline of the Science of Life

The macro-nucleus with the closely applied micro-nucleus lies in the middle section of the trunk. It is possible with some patience to separate, under the microscope, by sharp cuts the individual parts of the body, in which the head, the neck, and the posterior end-piece of the trunk are always non-nucleated, while the trunk itself contains the two kinds of nuclei. The result of the cross-section is that in every piece the ciliary motion is very much accelerated. All the pieces whirl about their axis and through the water with furious rapidity in the contracted condition. The enormous augmentation of ciliary activity gradually passes away, and then every piece behaves exactly as it behaved when in connection with the whole organism. The nucleated trunk continues its metabolic movements, twitching now forward, now backward, by changing the direction of the ciliary stroke ; the neck at times stretches far out (Fig. 252, d) and gropes restlessly about, although it possesses neither head nor trunk, and at times contracts like a rubber cord (Fig. 252, c) ; the head, being now free from the trunk, runs about over the particles of mud in the water like an independent individual by means of exactly the same ciliary motions as in the

uninjured organism (Fig. 252, e). In short, as regards its movements, every piece behaves exactly as when in connection with the body of the normal Lacrymaria. Upon stimulation, contraction of the myoids and acceleration of the ciliary stroke take place in FIG. 252. — Section of Lacrymaria olor. The black lines indicate the cuts. all pieces, the cilia causing the rotation upon the axis, exactlyras happens upon stimulation in the uninjured protist. In the nonnucleated pieces, this normal motion continues, as a rule, for nearly a day. Then the difference between the non-nucleated and the

nucleated pieces, appears, the former perishing, while the latter regenerate themselves into complete individuals. The behaviour of non-nucleated pieces of cells may, therefore, be summarised in the statement, that, after the passage of a stage of excitation caused by the stimulation of the operation, every piece continues to carry out the movements peculiar to it in the uninjured organism and to react to stimuli in the same manner as before the operation. The normal character of the movements is not changed until the appearance of the phenomena of necrobiosis, which affect the non-nucleated protoplasm and lead to death.

It appears in all such experiments that, after the passage of a stage of excitation caused by the operation, the movements of nonnucleated pieces of protoplasm continue for a long time, frequently for several days, completely unchanged ; they undergo disturbances only in the course of the necrobiosis of the piece and finally cease. The facts discovered by Hofer agree completely with this. If, however, the normal movement of the protoplasm continues for days after the removal of the nucleus, the nucleus cannot be a regulating centre for the movement, and thus the theory falls.

2. Nucleus and Protoplasm as Links in the Metabolic Chain of It appears from the above discussion that the later views upon the dominance of the nucleus in the cell, in whatever form they are presented, are as little justified as the earlier ideas, which recognised the protoplasm alone as the essential bearer of life. Everything suggests that the truth lies between the two, i.e., that neither the nucleus nor the protoplasm alone plays the chief role in the life of the cell, but that the two are concerned equally in the inauguration of vital phenomena.

All the experiments and observations so far upon the relations of the nucleus and the protoplasm show that this view is the correct one. It would lead too far to present all the facts bearing upon this question ; only the more important ones will here be noticed. The first and most significant one is the phenomenon, already mentioned and confirmed by all past vivisection-experiments upon a great variety of cells, that after a longer or shorter time nonnucleated protoplasmic masses invariably perish, just as do nuclei deprived of protoplasm. Unquestionable proof is thus afforded that the vital phenomena of the cell come about only through the undisturbed correlation of the two parts of the cell. That this correlation is a metabolic correlation is a priori evident, since vital phenomena are merely the expression of cell-metabolism. But this fact is proved by special facts relative to phenomena that occur up to the time of death in protoplasmic masses deprived of

a nucleus. During the frequently long time that elapses between the removal of the nucleus and the death of the enucleated protoplasmic mass, certain metabolic phenomena gradually disappear, while many activities continue even until the last moment before death. The disappearance of phenomena shows at once that by the removal of the nucleus the metabolism of the protoplasm has undergone a disturbance. One group of phenomena that disappear relates to the treatment of ingested food, and may be especially well observed in the naked protoplasm of Rhizopoda. If in a Polystomella, whose delicate, snail-like, calcareous shell is filled with a protoplasmic body that is usually uninucleated, a piece of the shell containing nonnucleated protoplasm be skilfully cut off, after some time the protoplasm forms again wholly normal pseudopodia, and for some days behaves like an uninjured Polystomella. Small Infusoria, which serve the organism as food, are still caught by the pseudopodia, which latter are covered with a delicate viscous secretion ; and under certain circumstances these Infusoria can even be killed by the action of the pseudopodial protoplasm surrounding them ; but no digestion takes place.1 The same observation can be made readily upon large Radiolaria, like Thala&sicolla, which can with ease be deprived of its central capsule containing the nucleus. After this operation the large non-nucleated protoplasmic body behaves like a complete Thalassicolla. The pseudopodia hold fast the swimming food-Infu&oria and surround them with their protoplasm. The Infusoria are killed and sometimes even altered in form, but complete digestion never takes place.2 Hofer ('89-'90) observed the same thing in large specimens of Amceba. When he divided under the microscope Amcebce that had devoured Infusoria, so that the latter were present in the nucleated as well as in the non-nucleated half of the protoplasm, those in the latter half underwent feeble digestion at first and then ceased to be affected, while those in the nucleated half were completely digested, as in an uninjured Amceba. It follows from all these experiments that the assimilation of ingested food ceases in the protoplasm after the extrusion of the nucleus.

As with the consumption, so the production of certain substances by the protoplasm ceases after removal of the nucleus. A nonnucleated protoplasmic mass of Polystomella no longer excretes calcium carbonate to complete its calcareous shell, while nucleated pieces repair an imperfection in the shell immediately by laying down new calcareous masses at the wounded place.3 The secretion of slime by the naked protoplasm of Amceba, as Hofer ('89-'90) has shown, is not observed in non-nucleated masses ; hence after enucleation such pieces float in the water, while nucleated pieces, like uninjured Amcebce, immediately attach themselves again to 1 Cf. Verworn ('88). 2 Cf. Verworn ('91). 3 Cf. Verworn ('88).

the bottom by means of the delicate layer of slime, and continue creeping. In non-nucleated pseudopodia of Difflugia at first a secretion of slime takes place, but it soon ceases, and after a few hours these protoplasmic masses likewise lose the power of attaching themselves.1 Finally, the loss of the power of producing cellulose in the formation of a cellwall, which Klebs ('87) observed in plantcells, is a very characteristic phenomenon. In his experiments Klebs made use of the fact that harmless solutions of substances that extract water cause the protoplasmic body of the plant-cell to contract and to break up into separate protoplasmic globules, a phenomenon that is termed by botanists " plasmolysis. " If he put threads of Zygnema or Spirogyra into a 16 per cent, solution of cane-sugar, the protoplasmic body of the cell in many cases broke into two or more globules, of which one contained the single nucleus. Both nucleated and non-nucleated pieces continued to live, in many cases the latter even for six weeks. But during this time a profound difference in the two was shown : the nucleated pieces immediately surrounded themselves with a new cellulose membrane, while the non-nucleated pieces always remained naked. It follows from this experiment that the nucleus with its metabolism takes an essential part in the formation of cellulose. But the experiment is especially interesting from the fact that very recently it has received a desirable completion by another experiment, which Demoor (' 95) has performed upon the cells of Spyrogyra.

In a manner analogous to that in which by means of vivisectionoperations the influence of the nucleus upon the protoplasm was excluded, Demoor succeeded by means of the suitable application of various agents, such as chloroform, hydrogen, cold, etc., in bringing to a standstill the life of the protoplasm while the nucleus remained still active ; in other words, the activity of the protoplasm was excluded. The result was that the nucleus remained living for a considerable time undisturbed, just as after the exclusion of the nucleus the protoplasm shows normal vital phenomena for a considerable time. In Demoor's experiments the vital activity of the nucleus expressed itself just as in the normal life of the cell, pre-eminently by the phenomena of nuclear division. The nucleus proceeded to divide as normally, and to form the well-known complex mitotic figures, and soon two nuclei appeared and separated from one another.2 While, however, in the undisturbed cell upon the separation of the two nuclei in the protoplasm a new cellulose membrane is always formed immediately, completing the division of the whole cell into two daughter-cells, in Demoor's experiments the formation of such a membrane was invariably absent, although the nucleus still con-

tinued to show its normal vital phenomena. While, therefore, the experiments of Klebs prove that the nucleus is necessary for the formation of cellulose, those of Demoor show that the protoplasm also takes part in its production. In other words, cellulose can be formed only by the combined action of nucleus and protoplasm. Besides these experimental results, a considerable number of morphological observations upon very different kinds of cells exist, all pointing toward an active exchange of substance between nucleus and protoplasm. Of great interest are the positions of the nucleus relative to certain substances that are produced or taken in by the cell, which have been demonstrated by Haberlandt upon plant-cells and by Korschelt upon animalcells. Haberlandt's investigations ( '87, '89) have reference to the phenomena of growth of the cell-membrane. In a wide range of material he has established the fact that in certain cases the nucleus exists at the place at which growth-processes are localized. Such cases are the following: where local growth of the cell-wall is necessary to its final form, such as in the thickening on the

FIG. 253. — A, An epidermis-cell of a foliage leaf of Luzula maxima. The nucleus lies in the middle of the cell. B, Epidermis-cells of a leaf of Cypripedium insigne. The upper cell-wall is thickening ; the nuclei lie upon it. C, Epidermis-cell of a leaf of Aloe vermcosa. A swelling is being formed upon the upper cell-wall; upon it lies the nucleus. (After Haberlandt.) outer side of epidermal cells, in the formation of ridges upon the guard-cells of stomata, and in the rudiments of root-hairs that develop by apical growth at the growing point of roots ; and where regeneration of an artificially injured cell-wall takes place; in brief, wherever a special development of material for the cellwall takes place (Figs. 253 and 254). But before the beginning and after the cessation of these various phenomena of growth the nucleus takes no definite position in the cell (Fig. 253, A),

These comprehensive observations of Haberlandt are paralleled by the striking zoological researches of Korschelt ('89). Korschelt has studied chiefly the ova and secreting-cells of insects. In the eggtubes of the ovaries of Dytiscus marginalis, a large water-beetle, the ova are arranged in succession like a string of pearls and separated from one another by a so-called nutrient chamber. This chamber consists of cells which produce and give off nutrient material to the ova. The behaviour and the position of the nuclei of the ova toward this nutrient material is very characteristic (Fig. 255). From the chamber the nutrient material extends into the

ovum in the form of a granular mass and there disposes itself in such a manner that it comes into very close contact with the nucleus. But the most interesting fact is that which makes the activity of the nucleus toward the nutrient material apparent, namely, that the former sends pointed, pseudopodium-like processes into the granular mass where the latter touches it, and only in this direction, and thus very greatly place of contact with the nutrient material. If the latter completely surrounds the nucleus, the whole surface shows pseudopodium-like processes. Korschelt describes a similar phenomenon, especially as regards the nucleus, in a whole series of arthropod and ccelenterate ova. The interesting behaviour of the nuclei in secreting - cells toward the secreted substances forms a counterpart to these phenomena of the ingestion of substance on the part of the nucleus. Here certain relations exist toward the substances produced, which are wholly analogous to those existing in ova toward ingested substances. In the eggs of certain waterbugs, Nepa and Ranatra, there occur peculiar chitinous appendages, the so-called eggrays, which are formed by cells especially differentiated for this purpose. These cells, of which each two unite into a single cell with two nuclei, termed by Korschelt a douUe cell, assume a considerable size and secrete within their body the

FIG. 254. — A, A row of cells from a root of Pisum sativum. Upon the right side three stages in the formation of a root-hair are shown ; the nucleus lies at the places in question. B, Three cells from the root of Cucurbita pepo. A root-hair is beginning to form upon each cell ; the nucleus lies at the place where the hair projects. C, Root-hair of Cannabii sativa. The nucleus lies at the tip of the hair, where the growth is taking place. (After Haberlandt.)

of the two nuclei in this process is very characteristic (Fig. 256, 7). They send out toward the middle, where the secretion is taking place, numerous, frequently branched, pseudopodium-like processes, which increase the nuclear surface upon this side very considerably, while the rest of the surface remains smooth. Such enlargements of the surface of nuclei are wide-spread in the secretingcells of insects (Fig. 256, II), and show that the exchange of substance between protoplasm and nucleus in secretion must be very active. Corresponding to this is the fact observed by Heidenhain ('83), that the nuclei of the cells of the salivary glands behave essentially differently in the state of rest and that of extreme secretion; in rest they send out pointed processes into the surrounding protoplasm, while after continued stimulation they are spherical and possess a smooth surface (Fig. 257). Further, Baum ('86) has found that the nuclei of resting gland-cells stain much more deeply with nuclear stains than the nuclei of gland-cells that have secreted strongly — a sign that the chromatic nuclein must be destroyed in secretion. Lily Huie ('97) has also recently

discovered very profound changes in the nucleus during increased activity of the cell in the secreting-cells of the insecteating marsh-plant Drosera, when the latter is fed with egg-albumin. Greenwood ('96) has likewise observed in the colonial infusorian Carchesmm nuclear changes which develop parallel with the nutrition of the cell. The phenomena of fatigue which Hodge, Lugaro, Mann and others have observed in ganglion-cells during excessive activity and with which we have already become acquainted,1 belong in the same category ; and the same is true of O. Hertwig's observation ( '84) that in eggs that are rich in yolk, the nucleus always moves toward the place where there is the greatest accumulation of protoplasm. Finally, in a wide variety of cells a remarkable change in the size of the nucleus during cell-life 2 can be observed, which can be brought about

only by the nucleus receiving substances from the protoplasm and giving off others to it. Usually, on account of the existence of a nuclear membrane, an exchange of liquid substances only is possible between nucleus and protoplasm. But in many cases where because of the failure or disappearance of such a membrane solid masses may be exchanged, many observers, such as Frommann, Auerbach, Leydig, Brass, Stuhlmann, and others, have observed on the part of the nucleus a direct ingestion or extrusion of granules and flakes. In certain stages in the course of development of many cells there even occurs regularly a disintegration of the nucleus into many small particles, which are resorbed by the protoplasm. Thus, we recall the be-

FIG. 255. — Ovum of Dytiscus marginalia with two nutrient cells above it. Nutrient material is passing from the nutrient cells into the ovum, and the nucleus of the latter is sending out pointed pseudopodia toward this material. (After Korschelt.) haviour of the nuclei observed by R. Hertwig ('88-'89) in the conjugation of ciliate Infusoria : l the fairly large macronucleus breaks up into numerous separate particles and becomes completely re-

FIG. 256.— Form of the nucleus in secreting-cells. 7, Double cells with two nuclei from the eggfollicles of Nepa cinerea. Between the two nuclei a mass of chitin is being secreted for the formation of an egg-ray ; the nuclei have extended pseudopodia toward this side, so that the secreted (granular) mass is circularly enclosed by the nuclear pseudopodia. II, Secreting-cells from the spinning-glands of caterpillars. (After Korschelt.) .sorbed ; the micronucleus grows by the ingestion of substance, differentiates, divides, and gives rise to a new micronucleus and a

FIG. 257.— Cells of salivary glands. A, At rest. The nuclei are star-shaped. B, After stimulation of the gland. The nuclei are round. (After Heidenhain.) new macronucleus, which undergo a very considerable increase in .size. It is unnecessary to extend still farther the enumeration of facts. From the experiments and observations presented it is evident that between the protoplasm and the nucleus a mutual exchange of substance takes place, without which neither of the two parts of the cell can continue to exist. In other words, both nucleus and protoplasm take part in the metabolism of the whole cell and are indispensable to its continuance.

1. The Mechanics of Cell-metabolism a. Scheme of Cell-metabolism With the inclusion of the nucleus and the protoplasm in the metabolic chain the mechanics of cell-metabolism assumes extraordinary complexity. With our lack of knowledge of the special chemical processes in living substance we can at present form no idea of the metabolism of the biogens in the type of cell possessing a differentiated nucleus and protoplasm, nor of the share taken by the two parts of the cell in the various components of this metabolism ; nor do we even know whether we must not assume the existence of a large number of different kinds of biogens in the nucleus and in the protoplasm, whose metabolism is closely interwoven with one another. Although we must leave the solution of all these questions to the future, upon the basis of our present knowledge we can at least form a picture of the great complexity of the metabolic machinery by means of a graphic scheme showing the mutual relations of the surrounding medium, the protoplasm and the nucleus.

For this purpose it is necessary first to become acquainted with certain new facts. Although the non-appearance of certain phenomena in non-nucleated protoplasmic masses on the one hand, and in nucleated cells with the protoplasm excluded on the other, shows that as regards many metabolic processes the nucleus and the protoplasm are greatly dependent upon each other, with reference to many other phenomena there appears from some experiments a certain independence of the two. For example, certain processes continue in the protoplasm for a considerable time even after the removal of the nucleus. Among these processes two groups may be distinguished. First, since the nucleus is continually giving off substances to the protoplasm, there always occur in the latter certain quantities of those substances, which we shall term in brief nuclear substances, which cannot be removed with the removal of the nucleus. Hence certain processes, for the occurrence of which the nuclear substances are absolutely necessary, are still able to continue at the expense of those present in the protoplasm since a time previous to the enucleation.

They cease only with the consumption of these substances. These processes constitute the first group. Those constituting the second group are not immediately dependent upon the presence of nuclear substances. That such processes exist is shown by the observations made byKlebs ('87) in plasmolysed cells of Spirogyra, which Gerassimoff ('92) has completely confirmed. When by plasmolysis in a 16 per cent, solution of cane-sugar Klebs caused the cells of a thread of Spirogyra to break up into protoplasmic lumps, bits of protoplasm frequently appeared that possessed shreds of the chlorophyll-band but no nucleus. By employing narcotics upon cells of Spirogyra that were undergoing division Gerassimoff ('97) has recently obtained cells completely destitute of a nucleus. Under favourable conditions these non-nucleated masses of protoplasm in Klebs's experiments continued to live for weeks. As has already been seen, in contrast with nucleated masses they had lost the power of forming a new cellulosemembrane. But they exhibited other vital phenomena unchanged. For example, when put into the dark, they consumed completely the starch that was contained in them, and, when in the light, they formed new starch in case they still possessed chlorophyll. In other words, the synthesis of starch from carbonic acid and water, and the further consumption of starch is in a certain degree independent of the influence of the nucleus. We say " in a certain degree," for, if the non-appearance of other phenomena caused by the removal of the nucleus has reached a certain extent, evidently the starch-building chlorophyll-bodies will share in the disorder ; they will experience changes, will form starch no longer, and will finally perish. In the case above mentioned this came in relatively late, frequently not until after several weeks.

In so far, therefore, as the metabolism of the chlorophyll-bodies is disturbed along with the disturbance of the whole metabolism by the removal of the nucleus, the formation of starch is in a certain sense, but only indirectly, dependent upon the nucleus. While non-nucleated masses of protoplasm in plant-cells, in case they still possess chlorophyll, split up carbonic acid and produce starch synthetically, they are also able to respire for a long time. The proof of the fact that respiration continues in such pieces to the same extent as in nucleated pieces or whole cells, is afforded by the following experiment.1 We place in a pendent drop in Engelmann's gas-chamber2 a number of nucleated and non-nucleated pieces of Infusoria together with uninjured individuals, and let a stream of washed hydrogen pass through the chamber from a Kipp's apparatus (Fig. 258) ; in a short time this forces out the air contained in the chamber. As a rule after five or ten minutes we see the non-nucleated and the nucleated pieces and the uninjured Infusoria begin to undergo granular disin- 1 Cf. Verworn ('91). 2 Cf. p. 283.

tegration. If then the hydrogen be very rapidly displaced by fresh air, it is frequently possible to prevent complete disintegration, which otherwise invariably appears after a short time. It follows from this that in non-nucleated protoplasmic masses, oxidation processes take place as in nucleated masses and uninjured cells. Respiration, therefore, is in a certain degree independent of the influence of the nucleus. This is completely confirmed by the experiments of Demoor ('95), who put the cells of Spirogyra into pure hydrogen and found that the protoplasm soon suspended all

FIG. 258. — /, Engelmann's gas-chamber. An annular space is closed below by a glass plate and above by a metal cover, the latter having in its middle a cover-glass for the examination of a pendent drop ; a a' are tubes which open into the cavity of the ring and serve for heating the latter by conveying warm water through it ; 66' are tubes which open into the glasscovered chamber and serve for the passage of gases ; the drop suspended upon the coverglass with its living contents is bathed by the gas in the chamber. II, Arrangement for investigation in pure hydrogen, a, Kipp's apparatus for the production of hydrogen ; 6, two wash-bottles for purifying the hydrogen ; c, microscope upon which is the gas-chamber containing the pendent drop.

vital phenomena, while the nucleus showed no disturbance and quietly proceeded to divide. It appears, accordingly, as if respiration were localized exclusively in the protoplasm, as if the nucleus took no direct part in the oxidation-processes. In viewof these discoveries in non-nucleated masses of protoplasm, it would be desirable to perform analogous experiments upon nuclei deprived of protoplasm, in order to find out whether certain metabolic processes continue undisturbed after the exclusion of the latter. But the decision of this question is beset with great difficulties, for the simple reason that it is not easy to find

in the nucleus any outwardly visible evidences of its metabolism. Nevertheless, it follows clearly from Demoor's experiments that the nucleus also exhibits vital phenomena after the exclusion of the protoplasm. Demoor narcotized cells of Spirogyra with chloroform so that the protoplasm was completely paralysed, and found that, notwithstanding, the nucleus went through all the stages of division undisturbed, and showed the characteristic changes that it exhibits in an uninjured cell in division. In the leucocytes of the frog the nucleus has the power of amoeboid motion, and Demoor was able to paralyse the protoplasm by the use of chloroform without interrupting the movements of the nucleus (Fig. 259). These discoveries show that individual processes take place in the nucleus in a certain measure independent of the influence of protoplasm. Naturally it cannot be decided at present whether these processes continue only because after the exclusion of the protoplasm there are still contained in the nucleus protoplasmic substances which must be consumed before the processes in question cease, or whether the latter are not directly dependent upon protoplasmic substances. Possibly both

FIG. 259.— Leucocyte from the frog in a state of chloroforn. narcosis ; the protoplasm is completely paralysed, while the nucleus still makes amoeboid movements. Cf. a, b, c, and d. (After Demoor.) cases are here realized; this appears very probable when it is borne in mind that the nucleus has direct metabolic relations with the external medium, without the mediation of the protoplasm. Without doubt there are substances that pass from the external medium through the protoplasm unchanged into the nucleus, to be employed there for metabolism. This is certainly the case with water in a certain quantity, which is absolutely necessary to every vital process. The water is able to diffuse continually through the cell-membrane into the protoplasm and through the nuclear membrane into the nucleus. It is possible that along with the water many substances which are dissolved in it also come from the outside into the nucleus to engage there in chemical transformation.