PHOTOREACTIONS IN PHYCOMYCES

Sporangiophores of Phycomyces in stage IV b have been stimulated by parallel light in test areas 0.2 mm. wide. The growth responses to large stimuli are very large, owing probably to light scattered within the specimen. For medium stimuli the sensitive zone coincides with the growth response zone obtained previously and excludes the region of maximum stretch. Sustained stimulations were used to elicit tropic responses. The bends formed travel away from the sporangium at a speed equal to the growth speed. Thus they remain very close to the stimulus when this is held at a constant level relative to ground but separate from it for stimuli programmed differently. The existence of a protoplasmic structure, the "inner wall," with the following properties is postulated: it is attached to the lower, non-growing part of the sporangiophore and grows by addition above the sensitive zone. It neither stretches nor twists in the sensitive zone. It is the seat of the light receptors and gives growth and tropic responses. The cell wall follows its bends by elastic stretch.

New Features of Protoplasmic Structure Observed in Recent Electron Microscope Studies

1. This article discusses current electron microscope specimen techniques (particularly thin sectioning) and various special aspects of protoplasmic structure. It is not a general review and deals only with subjects studied in this laboratory. Dimensions specified are approximate only. 2. As regards sectioning (which is inevitable for the study of fine cell structure in most complex tissues, by whatever microscope) electron microscope practice has two considerable advantages over light microscope practice: (a) osmium tetroxide solutions alone (without addition of dichromate, &c.) are excellent for cytoplasmic fixation; (b) after cutting, sections receive no further treatment (such as de-waxing and re- and de-hydration). Moreover, criticisms regarding vacuum drying of electron microscope specimens are irrelevant to electron microscope studies of sections. For high resolution, sections must be cut at 005 µ or less and examined without removal of embedding medium. Such sections are obtained by embedding in medium-hard plastics and cutting on a glass knife. Specially sharpened blades can be used instead of glass, but it is doubtful whether waxes can be substituted for plastics. 3. Many, possibly all, animal and plant flagella contain two similar central subfibrils surrounded by a ring of nine fibrils different in size and chemical composition from the central pair. As far as is known at present, mammalian sperm are unique (a) in containing a second concentric ring of nine sub-fibrils and (b) in possessing a double-helix sheath round the axial sub-fibrils of the tail. Bacterial flagella consist of single fibrils each equivalent to one of the component sub-fibrils of a multi-fibrillar flagellum. They often occur in bunches, but so far no intermediates have been found between these bunches and the sheathed ‘9 + 2’ flagella of animals and plants. Vertebrate striated muscle consists of sub-fibrils which are (very roughly) 100 Å thick, 250 Å centre to centre and which in resting amphibian muscle have a 400 A periodicity; in cross sections these sub-fibrils are packed solid (not in hollow cylinders) and in a fairly regular array. 4. Nuclear, cell, and mitochondrial membranes appear double in cross sections (150-300 Å thick); this may be due to the dissolving away of internal lipoid leaving two outer protein sheets, but none of these membranes is thin enough to contain simply a bimolecular lipoid layer. Electron microscope studies of striated cell borders confirm that in some sites there may be distinct filaments of variable length and in others closed ducts, or rods, covered distally by a smooth membrane. One border described contains distinct filaments which join basal mitochondria. It is not yet certain whether the complex internal ‘double-membranes’ of sectioned mitochondria arise from tubes, or paired sheets, or both. 5. When sectioned after freezing-drying or buffered osmic or formal fixation, the cytoplasm of many protein secreting cells in vertebrates is full of double membranes, like those of mitochondria, and of equally uncertain origin. Sections of many other cells show similar structures, varying in thickness from 75 Å--600 Å. There is some evidence that they are associated with cytoplasmic ribonucleic acid.

Protoplasmic Structure and Mitosis

1. The mitotic figure of the sea-urchin egg is most strongly birefringent at metaphase. During anaphase this birefringence decreases considerably, but the spindle and asters both grow in size. These changes have been investigated quantitatively by constructing curves of retardation and coefficient of birefringence across the mitotic figure, using techniques described in an earlier paper. 2. The decrease of birefringence in the spindle starts at the equator, and then moves, in the course of a few minutes, to either pole. Only when the decrease has reached the spindle poles does it begin in the asters, where it moves outwards from the centres. 3. These changes resemble the movements of the chromosomes, which also start at the equator in metaphase, and move in separate groups to the poles during t anaphase. By examining single eggs in qaphase up to the moment of fixation, and then staining them to show the chromosomes, it is established that the regons of, decreasing birefringence actually correspond to the position of the chromosomes. 4. Since the chromosomes are too small to be the direct cause of the decrease in birefringence, it is concluded that they are producing the decrease indirectly by initiating a structural change in the spindle and asters. 5. The possible mechanisms for this change are discussed. It is concluded that the chromosomes must be releasing an active substance, for which the term ‘structural agent’ is suggested. 6. The growth of the mitotic figure takes the form, in the spindle, of an increase in length, and in the asters, of an increase in size. It is accompanied by an increase in coefficient of birefringence, though this is to some extent masked by the decrease in birefringence referred to earlier. 7. The increase in coefficient of birefringence affects the whole mitotic figure from the very beginning of anaphase, and is not therefore relatable to the position of the chromosomes. For this reason it might be due to a number of merent mechanisms, but as it starts at the same moment as the decrease in birefringence, it is tentatively assumed to be due to the release of a second ‘structural agent’. 8. The increase in coefficient of birefringence is probably due to the orientation of new material. The decrease is more Uely to be due to changes in molecular and micellar arrangement; it would be consistent with a contractile mechanism in the spindle. 9. The implications of these findings are discussed in a concluding section.

Protoplasmic Structure and Mitosis

1. The present paper is the first of a series dealing with the birefringence of mitotic figures in the eggs of the sea-urchin Psammechinus miliaris. 2. Living eggs have been examined using time-lapse photography, and retardation curves for the mitotic figures constructed from densitometric measurements made on the film negatives. 3. In the case of the aster, an integral equation relating retardation and coefficient of birefringence can be formulated and solved exactly to give coefficient of birefringence. In the case of the spindle, coefficient of birefringence can only be calculated approximately. 4. In both asters and spindles, the coefficient of birefringence is nil at the centres,rises to a maximum at 5 or 6CL out, and then falls to a minimum at the equator of the spindle or the periphery of the aster. 5. The rise in coefficient of birefringence round the centre is not as sharp as might be expected, and there is some evidence that orientation is built up gradually over a distance of a few microns. 6. The fall in coefficient of birefringence away from the maximum is approximately an inverse square in the case of the spindle. In the aster it falls off somewhat more rapidly. Since the density of material does not vary from point to point, this fall must be due to changes in molecular and micellar arrangement, or to a decreasing proportion of oriented material. 7. The classical conception of the spindle and asters as structures built up of discrete fibrils radiating from the centres, would be expected, for geometrical reasons, to give an inverse square fall in proportion of oriented material. While, therefore, a homogeneous structure with varying molecular and micellar arrangement cannot be ruled out, it is possible that the mitotic figure consists of definite fibrils radiating from the centres. 8. Evidence from other sources supports this view, and suggests that the fibrils must be submicroscopic in size.