Substructure of the glomerular slit diaphragm in freeze-fractured normal rat kidney

In the renal glomerulus, the narrow slits between adjacent epithelial podocytes are bridged by a diaphragm (2, 8, 11). In rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde (TAG), it has recently been discovered that this diaphragm has a highly ordered, isoporous substructure (9). It consists of a regular array of alternating cross bridges extending from the podocyte plasma membranes to a centrally running filament. This zipperlike pattern results in two rows of rectangular pores, approximately 40 X 140 A in cross section, dimensions consistent with the proposed role of the diaphragm as an important filtration barrier to plasma proteins (6). In the present study, we found in freeze-cleaved and in freeze-etched normal rat glomeruli that the surface of the slit diaphragm has an appearance conforming to the pattern found in sectioned material.     

MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.     

THE PERMEABILITY OF GLOMERULAR CAPILLARIES TO GRADED DEXTRANS : Identification of the Basement Membrane as the Primary Filtration Barrier

Graded dextrans have been used as tracers to identify the primary permeability barrier(s) to macromolecules among the structural elements (endothelium, mesangium, basement membrane, epithelium) of the glomerular capillary wall. Three narrow-range fractions of specified molecular weights and Einstein-Stokes radii (ESR) were prepared by gel filtration: (a) 32,000 mol wt, ESR = 38 Å; (b) 62,000 mol wt, ESR = 55 Å; and (c) 125,000 mol wt, ESR = 78 Å. These fractions are known to be extensively filtered, filtered in only small amounts, and largely retained, respectively, by the glomerular capillaries. Tracer solutions were infused i.v. into Wistar-Furth rats, and the left kidney was fixed after 5 min to 4 h. The preparations behaved as predicted: initially, all three fractions appeared in the urinary spaces, with 32,000 > 62,000 » 125,000. The smallest fraction was totally cleared from the blood and urinary spaces by 2.5 h, whereas the intermediate and largest fractions were retained in the circulation at high concentrations up to 4 h. With all fractions, when particles occurred in high concentration in the capillary lumina, they were present in similarly high concentrations in the endothelial fenestrae and inner (subendothelial) portions of the basement membrane, but there was a sharp drop in their concentration at this level—i.e., between the inner, looser portions of the basement membrane and its outer, more compact portions. With the two largest fractions, accumulation of particles occurred against the basement membrane in the mesangial regions with time. No accumulation was seen with any of the fractions in the epithelial slits or against the slit membranes. Dextran was also seen in phagosomes in mesangial cells, and in absorption droplets in the glomerular and proximal tubule epithelium. It is concluded that the basement membrane is the main glomerular permeability barrier to dextrans, and (since their behavior is known to be similar) to proteins of comparable dimensions (40,000–200,000 mol wt). The findings are discussed in relation to previous work using electron-opaque tracers to localize the glomerular permeability barrier and in relation to models proposed for the functions of the various glomerular structural elements.     

A stereological study of the glomerular filter in the rat. Morphometry of the slit diaphragm and basement membrane

Kidney from normal male albino rats, of body weight 170-200 g, was fixed by arterial perfusion with buffered tannic acid-glutaraldehyde, and postfixed with osmium tetroxide. Random and isotropic ultrathin sections from 23 different glomeruli from five rats were mounted on slot grids for staining and electron microscopy. Prints of whole glomeruli at a magnification of 3,909 were analyzed by stereological methods. The mean glomerular volume was (8.048 +/- 0.474) X 10(5) mum3 if the glomeruli are treated as spheres. The area of the basement membrane was 0.281 +/- 0.017 mm2 per glomerulus, of which 0.184 +/- 0.011 mm2 represents peripheral basement membrane. The aggregate epithelial slit length per glomerulus was 65.19 +/- 3.84 cm, of which 48.69 +/- 2.87 cm represents epithelial slits abutting on the peripheral basement membrane. Assuming that a slit diaphragm is 390 A wide, and that the pores of the slit diaphragm represent 26% of its area, the mean pore area is 3.96 cm2, of which 2.96 cm2 represents the area of peripheral pores. These findings are discussed in the context of the hydrodynamic theory of glomerular ultrafiltration. We conclude that the porous substructure of the glomerular slit diaphragm is significant in determining the hydraulic conductivity of the glomerulus and hence also solute flux during ultrafiltration.     

Discovery of the congenital nephrotic syndrome gene discloses the structure of the mysterious molecular sieve of the kidney.

The molecular nature of the glomerular slit diaphragm, the site of renal ultrafiltration, has until recently remained a mystery. However, the identification of the gene affected in congenital nephrotic syndrome has revealed the presence of a novel protein, possibly specific for the slit diaphragm. This protein, which has been termed nephrin, is a transmembrane protein that probably forms the main building block of an isoporous zipper-like slit diaphragm filter structure. Defects in nephrin lead to abnormal or absent slit diaphragm leading to massive proteinuria and renal failure. The discovery of nephrin sheds new light on the glomerular filtration barrier, provides new insight into the pathomechanisms of proteinuria, and even opens up possibilities for the development of novel therapies for this common and severe kidney complication.     

ELECTRON MICROSCOPE AND EXPERIMENTAL INVESTIGATIONS OF THE NEUROFILAMENTOUS NETWORK IN DEITERS'' NEURONS : Relationship with the Cell Surface and Nuclear Pores

The assembly of filamentous elements and their relations to the plasma membrane and to the nuclear pores have been studied in Deiters' neurons of rabbit brain. Electron microscopy of thin sections and of ectoplasm spread preparations have been integrated with physicochemical experiments and differential interference microscopy of freshly isolated cells. A neurofilamentous network extends as a continuous, three-dimensional, semilattice structure throughout the ectoplasm, the "plasma roads," and the perinuclear zone of the perikaryon. This space network consists of ~90-Å wide neurofilaments arranged in fascicles which are interconnected by an exchange of neurofilaments. The neurofilaments consist of intercoiled ~20-Å wide unit-filaments and are associated through cross-associating filaments with other neurofilaments of the fascicle and with microfilaments. The ~20–50-Å wide microfilaments display intimate associations with the plasma membrane and with the nuclear pores. Electron microscopy of thin sections from glycerinated and heavy meromyosin-treated Deiters' neurons shows that actin-like filaments are present in the pre- and postsynaptic regions of synapses terminating on these neurons. It is proposed that the neurofilamentous space network serves a transducing function by linking plasma membrane activities with the genetic machinery of the neuron.     

ULTRASTRUCTURAL ORGANIZATION OF CHLOROPLAST THYLAKOIDS OF THE GREEN ALGA "OOCYSTIS MARSSONII"

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85–90 Å diameter particles. Adjacent membranes in stacked regions evidently share 170 Å diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 Å particles or only the upper portion of such particles, creating in the latter case images of 125–135 Å diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.     

Albuminuria associated with CD2AP knockout mice is primarily due to dysfunction of the renal degradation pathway processing of filtered albumin

Here we address the assumption that the massive intact albuminuria accompanying mutations of structural components of the slit diaphragm is due to changes in glomerular permeability. The increase in intact albumin excretion rate in Cd2ap knockout mice by >100-fold was not accompanied by equivalent changes in urine flow rate, glomerular filtration rate or increases in dextran plasma clearance rate, which demonstrates that changes in glomerular permeability alone could not account for the increase in intact albumin excretion. The albuminuria could be accounted for by inhibition of the tubule degradation pathway associated with degrading filtered albumin. There are remarkable similarities between these results and all types of podocytopathies in acquired and toxin-induced renal disease, and nephrotic states seen in mice with podocyte mutations.     

EXCITABLE MEMBRANE ULTRASTRUCTURE : I. Freeze Fracture of Crayfish Axons

Cross-sectioned and cross-fractured crayfish axons display regions in which axon and Schwann cell surface membranes are regularly curved and project into the axoplasm. At these regions (projections) the two membranes run precisely parallel, separated by a gap of 130–140 Å. Longitudinal fractures through the axons expose the inner fractured surface of either the internal (face A) or the external (face B) leaflet of axon and adjacent Schwann cell surface membranes. On both membranes the projections appear as elongated structures oriented with the long axis parallel to the long axis of the nerve fiber. On face A of the axon surface membrane they are seen as elongated indentations 0.5–1.2-µm long, 0.12–0.15-µm wide. The indentations contain parallel chains of globules. The chains repeat every 120–125 Å and are oriented obliquely in such a way that if one looks at the axon surface from the extracellular space, the axis of the chains is skewed counterclockwise to the long axis of the indentations by an acute angle (most often 55–60°). The globules repeat along the chain every 80–85 Å. Globules of adjacent chains are in register in such a way that the axis on which globules of neighboring chains are aligned forms an angle of 75–85° with the axis of the chains. The complex structure can be defined as a globular array with a rhomboidal unit cell of 80–85 x 120–125 Å. On face B of the axon surface membrane the complementary image of these structures is seen. The projections of the Schwann cell surface membrane also contain groupings of globules; however, these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. Several possible interpretations of the meaning of these membrane specializations could be proposed. They could be: (a) structures involved in the mechanism of excitation, (b) regions of presumed metabolic couplings, and (c) areas of cell-to-cell adhesion.     

Solute Flux Coupling in a Homopore Membrane

Our previous studies on solute drag on frog skin and synthetic heteropore membranes have been extended to a synthetic homopore membrane. The 150-Å radius pores of this membrane are formed by irradiation and etching of polycarbonate films. The membrane is 6-µm thick and it has 6 x 10⁸ pores cm^–2. In this study, sucrose has been used as the driver solute with bulk flow blocked by hydrostatic pressure. As before on heteroporous membranes, the transmembrane asymmetry of tracer solute is dependent on the concentration of the driver solute. Tracer sucrose shows no solute drag while maltotriose shows appreciable solute drag at 1.5 M sucrose. With tracer inulin and dextran, solute drag is detectable at 0.5 M sucrose. These results are in keeping with the previous findings on heteropore membranes. Transmembrane solute drag is the result of kinetic and frictional interaction of the driver and tracer solutes as the driver flows down its concentration gradient. The magnitude of the tracer flux asymmetry is also dependent on the size of the transmembrane pores.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : V. THE INFLUENCE OF THE THICKNESS OF DRIED COLLODION MEMBRANES UPON THEIR ELECTROMOTIVE BEHAVIOR

1. Experiments were carried out to decide whether or not the electromotive properties of dried collodion membranes depend upon their thickness. 2. A number of dried collodion membranes of varying thickness, 3–160 µ, were prepared from collodion preparations of different electrochemical activity. The characteristic concentration potentials across them were measured and the means of these values determined for each thickness. 3. The characteristic concentration potentials across dried collodion membranes are a function of their thickness. The thinnest membranes yield in all cases the lowest concentration potentials; increasingly thicker membranes give increasingly higher potential values, until a constant value is reached which is characteristic of the particular collodion preparation used. With electrochemically active collodion, characteristic concentration potentials approaching the thermodynamically possible maximum are obtained with membranes of only 10 µ thickness, thinner membranes giving appreciably lower values. With two rather inactive commercial collodion preparations the characteristic concentration potential increases from about 30 mv. for membranes 3 µ thick to about 42 mv. for 20 µ membranes; still thicker membranes do not show a significant increase in the potential values. With a highly purified collodion preparation the constant maximum value was found to be about 32 mv., 4 µ thick membranes giving only about 22 mv. 4. These results do not support the homogeneous phase theory as applied to the dried collodion membrane. They are readily compatible with the micellar-structural theory. Several special possible cases of the latter as applied to the dried collodion membrane are discussed.     

STUDIES OF EXCITABLE MEMBRANES : I. Macromolecular Specializations of the Neuromuscular Junction and the Nonjunctional Sarcolemma

The neuromuscular junctions and nonjunctional sarcolemmas of mammalian skeletal muscle fibers were studied by conventional thin-section electron microscopy and freeze-fracture techniques. A modified acetylcholinesterase staining procedure that is compatible with light microscopy, conventional thin-section electron microscopy, and freeze-fracture techniques is described. Freeze-fracture replicas were utilized to visualize the internal macromolecular architecture of the nerve terminal membrane, the chemically excitable neuromuscular junction postsynaptic folds, and the electrically excitable nonjunctional sarcolemma. The nerve terminal membrane is characterized by two parallel rows of 100–110-Å particles which may be associated with synpatic vesicle fusion and release. On the postsynpatic folds, irregular rows of densely packed 110–140-Å particles were observed and evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates. Differences in the size and distribution of particles in mammalian as compared with amphibian and fish postsynaptic junctional membranes are correlated with current biochemical and electron micrograph autoradiographic data. Orthogonal arrays of 60-Å particles were observed in the split postsynaptic sarcolemmas of many diaphragm myofibers. On the basis of differences in the number and distribution of these "square" arrays within the sarcolemmas, two classes of fibers were identified in the diaphragm. Subsequent confirmation of the fiber types as fast- and slow-twitch fibers (Ellisman et al. 1974. J. Cell Biol. 63[2, Pt. 2]:93 a. [Abstr.]) may indicate a possible role for the square arrays in the electrogenic mechanism. Experiments in progress involving specific labeling techniques are expected to permit positive identification of many of these intriguing transmembrane macromolecules.     

Albumin Antioxidant Response to Stress in Diabetic Nephropathy Progression

Background

A new component of the protein antioxidant capacity, designated Response Surplus (RS), was recently described. A major feature of this component is the close relationship between protein antioxidant capacity and molecular structure. Oxidative stress is associated with renal dysfunction in patients with renal failure, and plasma albumin is the target of massive oxidation in nephrotic syndrome and diabetic nephropathy. The aim of the present study was to explore the albumin redox state and the RS component of human albumin isolated from diabetic patients with progressive renal damage.

Methods/Principal Findings

Serum aliquots were collected and albumin isolated from 125 diabetic patients divided into 5 groups according to their estimated glomerular filtration rate (GFR). In addition to clinical and biochemical variables, the albumin redox state, including antioxidant capacity, thiol group content, and RS component, were evaluated. The albumin antioxidant capacity and thiol group content were reciprocally related to the RS component in association with GFR reduction. The GFR decline and RS component were significantly negatively correlated (R = –0.83, p<0.0001). Age, creatinine, thiol groups, and antioxidant capacity were also significantly related to the GFR decline (R = –0.47, p<0.001; R = –0.68, p<0.0001; R = 0.44, p<0.001; and R = 0.72, p<0.0001).

Conclusion/Significance

The response of human albumin to stress in relation to the progression of diabetic renal disease was evaluated. The findings confirm that the albumin molecular structure is closely related to its redox state, and is a key factor in the progression of diabetes nephropathy.     

Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.     

FLUIDITY OF THE SURFACE OF CULTURED MUSCLE FIBERS : Rapid Lateral Diffusion of Marked Surface Antigens

Fluorescent antibody fragments of anti-muscle plasma membrane antibody bound as small fluorescent spots when applied by micropipetting to cultured myotubes. The spots were observed to enlarge with time. The rate of enlargement of fluorescent spots was greater when fragments were applied than when divalent antibody was used. It was also greater at 23°–25°C than at 0°–4°C. With glutaraldehyde-fixed cells no increase in the size of the spots was seen. The observations are consistent with the spread of fluorescent spots due to diffusion of surface protein antigens within the plane of a fluid membrane. From measurements of spot size against time, a diffusion constant of 1–3 x 10^-9 cm² s^-1 can be calculated for muscle plasma membrane proteins of mol wt approximately 200,000. This value is consistent with other observations on the diffusion of surface antigens and of labeled lipid molecules in synthetic and natural membranes.     

Glomerular permeability in the bullfrog Rana catesbeiana

Filtration studies suggest similar size pores in the glomerular filters of mammals and amphibians. However, the glomerular wall in the bullfrog exhibits several structural features not found in mammals. The subendothelial space of the basement membrane is often greatly enlarged and infiltrated by cellular elements. The lamina densa of the basement membrane shows extensive variation in thickness and packing of its filaments. On the other hand, the epithelial slits in the bullfrog are closed by a slit diaphragm which appears similar in size and structure to the slit diaphragm in mammals. Horse spleen ferritin, a protein with a hydrodynamic radius of 61 A, was used as an ultrastructural tracer to determine whether the highly variable structure of the basement membrane renders this layer more permeable than its mammalian counterpart. Within 10 min after intravenous injection, ferritin was found throughout the basement membrane and often in clusters within the subepithelial layer adjacent to the slit diaphragm. Virtually no ferritin was found within the urinary space, podocytes, or cells of the proximal tubule. Ferritin distribution was the same in both superficial glomeruli and more deeply lying glomeruli regardless of the method of fixation. These results indicate that in the bullfrog the slit diaphragm is a principal filtration barrier to ferritin and thus to smaller plasma proteins.     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : II. Structural Details and Further Evidence for Intercellular Channels by Freeze-Fracture and Negative Staining

Low resistance junctions between axons of crayfish ganglia are studied by freeze-fracture and negative staining. In freeze-fracture, fracture planes that go through a junctional membrane expose two faces, both internal, called face A and face B. Face A belongs to the internal membrane leaflet and faces the gap. Face B belongs to the external membrane leaflet and faces the axoplasm. Face A displays pits, 60–100 Å in diameter, arranged in a hexagonal array with a unit cell of ~200 Å. An ~25 Å bump is frequently seen at the center of each pit. Some pits are occupied by a globule ~125 Å in diameter, which displays a central depression ~25 Å in size. Face B contains globules also arranged in a fairly regular hexagonal pattern. The center-to-center distance between adjacent globules is most frequently ~200 Å; however, occasionally certain globules are seen separated by a distance as short as ~125 Å. The top surface of the globules occasionally displays a starlike profile and seems to contain a central depression ~25 Å in diameter. In negatively stained preparations of membranes from the nerve cord, two types of membranes are seen containing a fairly regular pattern. In one, globules ~95 Å in diameter form a hexagonal close packing with a unit cell of ~95 Å. In the other, globules of the same size are organized in a larger hexagonal array with a unit cell of ~155 Å (swollen arrangement). Some of the globules forming the swollen arrangement are seen containing six subunits. The six subunits form a hexagon which is skewed with respect to the main rows of hexagons in such a way that the subunits lie on rows which make an angle of ~37° with the main rows.     

ELECTRON MICROSCOPY OF ORAL CELLS IN VITRO : II. Subsurface and Intracytoplasmic Confronting Cisternae in Strain KB Cells

Two special areas involving membranous components in strain KB cells were studied by electron microscopy. The first area described is that of the subsurface regions of two apposing cells in which flattened cisternae (one cisternae in each subsurface region) with membranes spaced 110–230 A apart were found in a confrontation alignment. The long dimension of the profiles of these cisternae ranges from 0.5 to 2 µ. At these intercellular contact areas, each cisterna is closely applied to the adjacent plasma membrane; the intervening space is 60–100 A. We have named the cisternae in these roughly symmetrical areas of cell contact the subsurface confronting cisternae. Communications between these cisternae and those of the rough-surfaced endoplasmic reticulum also were observed. The second area described is that of the intracytoplasmic confronting cisternae. These cisternae were observed as oval or round images about 0.3–1.4 µ in diameter, each image being composed of a pair of concentrically arranged confronting cisternae with membranes spaced 200–400 A apart. The apposing membranes of the two confronting cisternae are electron opaque, smooth, and free of ribosomes, whereas the unapposed membranes are less dense, scalloped, and associated with ribosomes. The spacing between the two intracytoplasmic confronting cisternae is 70–110 A.     

THE EFFECT OF UNILATERAL ULTRAVIOLET LIGHT ON THE DEVELOPMENT OF THE FUCUS EGG

1. When Fucus eggs which have been fertilized for a sufficient length of time are irradiated unilaterally with monochromatic ultraviolet light (λ2804 Å) of adequate dosage, 97–100 per cent form rhizoids on the halves of the eggs away from the source of radiation (see Figs. 1 and 2). 2. The responsiveness of the eggs increases gradually after fertilization and does not reach a maximum until about 7 hours at 15°C. (see Fig. 3). The first rhizoids begin to form in a population at about 12 hours after fertilization. The responsiveness remains maximal until at least 11 hours after fertilization. 3. It is suggested that the low responsiveness of a population of eggs at an earlier period is due to recovery from the effects of irradiation before the rhizoids begin to form. 4. The response of eggs to λ2804 Å is proportional, over a wide range, to the logarithm of the dosage (see Fig. 1). Dosage was regulated by the duration of exposure during the period of maximum response. 5. High dosages of λ2804 Å, of the order of 10,000 ergs per mm.², cause the rhizoids to form fairly precisely away from the source of radiation (see Fig. 2). Twice this dosage inhibits rhizoid formation altogether without causing cytolysis. 6. Other wave-lengths which have also been shown to be effective are: 3660, 3130, 2654, 2537, 2482, and 2345 Å. Only exploratory measurements have been made to test the effectiveness of these wave-lengths, but they show that much greater energy is necessary to obtain a strong response with λ3130 and 3660 Å, especially the latter. The wave-lengths shorter than 2804 Å, on the other hand, show the same order of effectiveness as λ2804 Å. Some may be more effective. 7. A beam of λ2804 Å which is incident on a single layer of Fucus eggs is completely extinguished at 2, 3, 6, or 6½ hours after fertilization. About 85 per cent of a beam of λ3660 Å is extinguished. The wave-length 3660 Å is thus not so completely absorbed as λ2804 Å, but the difference in proportion absorbed by the egg is not nearly so great as the difference in effectiveness.