Freeze-etch and histochemical evidence for cycling in crayfish photoreceptor membranes

Summary Freeze-etched rhabdoms and adjacent cytoplasmic organelles from crayfish compound eyes have been studied for evidence of photoreceptor membrane cycling. The protoplasmic leaflet face (PF) of split photoreceptor membrane of the microvilli is richly particulate. The particles (92±16 A in diameter in surface fractures; 70±9 A in cross fractures; density about 8000/m²) probably indicate rhodopsin molecule localization. Closely similar particles appear in membranes of pinocytotic vesicles, multivesicular bodies (MVB) and secondary lysosomes. In contrast other retinular cell membranes like plasma membrane remote from the rhabdom are quite distinct (60±23 A particle diameter, density ca 1000/m²). Histochemical tests for acid phosphatase demonstrate its presence in well-developed (but not early stage) MVBs, mixed lamellar vesicular bodies (LVB) and lamellar bodies. Density of PF particles decreases from 8000 in MVB to roughly 4500/m² in LVB indicating a degradative sequence from rhabdom to lamellar bodies. Membrane leaflet orientations show that primary endocytosis from microvilli must be followed by secondary endocytosis of fused coated vesicles to form MVB. Morphological evidence for photoreceptor membrane resynthesis has not been found yet in crayfish but some has been obtained in other crustaceans.This research was supported by grants from the U.S. National Institutes of Health (EY 00405), the National Geographic Society and the Japan Society for the Promotion of ScienceThe authors are grateful to Mr. Washioka of the JEOL Co. Research Laboratory for his essential help in effecting the freeze-etch preparations. They also thank Professor Ryoji Kikuchi of Tokyo Women's Medical College for welcome cooperation and hospitality as well as Dr. Karl Pfenninger of Yale University for his generous assistance in interpreting the freeze-etch data. Technical advice and help were also kindly provided on the acid phosphatase histochemistry by Professor Marilyn Farquhar and others in the Yale Cell Biology Section     

The eyes of mesopelagic crustaceans: I. Gennadas sp. (Penaeidae)

Summary The eye of the deep-sea penaeid shrimp Gennadas consists of approximately 700 square ommatidia with a side length of 15 n. It is hemispherical in shape and is located at the end of a 1.5 mm long eye stalk. The cornea is extremely thin, but the crystalline cone is well-developed. A clear zone between dioptric structures and the rhabdom layer is absent. A few pigment granules are found within the basement membrane; otherwise they, too, are absent from the eye of Gennadas. The rhabdom is massive and occupies 50 % of the eye. It consists of orthogonally oriented microvilli (the latter measuring 0.07 m in diameter) and is 75 m long. In cross sections adjacent rhabdoms, all approximately 8 m in diameter, form an almost continuous sheet and leave little space for retinula cell cytoplasm. In spite of a one h exposure to light, rhabdom microvilli show no disintegration or disruption of membranes. Vesicles of various kinds, however, are present in all seven retinula cells near the basement membrane. Bundles of seven axons penetrate the basement membrane. On their way to the lamina they often combine and form larger aggregations.The authors wish to thank the director of the Meat Industry Research Institute in Hamilton and his staff for the use of their electron microscope facilities     

The eyes of mesopelagic crustaceans

Summary The compound eyes of the mesopelagic euphausiid Thysanopoda tricuspidata were investigated by light-, scanning-, and transmission electron microscopy. The eyes are spherical and have a diameter that corresponds to 1/6 of the carapace length. The hexagonal facets have strongly curved outer surfaces. Although there are four crystalline cone cells, only two participate in the formation of the cone, which is 90–120 m long and appears to have a radial gradient of refractive index. The clear zone, separating dioptric structures and retinula, is only 90–120 m wide. In it lie the very large oval nuclei of the seven retinula cells. Directly in front of the 70 m long and 15 m thick rhabdom a lens-like structure of 12 m diameter is developed. This structure, known in only a very few arthropods, seems to be present in all species of Euphausiacea studied to date. It is believed that the rhabdom lens improves near-field vision and absolute light sensitivity. Rod-shaped pigment grains and mitochondria of the tubular type are found in the plasma of retinula cells. The position of the proximal screening pigment as well as the microvillar organization in the rhadbdom are indicative of light-adapted material. The orthogonal alignment of rhabdovilli suggests polarization sensitivity. Behind each rhabdom there is a cup-shaped homogeneous structure of unknown, but possibly optical function. Finally, the structure and the function of the euphysiid eye are reviewed and the functional implications of individual components are discussed.This study was begun during the 1975 Alpha Helix South East Asia Bioluminescence Expedition to the South Moluccan Islands     

The eyes of mesopelagic crustaceans

Summary In Streetsia challengeri left and right eyes have fused and become a single cylindrical photoreceptor, which occupies the basal half of a forward directed head projection. This unusual compound eye consists of approximately 2500 ommatidia, which are arranged in such a way that the animal has almost circumferential vision, but cannot look ahead or behind. It is thought that the eye operates on light-guide principles, and that the crystalline cones are the major dioptric component. Ommatidia in anterior-posterior rows show a greater overlap of visual fields than dorso-ventrally arranged ommatidia. Cone layer and retinula are separated by a 4 m thick screen-membrane, which contains tiny pigment granules of 0.15 m diameter. Cells of unknown function and origin, containing unusual multitubular organelles, are regularly found near the proximal ends of the crystalline cone threads. The twisted rhabdoms measure 18–20 m in diameter, and consist of microvilli 0.05 m in width, which belong to five retinula cells and which show no trace of disintegration. The position of interommatidial screening pigment, the density of retinula cell vesicles and inclusions, and the narrowness of the perirhabdomal space all suggest that the eyes have been light-adapted at the time of fixation for electron microscopy. The retinula cell nuclei lie on the proximal side of the heavily pigmented basement membrane. A tapetum or basal retinula cells are not developed. It is concluded that the eye optimally combines acuity with sensitivity, and that for distance estimation parallax may be important.Address until January 25th 1978: Scott Base, Ross Dependency, Antarctica (C/-Chief Post Office, Christchurch, New Zealand)     

Actin cytoskeleton is responsible for the change of cytoplasmic organization in root hair cells induced by a protein phosphatase inhibitor, calyculin A

Summary In root hair cells ofLimnobium stoloniferum, a protein phosphatase inhibitor, calyculin A (CA), at concentrations higher than 50 nM inhibits cytoplasmic streaming and induces remarkable morphological changes in the cytoplasm: the transvacuolar strands disperse and spherical cytoplasmic bodies emerge. The mechanism of the morphological changes of the cytoplasm induced by CA was studied by pharmacological analyses. The formation of spherical bodies in cells treated with CA was suppressed by the actin-depolymerizing and -fragmenting drugs latrunculin B and cytochalasin D at concentrations higher than 100 nM and 5 M, respectively. In contrast, 100 M propyzamide, a microtubule-depolymerizing drug, did not affect the formation of spherical bodies by CA. Interestingly, 60 mM 2,3-butanedione monoxime, an inhibitor of myosin, also suppressed the CA-induced formation of cytoplasmic spherical bodies. These results indicate that the actin cytoskeleton is intimately involved in the morphological changes of the cytoplasm induced by CA.Abbreviations APW artificial pond water - BDM 2,3-butanedione monoxime - CD cytochalasin D - DMSO dimethylsulfoxide - LB latrunculin B - Pro propyzamide     

Rhabdom degradation in white-eyed and wild-type crayfish after long term dark adaptation

Summary Rhabdom degradation has been compared in groups of wild-type (WT) and white-eyed mutant (WEM) crayfish exposed to a 1212 LD cycle after 1 month of continuous darkness. Light and electron microscopic quantitative analysis of the lysosome-related bodies (LRB) formed during rhabdom degradation show that WEM photore-ceptors produced few LRB during the first 12 h of light exposure, but the microvilli in the rhabdom became severely disrupted. An increase in LRB production began in the WEM after the initial light-dark transition, and continued during the first dark period, as well as throughout the second light cycle. Analysis of fluctuations in specific LRB types shows that multivesicular bodies (MVB) were the major contributor to the total organelle population seen in the WEM, and lamellar bodies (LB) showed a significant drop following light onset of the second light cycle.Wild-type crayfish maintained in long-term dark before exposure to cyclic lighting showed a characteristic increase in LRB production in the light and a decline in the dark. However, LRB persisted throughout the light period and did not show a characteristic decline during the second 6 h in the light. This exaggerated degradative response in the WT animals was repeated during the second light cycle.Abbreviations WEM white-eyed mutant - LRB lysosome-related bodies - MVB multivesicular bodies - LB lamellar bodies - CB combination bodies     

Rhabdom breakdown in the eye of Cirolana borealis (Crustacea) caused by exposure to daylight

Summary The effect of daylight on the compound eye was investigated in the deep-water crustacean isopod Cirolana borealis Lilljeborg. The animals were captured and fixed at night (dark-exposed, i.e. not exposed to light) and day (daylight-exposed), respectively. Changes in light and darkness have an effect on the retinula cells; the ultrastructure of dark-exposed eyes is characterized by well-preserved organelles and cytoplasm. The photoreceptor membranes covering the microvilli are regularly aligned, and the outline of the villi is smooth. Electron-dense pigment granules are evenly distributed in the cytoplasm of the retinula cell outside the rhabdom. Daylight-exposed eyes differ from the dark-exposed eyes in the following aspects: (i) the microvilli are disrupted, (ii) retinula-cell pigment is found in the rhabdom, and (iii) the cytoplasm of retinula cells is vesiculated. These results are interpreted as retinal damage caused by excess exposure to light.     

Endocytosis in cultured rat alveolar type II cells: effect of lysosomotropic weak bases on the processes.

We investigated the uptake of Lucifer yellow and surfactant complexed with gold (S-G) by isolated alveolar Type II cells. The fluid phase marker Lucifer yellow did not reach lamellar bodies (LB) even after prolonged incubation time, whereas S-G was internalized and found in LB. Treatment of Type II cells with lysosomotropic weak bases (NH4Cl and chloroquine) resulted in dilation of endosomes, lysosomes, and LB. The effect of these agents on LB resulted in disappearance of their lamellar organization, as detected by polarized light and electron microscopy. After incubation in lysosomotropic agent-free medium, endocytosis of Lucifer yellow and S-G in treated cells was mainly directed towards large vacuoles resembling either multivesicular bodies (MVB) or lysosomes. The possible relationship between LB, MVB, and lysosomes in freshly isolated as well as cultured alveolar Type II cells is discussed.     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

The differential effects of cytochalasin B on microfilament populations and cytoplasmic streaming

Summary Two distinguishable populations of microfilaments (mfs) can be identified in the radish root hair. Bundles of mfs are found throughout the cytoplasm, excluding the tip region of the hair. Single mfs occur only as a cortical array, specifically associated with the microtubules. Both mf populations are oriented parallel to the direction of streaming. Hairs grown in 5 g/ml cytochalasin B (CB) exhibit site-specific differential responses to the drug in both their streaming pattern and sensitivity of their mfs. Cytochalasin B elicits the following responses: 1. cytoplasmic streaming is reduced in all regions of the hair; 2. small particles (<1 m in diameter) still stream, whereas large particles (>1 m in diameter) no longer stream but exhibit an oscillatory or rotational motion; 3. filament bundles show increasing sensitivity to CB along the length of the hair; 4. single mfs show decreasing sensitivity to CB along the hair length. The effects of CB on cytoplasmic streaming can be related to its effects on both mf populations, thus suggesting that although mf bundles are probably involved in streaming in the sub apical and basal regions of the hair, single mfs are most likely involved in generating the slower, more irregular streaming patterns exhibited in the hair tip and CB-treated hair base.     

Ultrastructure of carpospores inGastroclonium clavatum (Roth)Ardissone (Rhodymeniales)

Summary Carpospores ofG. clavatum have been studied under the light and electron microscopes. They are wedge-shaped cells of 80–100 m at their longest diameters. The nucleus is an uncondensed structure provided with a regular outline and a large nucleolus. The plastids constitute heterogeneous populations of organelles differing in size and shape as well as in number and arrangement of the thylakoids. Multiplicating plastids are also present. The mitochondria are small but have well developed cristae. The Golgi apparatus consists of very numerous active dictyosomes. Starch is the main storage substance but some large lipid bodies are also present. Labyrinthine polysaccharide aggregations are present in the carposporial cytoplasm. Multilayered bodies constitute a sui generis very conspicuous cell component.     

Ultrastructure of the main excretory duct epithelium of the female mouse submandibular gland with special reference to sexual dimorphism

The fine structure of the main excretory duct epithelium (MEDE) of female mouse submandibular gland was investigated by scanning and transmission electron microscopy and the results compared with the previously established structure of male mouse MEDE. A comparative analysis of the subepithelial capillaries of both sexes was also performed. In this pseudostratified epithelium, principal cell-types were observed: types-I,-II,-III and basal cells. This differed significantly from male MEDE, where type-II and-III are absent and type-I cells are the most numerous. The latter cell-type had abundant mitochondria, a few lipid-containing granules, lysosomes in the infra-nuclear cytoplasm and well-developed basal infoldings. These cells were also characterized by abundant glycogen granules throughout the cytoplasm, many profiles of strands of smooth endoplasmic reticulum in the apical region, and lysosomes in the infra-nuclear region. Type-II cells were the second most numerous. Their most characteristic features were the presence of tubular vesicles which appeared to be invaginated from the plasma membrane, RER, SER, free ribosomes, a few peroxisomes with nucleoids, and primary lysosomes in extremely light cytoplasm. They had many mitochondria throughout the cytoplasm, except in the apical region, a few lipid-containing granules and no basal infoldings. Type-III cells were very few and were characterized by well developed basal infoldings, abundant free ribosomes, RER, SER, vesicles containing moderately dense material, and many lipid-containing granules. They also had many mitochondria throughout the cytoplasm, except apically. Basal cells had a large nucleus and the cytoplasm had few organelles. In the male continuous capillaries predominated in the subepithelial network, and capillary density per 200 m of epithelium (3.76±1.54) was lower than in the female, as was the number of fenestrae per 10 m of available endothelium (4.46±1.71). In the female, fenestrated capillaries predominated, and the capillary density per 200 m of epithelium was 6.76 (±1.54), and the number of fenestrae per 10 m of available endothelium was 4.91 (±1.77).     

Deposition of fusiform crystals without apparent diurnal rhythm at the growing edge of septa of the coral Galaxea fascicularis

Recent studies suggest a diurnal periodicity in the deposition of fusiform crystals by scleractinian corals. In order to check whether this is universally true in the Scleractinia, the surface structure of skeletons of Galaxea fascicularis (L.), collected at 3 h intervals over 1 day was observed with a scanning electron microscope. Fusiform crystals 0.3–3 m wide and 0.5–5 m long were found on the growing edges of septa of polyps collected at different times of day. There was no apparent diurnal change in the mean diameter of fusiform crystals. The size distributions of these crystals were almost the same by day (1200 h) and at night (2400 h). Small fusiform crystals which appeared to have been newly deposited were observed on septa collected both during the day and at night. The present study suggests that fusiform crystals are deposited continuously with no diurnal rhythm in G. fascicularis.     

Fine structure of the ommatidia and the occurrence of rhabdomeric twist in the dorsal eye of male Bibio marci (Diptera,Nematocera, Bibionidae)

Summary The ommatidia in the dorsal eye of male Bibio marci (March flies) are comprised of eight retinula cells (R1–8). In the distal region, the open rhabdomeres of retinula cells 1–6 are arranged in a symmetrically circular pattern with their microvilli directed radially. Immediately beneath the crystalline cone, cell 7 forms a rhabdomere that is about 1 m long and lies in the center of the circle formed by the rhabdomeres of cells 1–6. For the remaining length of an ommatidium it is replaced by the rhabdomere of retinula cell 8. The cell body of this retinula cell almost encloses its own rhabdomere by forming a deep invagination. Consequently, no ommatidial cavity is present. In the left eye rhabdomeres R 3, 5 and 6 first twist clockwise along their longitudinal axes, while rhabdomeres R1, 2, 4 and 8 twist counterclockwise. Opposite twisting is observed in the right eye. The twist rate varies along the length of the rhabdomeres. In a middle region of 60 m, within which the direction of twist does not change, the maximal twist rates are approximately 2°–5°/m in R1–6 and even higher in R 8. In a proximal region, the direction of twist is reversed, but the initial orientation of the microvilli not reestablished. Both the cross-sectional shape of the rhabdomeres and their geometric arrangement in the retinula change along with the twisting. It is substantiated that the rhabdomeric twist is not due to artifactual deformation.Supported by the Deutsche Forschungsgemeinschaft (SFB 4: E 2)The authors thank Dr. I. de la Motte for providing the material used in this study, Prof. H. Altner for critical discussion and Dr. M. Burrows for his attentive linguistic corrections     

The effects of abscisic acid on the maturation ofBrassica napus somatic embryos

Summary A comparison of embryos, cultured for increasing periods of time with and without abscisic acid (ABA), was undertaken to investigate, at the ultrastructural level, the influence of this growth regulator on the maturation of rapeseed (Brassica napus) somatic embryos. In the absence of ABA, the embryos germinated precociously while lipid bodies (LB), which were not numerous, soon degraded, as revealed by a depletion process associated with the appearance of morphologically mature glyoxysomes and an increase in the number of mitochondria. Moreover, a lack of protein bodies indicated that storage protein accumulation was not initiated under these conditions. On the contrary, the addition of ABA (10 M) induced marked modification of embryo metabolism. Indeed, ABA completely prevented precocious embryo germination and inhibited lipid reserve catabolism. Moreover, the formation of small vacuoles and proliferation of rough endoplasmic reticulum in their vicinity suggested the onset of storage protein accumulation. After 15 days in the presence of ABA, the embryos contained abundant lipid and protein bodies. Nevertheless, these somatic embryos were not exactly the same as their mature zygotic counterparts since differences were found in chloroplasts, amyloplasts, and nuclear structures. These observations suggest that additional factors might be required to obtain fully mature somatic embryos.Abbreviations ABA abscisic acid - ABM ABA medium - BM basal medium - LB lipid bodies - MS Murashige and Skoog (1962) - PB protein bodies - RER rough endoplasmic reticulum     

Multiple shoot-bud formation and plantlet regeneration on Castanea sativa Mill. seeds in culture

Primordial initiation and development of shoot-buds has been accomplished by using shoots derived from chestnut (Castanea sativa Mill) seedlings cultured with added 6-benzylaminopurine (BAP). Germination of chestnut seeds in the presence of BAP (4 – 40 M) stimulated varying numbers of shoot-buds in those areas of the main axis that were favorably altered. When excised single shoots from these treated seeds were subcultured on a fresh medium containing BAP (4 – 40 M) continual shoot production was observed. Bud growth and shoot elongation were stimulated by transferring cultures to a reduced concentration of BAP (2 M) plus indole-3-butyric acid (IBA 0.4 M). Plant regeneration occurred in the presence of IBA (0.8 M) after a preconditioning treatment in which naphthaleneacetic acid (NAA 50 M) and kinetin (k 2 M) were applied to the tissue culture shoots for 7 days in light.     

Immunocytochemical localization of reserve protein in the endoplasmic reticulum of developing bean (Phaseolus vulgaris) cotyledons

The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at-80° C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 m. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G     

Polarization sensitivity of individual retinula cells

Summary This paper elucidates the influence of the structure of a rhabdom on the polarization sensitivity of its retinula cells. The terminology polarization sensitivity (PS) and dichroic sensitivity () needs clarification. expresses the directional property of the local microvillar medium and is independent of the gross morphology of the rhabdom. The PS of a retinula cell is that found by single cell electrophysiology and depends strongly on the gross morphology of the rhabdom. Both and PS are ratios of the effects of theE vector of linear polarized light parallel to, to that perpendicular to the microvilli. From the theoretical analysis and its correlation with experiments the following is concluded.     

Fine structure of the ‘Wulst’ region of the domestic fowl in organotypic cultures

Summary Parts of the Wulst region of the chick embryo brain were maintained for 39 days in vitro. Processes of adjacent glial cell form zonulae occludentes and desmosomal junctions in the uppermost stratum of the cultures. Subjacent to this layer, in the neuropil, axodendritic synapses are abundant. 10–20 m below the surface the perikarya of glial cells and neurons are found. The latter form small clusters, plasma membranes of contiguous cells being directly apposed to each other. Axosomatic synapses terminate on the perikarya. Occasionally one terminal synapses on two nerve cells simultaneously. Two types of cilia arise from basal bodies in the cytoplasm of nerve cells. Type I is a slender protrusion of about 0.5 m in diameter, extending into the neuropil. On transverse sections it displays a 9 + 0 pattern of organization of paired micro tubules proximally, and an 8 + 1 configuration more distally. The length of the cilium is approximately 7 m. Type II cilia also originate in the neuronal cytoplasm. The structure of the proximal portion is identical with that of type I cilia. Toward the tip, however, the type II cilium is characterized by a bulbous enlargement which is filled with loosely folded membranes. The fine structural details of this cilium correlate closely to the outer segments of retinal photoreceptor cells during differentiation. The possible role of a light receptor in the Wulst region of birds, controlling biological rhythms, is discussed. Acknowledgements. The authors wish to thank Mrs. H. Frenk for her expert assistance in tissue culture and electron microscopic techniques     

Lack of effect of toxic aluminium concentrations on nitrogen fixation by nodulatedStylosanthes species

Summary Effects of three solution aluminium concentrations (0, 25, and 100M) on nitrogen fixation by well-nodulated plants ofStylosanthes hamata, Stylosanthes humilis andStylosanthes scabra are reported. Plants were inoculated with Rhizobium CB756 and grown for 21 days in an aluminium-free nutrient solution at pH 5.3 before imposition of the aluminium treatments.Nitrogen fixation was measured both by the increase in total nitrogen content of the plants and acetylene reduction in roots of plants harvested at 10 and 20 days after imposition of the aluminium treatments. Solution aluminium concentrations as high as 100M, had no detrimental effect on nitrogen fixation in any species.