Pterin pigment granules are responsible for both broadband light scattering and wavelength selective absorption in the wing scales of pierid butterflies

A small but growing literature indicates that many animal colours are produced by combinations of structural and pigmentary mechanisms. We investigated one such complex colour phenotype: the highly chromatic wing colours of pierid butterflies including oranges, yellows and patterns which appear white to the human eye, but strongly absorb the ultraviolet (UV) wavelengths visible to butterflies. Pierids produce these bright colours using wing scales that contain collections of minute granules. However, to date, no work has directly characterized the molecular composition or optical properties of these granules. We present results that indicate these granules contain pterin pigments. We also find that pterin granules increase light reflection from single wing scales, such that wing scales containing denser granule arrays reflect more light than those with less dense granule collections. As male wing scales contain more pterin granules than those of females, the sexual dichromatism found in many pierid species can be explained by differences in wing scale pterin deposition. Additionally, the colour pattern elements produced by these pterins are known to be important during mating interactions in a number of pierid species. Therefore, we discuss the potential relevance of our results within the framework of sexual selection and colour signal evolution.     

Thioredoxin-family protein EYE2 and Ser/Thr kinase EYE3 play interdependent roles in eyespot assembly

The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and is associated with the daughter four-membered (D4) microtubule rootlet, comprises an elliptical patch of rhodopsin photoreceptors on the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. EYE2 is positioned along, and adjacent to, the D4 rootlet in the absence of pigment granules. The eyespot pigment granule array is required for maintenance of the elliptical shape of both the overlying EYE2 and channelrhodopsin-1 photoreceptor patches. We propose a model of eyespot assembly wherein rootlet and photoreceptor direct EYE2 to an area of the chloroplast envelope, where it acts to facilitate assembly of pigment granule arrays, and EYE3 plays a role in the biogenesis of the pigment granules.     

Starch Granule Initiation in Arabidopsis Requires the Presence of Either Class IV or Class III Starch Synthases

The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.     

Schistosoma mansoni: ultrastructural studies on the esophageal secretory granules.

The posterior portion of the esophageal gland of Schistosoma mansoni produces a granule that is highly structured internally. Each granule consists of arrays of membrane-bound tubules enclosed by a membrane. Cytochemical tests indicate that the granules are not reactive for cytochrome c-oxidase but du react for macromolecular carbohydrates. It is believed that the granules are synthesized in the Golgi complex and are secreted at the base of the luminal amplifications of the esophagus. Colchicine treatment results in an accumulation of granules in the cyton region. Their physiological function is still undetermined, but it is hypothesized that they are involved with early stages of digestion of host red blood cells.     

The adrenal paraneurone: tubulin organization

When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.     

ORIGIN OF GRANULES IN POLYMORPHONUCLEAR LEUKOCYTES : Two Types Derived from Opposite Faces of the Golgi Complex in Developing Granulocytes

The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO₄. Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger (~800 mµ) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller (~500 mµ), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.     

Ultrastructural cytochemistry of complex carbohydrates in developing feline neutrophils

The feline species provides animal models for at least six congenital lysosomal disorders. Since knowledge of normal feline neutrophils is a prerequisite for studies of their abnormalities, the present report describes the morphology and cytochemistry of normal feline neutrophils and compares the subcellular distribution of sulfate- and vicinal-glycol-containing complex carbohydrates to that of peroxidase and acid phosphatase. Immature feline primary granules, formed in promyelocytes, were stained for peroxidase, acid phosphatase, sulfate, and vicinal glycols. During maturation, primary granules retained strong staining for peroxidase, but staining for vicinal glycols decreased, and acid phosphatase and sulfate reactivity was lost. Secondary granules formed in myelocytes lacked peroxidase, acid phosphatase, and sulfate staining, but stained intensely for vicinal-glycol-containing complex carbohydrates. No analogues of tertiary granules previously described in rabbits and humans were demonstrated in feline neutrophils. However, a new sequential staining technique for peroxidase and vicinal glycols has suggested the formation in myelocytes and late neutrophils of a third granule type that contained peroxidase, acid phosphatase, and vicinal glycols but lacked sulfate staining. Thus, the staining characteristics of primary and secondary granules in cats closely resembled those in humans and rabbits. The third (late-forming) type of granule has not previously been described in other species.     

The composition of pyrophosphate heavy metal detoxification granules in barnacles

A major detoxification mechanism involving the binding of excess metals to inorganic, insoluble metalliferous granules occurs in many invertebrates. X-ray microanalysis of granules extracted from the barnacles Elminius modestus Darwin and Semibalanus balanoides (L.) (Crustacea: Cirripedia) indicated that one granule type was predominant. Examples of this granule type usually contained Fe and P, or Zn and S, or a combination of all four elements as major constituents. The predominant form of P in the granules was pyrophosphate, P₂O⁴⁻₇, with oprthophosphate and other polyphosphates present in lower amounts. The S was present as organic S. Biochemical analysis showed high levels of Ca, Fe, Mg, K and Zn in the granules with Cu and Ag present in smaller quantities. Considerable variation was shown between granules.     

Degradation of phenol by aerobic granules and isolated yeast Candida tropicalis

Aerobic granules effectively degrade phenol at high concentrations. This work cultivated aerobic granules that can degrade phenol at a constant rate of 49 mg-phenol/g x VSS/h up to 1,000 mg/L of phenol. Fluorescent staining and confocal laser scanning microscopy (CLSM) tests demonstrated that an active biomass was accumulated at the granule outer layer. A strain with maximum ability to degrade phenol and a high tolerance to phenol toxicity isolated from the granules was identified as Candida tropicalis via 18S rRNA sequencing. This strain degrades phenol at a maximum rate of 390 mg-phenol/g x VSS/h at pH 6 and 30 degrees C, whereas inhibitory effects existed at concentrations >1,000 mg/L. The Haldane kinetic model elucidates the growth and phenol biodegradation kinetics of the C. tropicalis. The fluorescence in situ hybridization (FISH) and CLSM test suggested that the Candida strain was primarily distributed throughout the surface layer of granule; hence, achieving a near constant reaction rate over a wide range of phenol concentration. The mass transfer barrier provided by granule matrix did not determine the reaction rates for the present phenol-degrading granule.     

The cytomorphology of goblet cells of the fetal intestine. Studies of the large intestine of cattle (Bos primigenius taurus)

In the region of the base of the intestinal crypts undifferentiated goblet cells display a configuration and constellation of organelles and membrane structures that are indicative of their importance for function. These images at this stage of development deliver a scenario of the mechanism of secretory granule production: aggregates of protein vesicles from the "transitional elements" (PALADE) of the granular endoplasmic reticulum are, so to speak, rolled up on the trans side of the Golgi apparatus by inversion of peripheral membrane segments of the innermost Golgi lamellae, thereby forming corpuscles. The origin of the capsulated vacuoles, which contain vesicles as single elements or as conglomerates, is well established. Their capsule consists of a trilaminar external and external and internal membrane; between them lies condensed material of the Golgi apparatus. In the opinion of the present author, the development of the ensheathed vacuoles represents a basic, more general mechanism. In contrast, the further steps of synthesis, for the formation of secretory granules, are more heterogeneous. Condensation of the vesicles and the inner capsular membrane results in the formation of a prosecretory granule, which in the basic element in the process of secretory granule production. The prosecretory granules develop singly or by fusion with other granules to give primary secretory granules. The complexity of this mechanism of secretory granule formation, however, becomes evident when considering the apposition of capsulated vacuoles and prosecretory--primary--secondary secretory granules, of prosecretory and primary secretory granules as well as prosecretory granules and secondary secretory granules. Generally, primary granules show a tendency to become secondary secretory granules or to fuse with them. During maturation of the goblet cells the secretory granules fuse to form larger mucous bodies in the theca by fusion of the laminae of the membranes; a final product, there is a homogeneous mucous mass devoid of membranes.     

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.     

In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell

The ultrastructural localization of growth hormone and prolactin in cow anterior pituitary was studied by double immunocytochemical labeling using specific antibodies and protein A-gold particles of different sizes. The two hormones were found in specific somatotrophs and mammotrophs as well as in somatomammotropic cells which were multinucleated and predominantly arranged in clusters in the central area of the lobules. In these mixed cells the two hormones were packaged (a) in different granules of the same cell, (b) in the same granules where they were segregated in different portions of the granule content, or (c) in the same granules but evenly intermixed. The relative proportion of these three types of granules varied in somatomammotrophs of different animals. A single large Golgi complex was generally present in somatomammotrophs. Small, immature granules containing either growth hormone or prolactin or both hormones were found randomly distributed along Golgi stacks. This suggests that in these cells the two hormones are processed in the same Golgi cisternae and that mechanism(s) exist(s) to sort out the two hormones from each other.     

Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment

AIMS: To obtain biomass and porosity profiles for aerobically grown granules of different diameters and to determine a suitable range of granule diameters for application in wastewater treatment. METHODS AND RESULTS: Microbial granules were cultivated in an aerobic granulated sludge reactor with model wastewaters containing acetate, or ethanol plus acetate, or glucose as the main carbon source. Granules were formed by retaining microbial aggregates using a settling time of 2 min. Sampled granules had diameters ranging from 0.45 to 3 mm. Microbial biomass in the granules was detected with the nucleic acid stain SYTO 9 and confocal laser scanning microscopy. The thickness of the microbial biomass layer was proportional to the granule diameter, and had a maximum value of 0.8 mm. The thickness of the microbial biomass layer correlated with the penetration depth of 0.1 microm fluorescent beads into the granule. CONCLUSIONS: The microbial biomass and porosity studies suggest that aerobically grown microbial granules should have diameters less than a critical diameter of 0.5 mm, if deployed for wastewater treatment applications. This critical diameter is based on the assumption that whole granules should have a porous biomass-filled matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could contribute to the development of aerobic granulation technology for effective biological wastewater treatment.     

A Method of Culturing Rhizobium meliloti on Porous Granules to Form a Pre-Inoculant for Lucerne Seed

A method for the production of a granule inoculant of Rhizobium meliloti for lucerne seed ( Medicago sativa ) is described. The method enables large scale production of such an inoculant to be considered and is derived from the work previously reported (Fraser, 1966). Modifications have resulted in a granule with a higher initial count of rhizobia and with better survival on storage. An inoculum ranging from 200 000 rhizobia/seed from freshly prepared granules to 2000/seed from granules stored for 6 months at room temperature is possible.     

Human leukocytes as viewed by stereo high-voltage electronmicroscopy

Whole-mount preparations of adherent leukocytes were investigated by stereo high-voltage electronmicroscopy (HVEM) to determine the organization of the cytoplast in unstimulated, motile, and phagocytosing cells. A highly ordered structured cytoplast is revealed. All cytoplasmic organelles are held within an intricate network of fine strands, termed the microtrabecular lattice (MTL), which appears more complex in neutrophils than eosinophils or monocytes. In neutrophils, the tendency of the MTL to expand and contract during cell movement and the responding deformability of the granules appear to influence granule shape. This pleomorphism in granule shape is particularly prominent in exceptionally elongated neutrophils that have not established directionality and demonstrate the appearance of having two leading lamellipodia. Results suggest that the morphology of neutrophil granules is influenced by cell motility, and may account for the pleomorphic populations of granules observed by standard transmission EM. Examination of the cytoskeleton of these elongated cells after detergent extraction reveals separation of the centrosome into two solitary centrioles, with each centriole surrounded by an aster of microtubules. A complex network of microfilaments, intermediate filaments, and microtubules is integrated within a thin area of cytoplasm separating the two cell bodies. Interaction between the MTL, microfilaments, intermediate filaments, and microtubules probably influences granule translocation in these elongated cells. Phagocytosis stimulates a reorganization of the cytoplast; all organelles are found in more central areas of the cytoplasm, bordered by a thin area of hyaloplasm. The MTL appears to limit cytoplasmic granules to a compartment around phagocytic vacuoles, which probably provides the framework for efficient phagolysosome fusion.     

PtdIns(4,5)P2 is not required for secretory granule docking

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co‐clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high‐resolution total internal reflection imaging of EGFP‐labeled PtdIns markers or syntaxin‐1 at secretory granule release sites in live insulin‐secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin‐1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin‐1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P₂) by recruitment of a 5′‐phosphatase strongly inhibited Ca²⁺‐dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin‐1. Cell permeabilization by α‐toxin or formaldehyde‐fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P₂ accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin‐1 at the release site.      

Control of starch granule numbers in Arabidopsis chloroplasts

The aim of this work was to investigate starch granule numbers in Arabidopsis (Arabidopsis thaliana) leaves. Lack of quantitative information on the extent of genetic, temporal, developmental, and environmental variation in granule numbers is an important limitation in understanding control of starch degradation and the mechanism of granule initiation. Two methods were developed for reliable estimation of numbers of granules per chloroplast. First, direct measurements were made on large series of consecutive sections of mesophyll tissue obtained by focused ion beam-scanning electron microscopy. Second, average numbers were calculated from the starch contents of leaves and chloroplasts and estimates of granule mass based on granule dimensions. Examination of wild-type plants and accumulation and regulation of chloroplast (arc) mutants with few, large chloroplasts provided the following new insights. There is wide variation in chloroplast volumes in cells of wild-type leaves. Granule numbers per chloroplast are correlated with chloroplast volume, i.e. large chloroplasts have more granules than small chloroplasts. Mature leaves of wild-type plants and arc mutants have approximately the same number of granules per unit volume of stroma, regardless of the size and number of chloroplasts per cell. Granule numbers per unit volume of stroma are also relatively constant in immature leaves but are greater than in mature leaves. Granule initiation occurs as chloroplasts divide in immature leaves, but relatively little initiation occurs in mature leaves. Changes in leaf starch content over the diurnal cycle are largely brought about by changes in the volume of a fixed number of granules.