Subsequent to Its Endocytosis by Megakaryocytes, Factor V Is Trafficked to the [Italic]cis[/Italic]-Golgi Network Prior to Its Storage in α-Granules.

Human platelet-derived factor V originates from megakaryocyte endocytosis of its plasma counterpart. Circulating platelets do not endocytose plasma-derived factor V and little, if any, of this hemostatically relevant, and unique, cofactor protein is synthesized by megakaryocytes. Fibrinogen, another α-granule protein, is also endocytosed from plasma, whereas other similarly stored proteins are synthesized by megakaryocytes, e.g. vWF and P-selectin. Using CD34+ex vivo-derived megakaryocytes as a model, mechanisms defining megakaryocyte endocytosis of plasma-derived factor V and its intracellular trafficking to α-granules are being studied. Expression of the well-known megakaryocyte proteins αIIb, β3, vWF, and P-selectin increased in parallel concomitant with the loss of CD34 and increased cellular differentiation. Expression of these proteins was apparent within 4 days of cell culture and was maximal by day 10. In contrast, endocytosis of factor V by cells at earlier stages of megakaryocyte differentiation (day 7) occurred in the absence of αIIb and vWF expression. By day 10 in culture, all cells endocytosing factor V also expressed αIIb and vWF. Fibrinogen endocytosis occurred with the concomitant expression of αIIb/β3. The colocalization of fluorescently-labeled factor V and fibrinogen with various organelle-specific, fluorescent antibodies was determined by confocal microscopy using day 10 or 11 ex vivo-derived megakaryocytes. After a 1 hr endocytosis period, 87.8 ± 7.2% of the factor V colocalized with an antibody to Rab5, an early endosomal marker. Colocalization decreased over time such that only 5% of the Rab5 colocalized with factor V at 4 hrs. Endocytosed factor V also colocalized in a time-dependent manner with an antibody to GM130, a cis-Golgi element, consistent with the hypothesis that endocytosed, plasma-derived factor V is structurally modified to generate the unique platelet-derived molecule. After a 2 hr endocytosis period, 53.2 ± 23.0% of the GM130 specific antibody colocalized with factor V. Colocalization decreased 5-fold by 4 hrs. In contrast, little if any of the GM130 antibody colocalized with endocytosed fibrinogen, while >80% of the cis-Golgi element colocalized with vWF. After 19 hrs, substantial colocalization was also observed between endocytosed factor V and vWF (84.2 ± 3.7%) or P-selectin (54.8 ± 3.5%), which is consistent with their storage in α-granules and confirms flow cytometric analyses. The colocalization of endocytosed factor V and fibrinogen was also determined over time. These proteins colocalized quickly (1 hr) likely due to their uptake into early endosomes as suggested by previous studies in a megakaryocyte-like cell line. Colocalization reached a maximum and plateaued after endocytosis periods ≥ 8 hrs again consistent with their ultimate storage in α-granules. At endocytosis periods < 8 hrs, less factor V colocalized with fibrinogen consistent with the localization of factor V in the cis-Golgi network at these times. In conclusion, these combined observations suggest that following its endocytosis by megakaryocytes, factor V is taken up into early endosomes and trafficked through the Golgi complex prior to its storage in α-granules. Its transport through the Golgi is consistent with previous observations that platelet-derived factor V contains an O-linked glycosylation not found in its plasma counterpart.

The cytochemical demonstration of GERL in rat hepatocytes during lipoprotein mobilization.

When a semisynthetic diet containing 1% orotic acid (OA) is fed to rats, the endoplasmic reticulum (ER) of hepatocytes vesiculates and lipoprotein (LP) droplets accumulate within the vesicles. When clofibrate (ethyl chlorophenoxyisobutyrate, CPIB) is added to the orotic acid-rich diet, the ER cisternae reform and the LP is mobilized through the reconstituted ER. A remarkable restoration of normal hepatocyte ultrastructure occurs except for a few organelles. From their morphological appearance it was suggested that cisternae which became dilated with small LP particles were part of GERL, abnormally enlarged. The present communication validates this interpretation through ultrastructural cytochemistry which can distinguish GERL from the adjacent Colgi apparatus. GERL shows acid phosphatase (AcPase) but not thiamine pyrophosphatase (TPPase) activity. In contrast, the adjacent Golgi element shows thiamine pyrophosphatase but not acid phosphatase activity. From such cytochemical studies we have recently proposed that GERL in normal rat hepatocytes may be involved in transforming LP particles, by enzymes like lipases that were presumed to be present in this hydrolase-rich portion of smooth ER. In the situation studied in this communication, the addition of ethyl chlorophenoxyisobutyrate to the diet causes the release from the ER of large amounts of LP to the Golgi apparatus and to GERL. Apparently the capacity of GERL to metabolize LP is exceeded and lipid accumulates in the residual bodies.

GOLGI APPARATUS, GERL, AND LYSOSOMES OF NEURONS IN RAT DORSAL ROOT GANGLIA, STUDIED BY THICK SECTION AND THIN SECTION CYTOCHEMISTRY

New insights into the ultrastructure and phosphatase localizations of Golgi apparatus and GERL, and into the probable origin of lysosomes in the neurons of fetal dorsal root ganglia and the small neurons of adult ganglia have come from studying thick (0.5–1.0 µ) as well as thin (up to 500 A) sections by conventional electron microscopy. Tilting the thick specimens, by a goniometer stage, has helped to increase our understanding of the three-dimensional aspects of the Golgi apparatus and GERL. One Golgi element, situated at the inner aspect of the Golgi stack, displays thiamine pyrophosphatase and nucleoside diphosphatase activities. This element exhibits regular geometric arrays (hexagons) of interconnected tubules without evidence of a flattened portion (saccule or cisterna). In contrast, GERL shows acid phosphatase activity and possesses small cisternal portions and anastomosing tubules. Lysosomes appear to bud from GERL. Osmium deposits, following prolonged osmication, are found in the outer Golgi element. Serial 0.5-µ and thin sections of thiamine pyrophosphatase-incubated material demonstrate that, in the neurons studied, the Golgi apparatus is a continuous network coursing through the cytoplasm. Serial thick sections of acid phosphatase-incubated tissue suggest that GERL is also a continuous structure throughout the cytoplasm. Tubules of smooth endoplasmic reticulum, possibly part of GERL, extend into the polygonal compartments of the inner Golgi element. The possible physiological significance of a polygonal arrangement of a phosphatase-rich Golgi element in proximity to smooth ER is considered. A tentative diagram of the Golgi stack and associated endoplasmic reticulum in these neurons has been drawn.

A histochemical study of the development of the mantle-edge and shell in the freshwater gastropod, Helisoma duryi eudiscus (Pilsbry)

Glycogen and ribonucleic acid are present in the mantle-edge during the prehatching period and in the adult. The cubocolumnar epithelium contains the largest stores of glycogen. Ribonucleic acid is most abundant in the mantle-edge gland, and the mucous and mucoprotein cells. Mucopolysaccharides and glycoproteins occur within the mantle-edge epithelium, excepting the mantle-edge gland, and within the shell ground substance. Mucous glands and the sheath surrounding the organic plates are rich in sulfated mucopolysaccharides. Alkaline phosphatase and calcium could not be demonstrated during the prehatching stages. In the adult, alkaline phosphatase reactions are intense along the distal border of the cubocolumnar epithelium, and the basal borders of the epithelia of the mantle-edge gland, median lobe, and ventral lobe. Calcium carbonate occurs as spherules in the connective tissue, in the extrapallial fluid, and within the organic plates and crystalline layers of the shell. In the adult, lipids are most plentiful in the dorsal lobe epithelium and yellow body cells. Vitamin A occurs only within the cubocolumnar and yellow body cells. Cytochrome oxidase is present within the mantle-edge epithelium and, in terms of relative amounts, reflects the activity of the various lobes. Similarly, the size of the Golgi element can be correlated with the activity of the mantle-edge epithelia.

The Histochemistry of the Saccus Vasculosus of Notopterus Chitala (Teleostei)

The saccus vasculosus of Notopterus chitala is situated posteriorly to the pituitary gland. It consists of a number of loculi surrounded by blood sinusoids. The loculi open into a number of collecting channels which unite and ultimately drain into the third ventricle of the brain. The loculi are lined by a layer of pear-shaped ‘coronet cells’. The coronet cell has an apical protrusion provided with hairs, each ending in a globule. The coronet cells contain glycogen, especially in the apical protrusions and in the globules. Phospholipid, alkaline phosphatase, and mitochondria are concentrated in the apical protrusion and globules. There are also mitochondria round the nucleus. The Golgi element is not only present in the characteristic Golgi zone but occasionally round the nucleus as well. It has been stated by other authors that acid mucopolysaccharides are synthesized in the coronet cells of the rainbow trout and ‘secreted’ into the lumen of saccus vasculosus. However, acid mucopolysaccharides are not histochemically demonstrable in the coronet cells of Notopterus. The observations recorded in this paper indicate that the coronet cells in the saccus vasculosus of Notopterus are secretory and that glycogen is abundant in them.

Some Observations upon the Golgi Elements of the Cells of the Surgically Removed Human Anterior Pituitary

1. The lipid inclusions of the cells of the pars distalis of the human pituitary are not restricted to the zone occupied by the Golgi element. 2. No evidence was obtained to suggest that a canalicular system is invariably associated with the Golgi zone. The frequency with which such systems were observed appeared to depend on the mode of fixation and other factors.

On the Functional Morphology of the Alimentary Tract of Some Fish in Relation to Differences in their Feeding Habits: Cytology and Physiology

1. The free border of the intestinal absorptive cells of cyprinids is nearly similar to that in tetrapods. This border has been described for four other species of fish and the possible evolution of the border from a ciliated epithelium has been described. 2. The mitochondria of the absorptive cells are arranged in a supranuclear group of mainly rod-like bodies and a subnuclear group of mainly granular ones. 3. The mitochondria are coarser and the Golgi element more readily impregnated with osmium tetroxide in the carnivorous Gobio than in the omnivorous Rutilus or the herbivorous Cyprinus. In all three fish both organoids are associated with fat absorption. 4. The goblet cells secrete both mucus and zymogen. Their stomata are permanently open. 5. Granular cells are described in Rutilus and Cyprinus, and in seven other species of fish, but they are absent from Gobio. Their varying reactions to numerous cytological techniques are described. 6. Alkaline phosphatase is especially concentrated in the free border of the absorptive cells. It occurs along the whole length of the gut. Its possible role in the absorption of fat and glucose is discussed. 7. Lipase is secreted by the absorptive cells. Fat absorption occurs along the whole length of the intestine, but the second limb of the intestine is especially active in this respect. 8. Carbohydrases and lipases are more concentrated towards the anterior end of the gut, whereas proteinases are more abundant caudally. 9. Carbohydrases are richer in Cyprinus and Rutilus than in Gobio. 10. Zymogen and lipase secretions are more concentrated in Gobio (carnivorous) than in the other two species. 11. The pH of the intestine of all three fishes is round about neutral point. Free HCl is absent from all three.

Further Remarks on the Golgi Element

1. The Golgi element has been reinvestigated in the same kinds of cells as were the subject of the author's 1944 paper. 2. Two new methods have been used, namely, phase-contrast microscopy and an improved form of the sudan black technique, in which the tissues are postchromed at 60° C. 3. The Golgi element consists of separate bodies, spheroid in shape. These Golgi bodies may be simple (i.e. non-vacuolate), or may contain one or more vacuoles. The material of the simple Golgi body and of the externum of the vacuolate body is a lipoid that in some cases can be shown to contain lipine. The secretion-product of the Golgi body originates in the vacuole. 4. The opinion as to the structural plan of the Golgi element set out in the earlier paper has been confirmed in the main. There are, however, two exceptions to this: (a) The vacuole in the Golgi body does not invariably colour with neutral red, and this dye occasionally causes the appearance of vacuoles not present before, both within the Golgi region and in other parts of the cytoplasm. (b) ‘Diffuse lipoid’ is not a characteristic feature of the Golgi element.

Memoirs: The Structure and Chemical Composition of the Golgi Element

Structure 1. Reasons are given for believing that the methods used to produce the classical Golgi network cannot be relied upon to give an accurate picture of the structure of the Golgi element during life. 2. In this investigation reliance has been placed chiefly on vital observations and on the results of the formal-sudan-black technique. Sudan black is a colouring agent with an intense affinity for lipoids,1 whether ‘masked’ or not. 3. The Golgi element was studied in the following cells: (1) the primary spermatocyte and early spermatid of the common snail, Helix aspersa; (2) the absorptive cell of the intestinal epithelium of the smooth or common newt, Triturus vulgaris ; (3) the nerve cell of the anterior mesenteric ganglion of the rabbit, Oryctolagus cuniculus. 4. In its fully developed condition, the Golgi element of diverse cells consists of four parts: (1) the ‘neutral-red vacuoles’; (2) the dense lipoid-containing substance, generally in close relation to the vacuoles in the form of strands, ‘lepidosomes’, caps, crescents, rings, or complete investments; (3) the diffuse lipoid-containing substance, which fills all the space in the Golgi element not occupied by the other constituents; (4) the Golgi-product, which arises in the vacuoles and is the result of the synthesis achieved by the Golgi element.

Memoirs: The Golgi Element in the Cells of the First and Second Convoluted Tubules of the Cat Kidney

1. The Golgi element in the cells of the first and second convoluted tubules, of the cat kidney have been demonstrated by the Aoyama and Da Fano techniques. 2. In both sorts of tubules the Golgi element appears as a rod-like or granular, sometimes crescentic structure, solid rather than reticular. The Golgi element usually lies on the free side of the nucleus. 3. In the macula densa of the second convoluted or distal tubule the Golgi element is seen on the basal side of the nucleus, that is, in apparent reversal to the position in the other cells. This condition in the cat is similar to that which is found in the rabbit and has been found in one autopsied case of nephritis (human). 4. The reversal of the Golgi element in the cells of the macula densa suggests (a) that some substance from the contents of the tubule here passes across the cells of the distal tubule to the specialized cells at the glomerular root, (b) this process is the method by which the haemodynamics of the nephron are integrated. This proposal, much like that made by Goormaghtigh, is here suggested as a basis upon which physiology of the normal and diseased kidney might be investigated cytologically.