Ultrastructural localization of calcium in post-mortem bovine muscle: A cytochemical and X-ray microanalytical study

Summary Calcium localization was demonstrated in bovine longissimus muscle using the antimonate precipitation technique in combination with electron probe X-ray microanalysis. Samples were taken each hour during the first 24 h post-mortem, and then after a storage period of 8 and 15 days. For all sampling times analysed, heavy precipitates were seen in dense parts of nuclei and on N-lines of myofibrils. Up to 18–20 h post-mortem, deposits were observed in sarcoplasmic reticulum at the level of triads. In comparison with the earlier post-mortem samples, myoplasmic precipitates were strongly increased at 4 h post-mortem, and just before rigor onset, at 19 h where intermyofibrillar spaces were completely blackened and triads were no more visible. These localizations of precipitates were still observed up to 15 days post-mortem. At these storage times, myofibril disruptions were seen at the level of N-lines. Wavelength-dispersive and energy-dispersive spectrometric analyses indicated that significant amounts of calcium occurred in the dense precipitates observed.     

Calcium distribution in islets of Langerhans: a study of calcium concentrations and of calcium accumulation in B cell organelles.

Calcium concentrations of various pancreatic B cell organelles have been determined by X-ray microanalysis of areas of frozen sections of unfixed rat islets of Langerhans. Highest concentrations were detected in storage granules and in mitochondria, although calcium was also present in nuclei, in areas of endoplasmic reticulum and of cytoplasm. Accumulation of 45Ca by isolated organelles has been studied in homogenates and isolated subcellular fractions of rat islets of Langerhans. In the presence of a permeant anion (oxalate or phosphate), accumulation of 45Ca into mitochondria and microsomes was strongly stimulated by ATP. This net uptake was diminished during incubation of homogenates or of a mitochondria plus storage granule-rich fraction in the presence of cyclic AMP, dibutyryl cyclic GMP; 2:4-dinitrophenol or of ruthenium red. Investigations of the characteristics of 45Ca accumulation by homogenates prepared from storage granule-depleted islets showed no differences from those of normal islets, suggesting that the granules do not represent an important labile pool of calcium. With the exception of cyclic AMP and cyclic GMP none of the insulin secretagogues tested (glucose, leucine, arginine, adrenalin, noradrenalin, theophylline, glibenclamide) altered calcium accumulation by islet homogenates. On the basis of absolute calcium levels and of 45Ca uptake studies it is concluded that islet B cells contain a readily exchangeable mitochondrial calcium pool, and an endoplasmic reticulum pool containing a lower concentration of calcium which is also readily exchangeable. The storage granules, despite their high calcium content, do not appear to constitute a labile pool. It seems likely that the labile mitochondria and endoplasmic reticulum pools play a predominant role in the regulation of cytoplasmic free calcium levels, which may in turn be important in the regulation of rates of insulin secretion.     

Uptake of calcium by the endoplasmic reticulum of the frog photoreceptor

We studied retinal photoreceptors of Rana pipiens by using techniques designed to investigate calcium localization. Particularly useful were methods in which intracellular sites of calcium uptake were detected by incubation of saponin-treated isolated retinas in calcium-containing media, with oxalate present as a trapping agent. With these procedures, cell compartments accumulate deposits, which can be shown to contain calcium by x-ray microanalysis. Calcium accumulation was prominent in the rough endoplasmic reticulum in the myoid region. In addition, deposits were observed in agranular reticulum and in certain Golgi- associated compartments of the myoid region, in mitochondria, in axonal reticulum, and in agranular reticulum of presynaptic terminals. Calcium was also detected in the endoplasmic reticulum of retinas fixed directly upon isolation, by a freeze-substitution method. The factors influencing accumulation of calcium in the endoplasmic reticulum were evaluated by a semiquantitative approach based on determining the relative frequency of calcium oxalate crystals under varying conditions. Calcium accumulation was markedly enhanced by ATP. Studies with a nonhydrolyzable ATP analogue (adenylyl- imidodiphosphate ) and with inhibitors of the sarcoplasmic reticulum Ca2+-Mg2+ ATPase (mersalyl and tetracaine) indicated that this ATP-dependent calcium uptake reflects an energy-dependent process roughly comparable to that in the sarcoplasmic reticulum.     

Ultrastructural visualization of lipids in trypanosomatids

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis. T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.     

Ultrastructural Localization of Calcium in the Presumptive Ectodermal Cells in Gastrulae of the Newt, Cynops pyrrhogaster, as Revealed by Cytochemistry and X-Ray Microanalysis

The ultrastructural localization of calcium in the presumptive ectodermal cells of gastrulae of the newt, Cynops pyrrhogaster , was examined by cytochemical methods and X-ray microanalysis (XMA). The cells were fixed with solutions that contained potassium oxalate, potassium ferricyanide and potassium pyroantimonate to preserve the localization of intracellular calcium in situ and for the analysis of electron density due to calcium. Electron-dense deposits associated with the localization of calcium were observed under the electron microscope. Specificially, pigment granules, round vacuoles, endoplasmic reticulum and mitochondria as well as the extracellular matrix were observed to contain calcium. In addition, XMA clearly demonstrated the localization of calcium in all of these electron-dense organelles and yolk granules.     

Experimental study of follicle formation in suppressed parathyroid glands of Mongolian gerbils.

Parathyroid follicle formation was studied in Mongolian gerbils subjected to different concentrations of calcium in vivo and in vitro, using light and electron microscopic methods, including the potassium pyroantimonate technique and x-ray microanalysis for identification of cations. Follicles were frequent at high calcium concentration, but sparse at intermediate and low levels of calcium. Two main types of follicle were differentiated: "degenerative follicles" containing cellular debris and lined by smooth-surfaced epithelium which occasionally showed degenerative changes; and "secretory follicles" characterized by amorphous and granular contents, and an epithelium possessing microvilli and cytoplasmic projections. Amorphous masses were also seen in dilated intercellular spaces and in dilated cisterns of rough endoplasmic reticulum in the follicle epithelium. Calcium-containing precipitates were found in degenerating chief cells, and between degenerating cells and follicles. Parathyroid follicles are believed to be formed by degeneration of suppressed chief cells (degenerative follicles), and by secretion of hormonal and/or other substances into dilated intercellular spaces which progressively increase in size to form follicular cavities (secretory follicles), thereby possibly reducing the level of metabolically active parathyroid hormone. Functional suppression is believed to underlie the development of parathyroid follicles.     

Ultrastructural Visualization of Lipids in Trypanosomatids1

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis, T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.     

Accumulation of cadmium, lead and strontium, and a role of calcium oxalate in water hyacinth tolerance

Eichhornia crassipes plants brought from the River Nile were cultured in jars containing river water supplemented with various concentrations of Cd, Pb, and Sr (0 to 100 μg cm-3), added simultaneously. Treatment continued for 20 d during which each cultivation solution was being replaced with fresh one every 3 d. The growth of Eichhornia was drastically retarded at heavy metal concentrations higher than 15 μg cm-3. At concentrations 15 or 25 μg cm-3, the accumulation of Cd and Pb to levels several times higher than those in control plants was found. More than 50 % of the uptaken metals were retained by roots alone. Leaves and leaf petiols received around 30 and 20 % of the accumulated metals, respectively. X-ray microanalysis indicated the presence of the three heavy metals in Ca oxalate crystals. Content of metals in the crystals increased progressively over time of exposure in a way similar to those in whole plant tissues. These results suggest a possible role for Ca oxalate crystalization in toxic heavy metal deposition and thus tolerance by Eichhornia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and properties of plasma membrane from smooth muscle

A generalized approach to obtain relatively pure fractions of plasma membrane from smooth muscle tissues for studying calcium transport is described. The use of various markers for cellular membranes to establish the purity of various fractions is critically considered. Plasma membranes from rat myometrium have been isolated in a purity estimated to be 95-99%. Plasma membrane purifications to 70-80% have been achieved from rat mesenteric arteries and veins, canine tracheal smooth muscle, rabbit intestinal muscle, rat vas deferens, rat fundus, and dog gastric corpus. The ATP-dependent transport of Ca is correlated with the distribution of plasma membrane markers. Ca gradient of greater than 1000-fold have been achieved. ATP-dependent active Ca transport by plasma membranes could sometimes be stimulated by oxalate or phosphate. Anion activation of Ca active transport is not a marker for endoplasmic reticulum. In some smooth muscles (e.g., rat vas deferens) ATP-dependent Ca uptake did not correlate exclusively with the distribution of plasma membrane markers. Instead, the correlation seemed to be with NADPH-cytochrome reductase EC 1.6.2.5 activity (putative endoplasmic reticulum marker) as well as with plasma membrane markers. In all smooth muscles, active Ca transport appears to be a property of the plasma membrane; in some it may also be a property of the endoplasmic reticulum. Mitochondria actively transport Ca, but in most systems studied to date, the Km for Ca2+ for this transport is higher than that for plasma membrane. Thus the plasma membrane may be the major physiological mechanism of active transport for Ca out of cytoplasm of smooth muscle cells. In two plasma membrane fractions (from rat myometrium and mesenteric arteries) it has been possible to demonstrate the existence of an Na-Ca exchange system. Its contribution to lowering cytoplasmic Ca is unknown.     

Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

The effects of Al on red spruce ( Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereological and microscopic methods. Exposure to Al for 24–48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al. Using energy-dispersive X-ray microanalysis on sections of freeze-substituted cells that had no obvious disruption in cytoplasmic or cell wall structure, Al (always in the presence of P) was detected in dense regions in cell walls, cytoplasm, plastids and vacuoles after 48 h exposure to Al. Stereological quantification of spruce cell structure showed that, after 24 h of Al treatment, intact cells had increased vacuolar and total cell volume, but the nuclear volume did not change. In addition, Al treatment resulted in increased surface area of Golgi membranes and endoplasmic reticulum. The biochemical and ultrastructural alterations in red spruce cells, in combination with the presence of Al in cellular organelles of visually intact cells, suggest that Al movement occurred across the plasma membrane without major cellular disruption. Detailed short-term time course studies are needed to determine if intracellular Al in these cells results from its passage into cells through submicroscopic lesions in the plasma membrane or it is taken up into the symplast through the intact membrane by an active, but slow, process.     

Metabolomics of the human aqueous humor

Introduction

The optical elements of the eye—cornea, lens, and vitreous humor—are avascular tissues, and their nutrition and waste removal are provided by aqueous humor (AH). The AH production occurs through the active secretion and the passive diffusion/ultrafiltration of blood plasma. The comparison of the metabolomic profiles of AH and plasma is important for understanding of the mechanisms of biochemical processes and metabolite transport taking place in vivo in ocular tissues.

Objectives

The work is aimed at the determination of concentrations of a wide range of most abundant metabolites in the human AH, the comparison of the metabolomic profiles of AH and serum, and the analysis of the post-mortem metabolomic changes in these two biological fluids.

Methods

The quantitative metabolomic profiling was carried out with the use of two independent methods—high-frequency ¹H NMR spectroscopy and HPLC with high-resolution ESI-MS detection.

Results

The concentrations of 71 most abundant metabolites in blood serum and AH from living patients and human cadavers have been measured. It has been found that the level of ascorbate in AH is by two orders of magnitude higher than that in serum; the levels of other metabolites are either similar to that in serum, or differ from that by a factor of 2–5. The post-mortem metabolomic composition of both serum and AH undergoes rapid and strong changes.

Conclusion

The differences between the metabolomic profiles of AH and serum for majority of metabolites can be attributed to the metabolic activity of the ocular tissues leading to the lack or excess of some metabolites, while the high concentration of ascorbate in AH demonstrates the activity of ascorbate-specific pumps at the blood-aqueous border. The post-mortem metabolomic changes are caused by the disruption of the major biochemical cycles and cell lysis. These changes should be taken into account in the analysis of disease-induced changes in post-mortem samples of the ocular tissues.

     

The endoplasmic reticulum network in the ascidian egg: localization and calcium content

In contrast with most other eggs, where the endoplasmic reticulum is mixed with many other organelles, in ascidians, continuous sheets and tubes of endoplasmic reticulum constitute the only prominent organelle in the immediate layer (0.5-1μm) beneath the plasma membrane, and occupies 16–20% of the cortical volume. We took advantage of this unusual stratification of the organelles in the ascidian egg, to carry X-ray microanalysis. Our measurements provide the first estimate of the calcium content of the endoplasmic reticulum network in an egg, and show it is the main calcium store.     

Biochemistry Changes That Occur after Death: Potential Markers for Determining Post-Mortem Interval

Death is likely to result in very extensive biochemical changes in all body tissues due to lack of circulating oxygen, altered enzymatic reactions, cellular degradation, and cessation of anabolic production of metabolites. These biochemical changes may provide chemical markers for helping to more accurately determine the time since death (post-mortem interval), which is challenging to establish with current observation-based methodologies. In this study blood pH and changes in concentration of six metabolites (lactic acid, hypoxanthine, uric acid, ammonia, NADH and formic acid) were examined post-mortem over a 96 hour period in blood taken from animal corpses (rat and pig) and blood from rats and humans stored in vitro. The pH and the concentration of all six metabolites changed post-mortem but the extent and rate of change varied. Blood pH in corpses fell from 7.4 to 5.1. Concentrations of hypoxanthine, ammonia, NADH and formic acid all increased with time and these metabolites may be potential markers for post-mortem interval. The concentration of lactate increased and then remained at an elevated level and changes in the concentration were different in the rat compared to the human and pig. This is the first systematic study of multiple metabolic changes post-mortem and demonstrates the nature and extent of the changes that occur, in addition to identifying potential markers for estimating post-mortem interval.     

An X-ray, microanalytical and ultrastructural study of cadmium absorption and transport in rat liver

Accumulation of cadmium in the liver was demonstrated by X-ray microanalysis in every type of experiment, i.e. after injecting Cd into the ligated intestine and after the peroral acute single and combined, subchronic and chronic administration of Cd. Half an hour after its injection, Cd was localized diffusely in the liver; one hour after injection its increased accumulation in the cells caused generalized changes in the endoplasmic reticulum, mitochondria and nuclei. In acute and chronic peroral tests, the hepatocytes of the intermedial and peripheral zone of the lobes were the main storage region. After an acute dose of Cd, the cells in the centrolobular zone were hydropic, or single-cell necrosis developed; after the longer effect of combined doses the latter was manifested as centrolobular focal necrosis. Cd was not demonstrated in the lesions. Chronic administration did not lead to manifest severe degenerative changes in the liver. Accretions in the mitochondria and on the membranes of the endoplasmic reticulum were identified by means of X-ray analysis with cadmium peaks. Cadmium showed up clearly as L alpha- and L beta-lines at 3.135-3.320 keV. We presume that cadmium is bound in the ribosomes of the endoplasmic reticulum, as well as the mitochondria, and is released by the invagination of swelling mitochondria of the peripheral hepatocytes into Disse's spaces.     

Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.

The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.     

Evidence that basal secretion of relaxin by individual cultured large luteal cells is influenced by mobilization of intracellular calcium: analysis by a reverse hemolytic plaque assay.

Ca2+ redistribution from an intracellular site(s) is a key biochemical event associated with relaxin (RLX) secretion by large luteal cells (LLCs) of porcine origin. However, the functional significance of internal stores of Ca2+ to basal rates of RLX secretion is not well understood. In addition, the identity of the intracellular storage site(s) for Ca2+ within LLCs is not known, nor is it clear if all RLX-releasing LLCs are equally dependent on this pool. In the present study, release of RLX from 24 h cultured luteal cells derived from early pregnant swine was monitored by a reverse hemolytic plaque assay (RHPA). Incubation of cultures in the presence of graded concentrations of thapsigargin (1 nM-1 microM), a plant sesquiterpene lactone that inhibits endoplasmic reticulum Ca(2+)-ATPase and thereby increases cytosolic Ca2+ concentrations, resulted in a dose-related increase in basal RLX secretion. The stimulatory effect of thapsigargin on RLX production was not abrogated by culture in Ca(2+)-free medium. Suppression of Ca2+ release from the endoplasmic reticulum of LLCs, achieved by incubating monolayers in medium containing dantrolene (1-100 microM), resulted in dose-related inhibition of basal RLX release. Taken together, these results suggest that the endoplasmic reticulum serves as a major storage site for Ca2+ redistribution within LLCs and, furthermore, that mobilization from this site is functionally coupled to basal secretion of RLX.     

Effects of cholinergic and α-adrenergic agonists on the monovalent ion content of rat submandibular gland acinar cells studied by X-ray microanalysis

 The effects of cholinergic and α-adrenergic stimulation (in vivo and in vitro) on the monovalent ion content of rat submandibular gland acinar cells were evaluated at the subcellular level by X-ray microanalysis. Fragments of glands or enzymatically dispersed acini were slam-frozen and cut into ultrathin cryosections. Spectra were collected from secretory granules, nucleus, the basal cytoplasm containing endoplasmic reticulum and the apical cytoplasm identified between secretory granules. No significant changes in Na and Cl content were observed after the isolation of acini, but the K concentration decreased compared with cells from in situ glands. The Cl and K content in all four compartments studied decreased significantly after cholinergic stimulation both in vivo and in vitro but in a more restricted fashion after α-adrenergic stimulation. Our findings indicate that: (1) the physiological mechanisms regulating the monovalent ion composition of submandibular cells are relatively well preserved in isolated acinar cells; (2) the results from in vivo experiments are in good agreement with those from in vitro experiments; and (3) the effects of cholinergic and α-adrenergic stimulation on the K⁺ and Cl^–efflux at the subcellular level are similar but the response is generally less with α-adrenergic stimulation. Accepted: 24 April 1997     

ARCHITECTURE AND NERVE SUPPLY OF MAMMALIAN SMOOTH MUSCLE TISSUE

Smooth muscle tissue from mouse urinary bladder, uterus, and gall bladder has been studied by means of the electron microscope. The smooth muscle cells are distinctly and completely separated from each other by a cytolemma comparable to the sarcolemma of striated muscle. The tissue is thus cellular and not syncytial. With this evidence, supported by electron microscopy of other tissues, we question the existence of true syncytia in animal tissues. Individual cell membranes necessary for the electrophysiologic events exist in smooth muscle, and its nerve and conduction in a tissue such as uterus or bladder can occur at the cellular level as well as at the tissue area level. The smooth muscle cell contains myofilaments, nucleus, endoplasmic reticulum, mitochondria, Golgi complex, centrosome, and pinocytotic vesicles. These structures are described in some detail, and their probable interrelations and functions are discussed. The autonomic nerves innervating smooth muscle cells are composed of axons and lemnoblasts. The axon is suspended by the mesaxon formed by the infolded plasma membrane of the lemnoblast. The respective plasma membranes separate axon and lemnoblast from each other and from surrounding muscle cells. The axons of autonomic nerves never penetrate the plasma membrane of the muscle cell, but pass or intrude into muscle cell pockets, forming a contact between axonal plasma membrane and smooth muscle plasma membrane. The lemnoblast shows well developed endoplasmic reticulum with Palade granules, mitochondria, and a long, elliptical nucleus. The axon contains neurofilaments, mitochondria, and synaptic vesicles; the quantity of the latter two being significantly greater in the periphery of lemnoblasts and near axon-muscle contact regions. We regard the contact regions as the synapses between the autonomic nerves and the smooth muscle cells.     

A morphometric analysis of the redistribution of organelles in columella cells in primary roots of normal seedlings and agravitropic mutants of Hordeum vulgare

The redistribution of organelles in columella cells of horizontally-oriented roots of Hordeum vulgare was quantified in order to determine what structural changes in graviperceptive (i.e., columella) cells are associated with the onset of the root gravicurvature. The sedimentation of amyloplasts is the only major change in cellular structure that correlates positively with the onset of root gravicurvature, which begins within 15 min after re-orientation. There is no consistent contact between sedimented amyloplasts and any other organelles. Nuclei are restricted to the proximal ends of columella cells in vertically-oriented roots, and remain there throughout gravicurvature after roots are oriented horizontally. Root gravicurvature does not involve significant changes in (1) the volume of columella cells, (2) the relative or absolute volumes of organelles in columella cells, or (3) the distribution of endoplasmic reticulum (ER). The size, number and sedimentation rates of amyloplasts in columella cells of non-graviresponsive roots of mutant seedlings are not significantly different from those of graviresponsive roots of normal seedlings. Similarly, there is no significant difference in (1) cellular volume, (2) distribution or surface area of ER, (3) patterns or rates of organelle redistribution in horizontally-oriented roots, (4) relative or absolute volumes of organelles in columella cells of graviresponsive and non-graviresponsive roots. These results suggest that the lack of graviresponsiveness by roots of mutant seedlings is probably not due to either (1) structural differences in columella cells, or (2) differences in patterns or rates of organelle redistribution as compared to that characteristic of graviresponsive roots. Thus, the basis of non-graviresponsiveness in this mutant is probably different from other agravitropic mutants so far studied.     

The secretion of collagen by insects: uptake of [3H]proline by collagen-synthesizing cells in Locusta migratoria and Galleria mellonella.

The uptake of [H3]proline by collagen-secreting cells of the locust, Locusta migratoria, and wax-moth, Galleria mellonella, has been investigated by electron autoradiography. The locust cells are around the ejaculatory duct and they secrete collagen in the young adult male, while the wax-moth cells are those which produce the dorsal mass of connective tissue on the abdominal nerve cord during the late pupal stage. The cells were exposed to [H3]proline either by injection of the [3H]proline into the insect, or as a pulse while the tissue was maintained in a culture medium. The tissues were fixed at differeing experimental times after exposure to the [3H]proline. The resulting electron autoradiographs were subjected to quantitative analysis, and the silver grain distribution was determined as the relative number of grains per unit area over a series of tissue compartments. When the results of this analysis for the matrix, rough endoplasmic reticulum and Golgi complexes of the two tissues were plotted against experimental time, it was seen that the relative number of grains per unit area over the rough endoplasmic reticulum decreases while that over the matrix increases; statistical analysis has shown that these changes are significant. For the Golgi complexes, however, the theoretical variances are much greater, due to the small relative area occupied by this organelle. There is little evidence for anything other than random sampling fluctuations in the relative numbers of grains per unit area, and hence it is unlikely that the time course of the label over the Golgi complexes follows that over the rough endoplasmic reticulum. The conclusions drawn from these experiments are firstly that a large portion of the labelled protein passes straight from the rough endoplasmic reticulum to the matrix, but that a smaller portion of the labelled material might pass from the rough endoplasmic reticulum to the Golgi complexes and thence to the matrix. It is assumed that collagen comprises most of the protein which passes straight from the rough endoplasmic reticulum to the matrix, and while there is no evidence to exclude collagen from the material passing through the Golgi complexes, it is probable that other proteins and glycosaminoglycans are also present in this labelled material. These conclusions about the intracellular pathway for collagen secretion are similar to those derived from recent studies of some vertebrate fibroblasts. There is, however, conflicting opinion about the intracellular pathway of collagen and it is pointed out that there is diversity in collagen-synthesizing cells, which may account for the differences in the intracellular pathways for collagen secretion which have been proposed.