Endocytotic pathways at the ruffled borders of rat maturation ameloblasts

Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytic vacuoles ( pinosomes ) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes , which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.     

AUTOPHAGIC VACUOLES PRODUCED IN VITRO : II. Studies on the Mechanism of Formation of Autophagic Vacuoles Produced by Chloroquine

Continuous phase-contrast observations have been made on macrophages following exposure to chloroquine. The initial abnormality is the appearance in the Golgi region of small vacuoles with an intermediate density between that of pinosomes and granules. Over the course of 1–2 hr these vacuoles grow larger and accumulate amorphous material or lipid. Pinosomes or granules frequently fuse with the toxic vacuoles. Chloroquine derivatives can be seen by fluorescence microscopy; the drug is rapidly taken up by macrophages and localized in small foci in the Golgi region. Chloroquine continues to produce vacuoles when pinocytosis is suppressed. Electron microscopic studies of chloroquine effects on macrophages preincubated with colloidal gold to label predominately pinosomes or granules suggest that toxic vacuoles can arise from unlabeled organelles. Later vacuoles regularly acquire gold label, apparently by fusion, from both granules and pinosomes. L cells also develop autophagic vacuoles after exposure to chloroquine. Smooth endoplasmic reticulum apparently is involved early in the autophagic process in these cells. Information now available suggests an initial action of chloroquine on Golgi or smooth endoplasmic reticulum vesicles, and on granules, with alterations in their membranes leading to fusion with one another and with pinosomes.     

Endocytotic pathways at the ruffled borders of rat maturation ameloblasts

Summary Using horseradish peroxidase (HRP) as a soluble protein tracer, electron microscopic studies were carried out in order to analyze endocytosis in the ruffle-ended ameloblasts of rat incisors. Accumulated HRP was initially incorporated from the ruffled border into the cytoplasm by means of pinocytotic vacuoles (pinosomes) and pinocytotic coated vesicles. The majority of the HRP was taken up by the large number of pinosomes, which then formed large endocytotic vacuoles by fusing either with each other or with preexisting endocytotic vacuoles. As time passed HRP accumulated, not in the pinosomes and ruffled border but in the endocytotic vacuoles and multivesicular bodies. Frequent connections between HRP-labeled coated vesicles and these cytoplasmic bodies indicate that these vesicles serve as an HRP carrier. These findings strongly suggest that ruffle-ended ameloblasts actively absorb soluble proteins from the enamel matrix during enamel maturation.     

Acidification of phagosomes is initiated before lysosomal enzyme activity is detected

We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC- RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH approximately 6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH approximately 5.6 in less than 10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of approximately 6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to approximately 5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.     

RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells

The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.     

Taenia crassiceps: temperature, polycations, polyanions, and cysticercal endocytosis

The effect of various temperatures, poly-L-lysine, and poly-L-glutamic acid on endocytosis of smooth micropinocytotic vesicles (pinosomes) in the tegument of the cysticercus of Taenia crassiceps has been investigated stereologically. The temperature regimes used were 0, 5, 10, 20, 30, and 40 C. Maximum volume, surface density, and number per unit volume were found at 40 C, and minimum surface-to-volume ratio and numbers at 10 C. At 10 C, mean pinosome volume and mean surface area per pinosome were maximal, but volume and surface density did not differ significantly from 40 C. It is proposed that this anomalous finding for 10 C incubations was due to this being a critical temperature at which a slower rate of pinosome formation was compensated for by the formation of larger individual pinosomes. Poly-L-lysine was shown to be a stimulant of pinosome formation, leading to a significant increase in numbers per unit volume. However, volume and surface density, surface-to-volume ratio, mean volume, and mean surface area per pinosome were not significantly different in poly-L-lysine-incubated samples, when compared to controls (fresh from the mouse) or incubations in medium only or samples returned to medium after poly-L-lysine incubation, the only exception being surface to volume ratio and mean volume of pinosomes in the 75-min incubation. These anomalous results were explained by a marked reduction in the form ellipse values, which indicated the production of more elliptical-shaped pinosomes under poly-L-lysine stimulation. Incubation in poly-L-glutamine acid did not have any significant effect at any incubation time.     

Taenia crassiceps: Basic mechanisms of endocytosis in the cysticercus

The effects of glucose, yeast extract, fetal bovine serum albumin, and ruthenium red on endocytosis of smooth micropinocytotic vesicles (pinosomes) in the tegument of the cysticercus of Taenia crassiceps have been investigated stereologically. Glucose has been shown to stimulate pinocytosis, whether it was used alone or in combination with yeast extract or bovine serum albumin. Yeast extract was a stimulant of endocytosis. Bovine serum albumin was the most potent stimulant of all the substances investigated in this study. Although the time of incubation in ruthenium red was the same for all incubation experiments, varied numbers of ruthenium red-containing pinosomes were observed in different experiments. The role of ruthenium red as a stimulant and/or initiator of endocytosis and the possible explanations for differences in ruthenium red uptake are discussed.     

CISPLATIN INHIBITS PINOCYTOSIS INDICTYOSTELIUM DISCOIDEUM

The effects ofcis-diamminedichloroplatinum(II) [cisplatin], a potential anticancer drug, were studied on pinocytotic functions in the cellular slime mouldDictyostelium discoideumby administering FITC-dextran as a fluid phase marker. Cisplatin treatment at a concentration of 100 and 200 μg/ml for 1 h causes inhibition in pinocytotic uptake in growingDictyosteliumcells in a dose-dependent manner. Cisplatin treatment induced the association of more actin with the cell cortex, thereby presumably restricting the flexibility of the cell membrane and inhibiting the formation of pinosomes. Ultrastructural analysis of cisplatin-treated cells showed a lower number of pinosomes. These results have been discussed in the light of cisplatin's known actions that affect various cellular functions.     

Role of Contractile Microfilaments in Macrophage Movement and Endocytosis

PHAGOCYTOSIS of bacteria and other large particles and pinocytosis of colloids—two processes collectively termed endocytosis—are among the characteristic properties of macrophages. When mouse peritoneal macrophages in culture are observed by phase contrast microscopy, most small endocytotic vesicles (pinosomes) are seen to be formed in the region of ruffled membrane activity, usually in a pseudopod¹. The phase-lucent pinosomes move rapidly towards the Golgi region where they unite with phase-dense granules to form secondary lysosomes. Although there is evidence that both phagocytosis and pinocytosis in macrophages have a high temperature coefficient and require metabolic energy¹, the mechanism of endocytosis is unknown. Clearly, movement of the plasma membrane and directional movement of pinosomes is involved. During the past few years attention has been drawn to the apparent association in many cells between movement and the presence of contractile microfilaments of about 50 Â diameter^2,3. Some of these are actin-like and can bind heavy meromyosin to give distinctive “arrowhead” structures in electron micrographs⁴. One of us (S. de P., in preparation) has found that the peripheral or cortical cytoplasm of macrophages contains a network of microfilaments, some of which may be inserted into the plasma membrane. These filaments bind heavy meromyosin (Figs. 1 and 2) and details of their structure and disposition will be published later.     

Quantification of endocytosis-derived membrane traffic

The main data covered by this article have been summarized in Table I. A fairly uniform picture is obtained for endocytosis-derived membrane transfer and compartmentation. This may be due to the limited amount of information and the resulting low resolution. Data on mainly three cell types are presented: macrophages, fibroblasts and amoebae. The data vary as much for one cell type as between different cells. Therefore, no possible differences related to cell function emerge. More detailed data, for more cell types, may change the picture. The values for cell surface area, although significantly different in absolute terms (column S in Table I), are rather similar when related to cell diameter, all being about 3-fold in excess of the surface area of the smooth sphere of comparable volume (column xi in Table I). The rate of plasma membrane internalization for macrophages and amoebae both professional phagocytes, is about 2 cell surface area equivalents per h or more. This may be somewhat higher than for fibroblasts (column PM/h in Table I). The average residence time for membrane on the cell surface, therefore, is about 30 min. A most interesting finding seems to be the rather uniform values obtained for the average size (volume weighted) of primary pinosomes, being about 0.3 micron in diameter (column phi-Internalization in Table I). Due to their rapid increase in size as a result of fusion (cf. Fig. 2), it has not been feasible to directly measure the size of primary pinosomes by morphometric means. The values in Table I, give no information on the size distributions of primary pinosomes and on whether these consist of one or more size classes. The steady-state average diameter of pinosomes is noticeably larger than that of primary pinosomes (column phi-pinosomes in Table I; cf. Table II for Acanthamoebae). The corresponding decrease in surface-to-volume ratio can make about 50% of pinosomal membrane available for recycling directly from this membrane compartment. Membrane recycling from the pinosomal compartment occurs after an average residence time of about 3 min for macrophages and 4-6 min for fibroblasts (column tau-pinosomes in Table I). The relative pool size of intracellular membranes participating in shuttling to and from the cell surface is significantly different for animal cells and amoebae (column rho in Table I). For macrophages, fibroblasts, CHO cells, and mast cells, this intracellular membrane pool amounts to about 10-20% the plasma membrane area, compared to 150-200% in the case of amoebae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microinjection of cultured cells using red-cell-mediated fusion and osmotic lysis of pinosomes: A review of methods and applications

Proteins and other macromolecules can be injected into cultured cells by several different methods. Here we review the strengths and limitations of two of these methods, red-cell-mediated microinjection and osmotic: lysis of pinosomes, and indicate how they may be successfully applied to the study of cultured cells.     

Transport of pinocytic vesicles in the eye of a snail,Helix aspersa

The heads of small adult snails, Helix aspersa, were injected with horseradish peroxidase (HRP) for one to five hours before extirpating the eyes and preparing them cytochemically for electron microscopy. There was internalization of tracer by pinocytic vesicles (pinosomes) at the bases of types-I and -II sensory cells, ganglion cells and, in lesser amounts, by pigmented supportive cells. Vesicles and vacuoles filled with HRP were transported in two directions: lensward as far distad as the ends of the cells (retrograde) and brainward down the optic nerve (anterograde). We believe that the numerous reacted vacuoles in the cell somata are formed by fusion of vesicles, tubules and C-shaped organelles filled with tracer; we present evidence that they become secondary lysosomes. Sensory cell type II possesses more HRP-reacted vacuoles distally than the other retinal cells. Other vesicles are also described. There was no uptake of tracer by the distal ends of the retinal cells following injection HRP into the hemolymph. The swelling of the optic nerve, immediately behind the eye, contains more HRP-filled pinosomes and vacuoles than does the nerve below the dilatation. The significance of endocytosis and transport of pinosomes within the eye and down the optic nerve is discussed.     

Prelysosomal acidic vacuoles in Dictyostelium discoideum

We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.     

Characterization of pinocytic vesicles from CHO cells: resolution of pinosomes from lysosomes by analytical centrifugation

Pinocytic vesicles (pinosomes) and lysosomes from suspension cultured, Chinese hamster ovary (CHO-S) cells have been resolved as two non-overlapping organelle populations by analytical centrifugation in Percoll gradients. Pinosomes were labeled with either horseradish peroxidase (HRP), a fluid phase content marker, or by radioiodination by pinocytosed lactoperoxidase (LPO). CHO-S cell lysosomes followed by three different marker enzymes and electron microscopy behaved as a single, dense organelle population. Pinosomes were partially resolved from plasma membrane, a less dense organelle population.     

Effect of the amino acid analog hadacidin on intracellular membrane flow and the cell surface in Dictyostelium discoideum

We examined the effect of the amino acid analog hadacidin (N-formyl-N-hydroxy glycine) on the process of endocytosis in the slime mold Dictyostelium discoideum. Endocytosis was followed using iron-dextran and transmission electron microscopy. In cells taken from the mid-log growth stage, iron-dextran was found to be distributed in small, medium, and large vesicles at a density lower than that present in the incubation medium, thus suggesting the fusion of small, iron-dextran-containing pinosomal vesicles with intracellular vesicles not containing iron-dextran. In cells treated with hadacidin, more small vesicles were present than in untreated cells, there being a reduction in the number of larger-sized vesicles; in these vesicles, iron-dextran was present at a density similar to that of the medium. This result is consistent with the conclusion that, while pinocytosis had continued, the fusion of vesicles and dilution of the vesicle contents had been inhibited. Also, the large number of small pinosomal vesicles in the drug-treated cells suggested that the recycling of vesicles to the surface had been inhibited. The observation that pinocytosis but not recycling continued after drug treatment raised the question of the origin of the membrane needed for the formation of pinosomes. Measurements of the cell surface revealed no difference between drug-treated and untreated cells, indicating that, when the membrane was internalized for pinosomes, the cell size remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Movements and other distinguishing features of small vesicles identified by darkfield microscopy in living macrophages

Perinuclear vesicles (estimated diameter less than 0.15 micron), too small to be seen in living mouse macrophages by direct phase-contrast microscopy, could be detected by darkfield microscopy thanks to their rapid non-saltatory movements at 37 degrees C, contrasting with the slower saltations of accompanying phase-visible larger vesicles (0.25-0.5 micron, presumed secondary lysosomes). The movements of these 'small visicles' also differed from those of the 'larger visicles' in their responses to changes in temperature, and to chemical agents known to inhibit both the saltations of secondary lysosomes and the latter's fusion with phagosomes. Thus the 'larger vesicles' stopped moving at 25 degrees C, the small ones did not; both stopped at 18 degrees C. The 'small vesicles' continued to move actively after cell uptake of the polyanion poly-D-glutamic acid, while the saltations of the 'larger vesicles' were markedly slowed; both sets of vesicles stopped after uptake of ammonium chloride. Degranulation of the small vesicles paralleled that of the larger, while simultaneously observed preformed pinosomes (labelled with fluorescent wheat germ agglutinin (WGA) appeared to be unaffected. On the basis also of refractivity, location and speed the 'small vesicles' are considered not to be pinosomes, but probably to be lysosomes. The question of whether they are a subgroup of small immature secondary lysosomes or primary lysosomes (0.05-0.08 micron) is discussed. The broad spectrum of movement inhibited by ammonia in macrophages raises the possibility that this weak base inhibits movements of all lysosomes. Further characterization of these 'small vesicles' requires their relation to be defined to the small particles in other cell types (especially in axoplasm) which have been detected by video-enhanced microscopy.     

Age-related change in the degradation rate of ovalbumin microinjected into mouse liver parenchymal cells

Change in the degradation rate of ovalbumin (OVA) microinjected into liver parenchymal cells isolated from mice of various ages was studied. OVA was injected by osmotic lysis of pinosomes and the amount of OVA was determined by immunoblotting using purified antibody to OVA. Cellular activity as judged by rates of protein synthesis and degradation of pulse-labeled proteins was not affected by the injection. To localize injected OVA in the cells, cell extracts were fractionated by differential centrifugation and the amount of OVA and the activity of beta-D-galactosidase as a lysosomal marker enzyme of each fraction were determined. The ovalbumin was mostly found in the soluble fraction, while the beta-D-galactosidase activity was found in the particulate fraction, indicating that the ovalbumin was spread in the cytosol but was not present in the pinosomes or lysosomes. The average half-lives of OVA were 106, 113, and 164 h in the cells from young (3.5-6.5 months), middle-aged (13.5-20.0 months), and old (24.5-30.5 months) mice, respectively. Thus, the half-life of ovalbumin in the cells of senescent mice was about 50% longer than that in the cells of young or middle-aged mice. These results are in good agreement with those of our previous investigation, which showed that the half-life of inactivation of horseradish peroxidase was extended by about 50% in the hepatocytes from old mice (Ishigami and Goto, 1988, Mech. Ageing Dev., 46, 125-133).     

A well-characterized plasma membrane fraction obtained from mouse peritoneal macrophages

A new method was developed to isolate a plasma membrane fraction from lipopolysaccharide-stimulated mouse peritoneal macrophages. Colchicine treatment was followed by sucrose density-gradient centrifugation. Total yield of Na,K-ATPase, a marker of plasma membrane, was 60 +/- 1% with the specific activity of 37 +/- 3 mumol of Pi/mg of protein/h. The preparation contained 1 +/- 1% pinosomes, 2 +/- 1% lysosomes, 17 +/- 2% endoplasmic reticulum, 6 +/-1% mitochondria, and a negligible number of nuclei, as judged by distribution of markers.     

pH changes in pinosomes and phagosomes in the ameba, Chaos carolinensis

Changes in pH are measured in pinosomes and phagosomes of single specimens of the giant, free-living ameba, Chaos carolinensis. Measurements of pH are made microfluorometrically, as previously described (Heiple and Taylor. 1980. J. Cell Biol. 86:885-890.) by quantitation of fluorescence intensity ratios (Ex489nm,/Ex452nm, Em520- 560nm from ingested fluorescein thiocarbamyl (FTC)-ovalbumin. After 1 h of pinocytosis (induced in acid solution), FTC-ovalbumin is found in predominantly small ( less than or equal to 5 micrometers in diameter), acidic (pH less than or equal to 5.0-6.2) vesicles of various shape and density. As the length of ingestion time increases (up to 24 h), the probe is also found in vesicles of increasing size (up to 100 micrometers in diameter), increasing pH (up to pH approximately 8.0), and decreasing density. Co-localization of fluorescein and rhodamine fluorescence, after a pulse-chase with fluorescein- and rhodamine- labeled ovalbumin, suggests vesicle growth, in part, by fusion. The pH in a single phagosome is followed after ingestion of ciliates in neutral solutions of FTC-ovalbumin. A dramatic acidification (delta pH greater than or equal to - 2.0) begins within 5 min of phagosome formation and appears to be complete in approximately 20 min. Phagosomal pH then slowly recovers to more neutral values over the next 2 h. pH changes observed in more mature populations of pinosomes within a single cell may reflect those occurring within a single phagosome. Phagosomal and pinosomal pH changes may be required for lysosomal fusion and may be involved in regulation of lysosomal enzyme activity.