Ultrastructural localization of anionic phospholipids in skeletal muscle plasma membrane

Summary A cytochemical study of acid phosphatase (AcPase) in the lateral prostate of the rat was performed to investigate whether AcP-ase in the secretory apparatus can be distinguished from AcP-ase in lysosomes and their related structures. Two types of AcP-ase were observed in the rat lateral prostate. One was found in the secretory apparatus (Golgi saccules and some Golgi vesicles, condensing and secretory vacuoles), and reacted well with naphthol AS-BI phosphate (N AS-BI P) as substrate; the other was found in the lysosomes and Golgi-associated endoplasmic-reticulum-lysosome system (GERL)-like structure, and reacted well with -glycerophosphate (GP) as substrate. Although the AcP-ase which reacted well with N AS-BI P was also observed in certain portions of pleomorphic lysosomes, it was concluded that it was the same as the AcP-ase found in the condensing and secretory vacuoles, since a lysosome engulfing a condensing vacuole was often observed. Therefore, it is concluded that the AcP-ase in the secretory apparatus in the rat lateral prostate is different from the AcP-ase in lysosomes. Condensing vacuoles appear to originate from particular portions of Golgi saccules, but not from the GERL or GERL-like structure, at least in the rat lateral prostate.     

Lysosomes of root tip cells in corn seedlings

Summary Nine acid hydrolases are present in lysosomes which are found in the mitochondrial fraction of a cell-free extract prepared from root tips of corn seedlings.Light and heavy lysosomes can be distinguished. The latter are sedimentable in a sucrose-medium, the former only in sorbitol-medium. The fraction of heavy lysosomes is in turn composed of at least three populations of lysosomes differing in density and enzyme content.Light lysosomes are membrane-bound particles with diameters from 0.3 to 1.5 . Electron micrographs of frozen-etched tissue and isolated particles provide evidence that light lysosomes are identical with small vacuoles. This type of lysosome is characterized by presence of transaminases in addition to that of hydrolases. Heavy lysosomes are small spheres (diameters from 0.1–0.3 ) with membranes resembling those of vacuoles and of the endoplasmic reticulum. These lysosomes are characterized by high specific activities of two oxydoreductases known to occur also in the membranes of the reticulum.The different types of particles are thought to represent stages of the development of the lysosomal apparatus; according to this hypothesis the large vacuole of parenchymatous cells represents the end product of this process.     

Ultrastructural distribution of NADPase within the Golgi apparatus and lysosomes of mammalian cells

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Are lysosomal enzymes involved in rapid damage in vertebrate muscle cells?

Summary Experiments with lysosomotropic agents suggest that the sarcotubular system subserves some of the functions of the lysosomal apparatus in frog skeletal muscle. Dinitrophenol or A23187 trigger lysosome labilization and myofilament damage in mammalian cardiac muscle. Lysolecithin labilizes isolated liver lysosomes, but has no action following phospholipase A₂ activation in vivo. Zinc ions or a pH_i of 7.5 do not protect against myofilament damage. In fractions from mammalian cardiac muscle, calcium and calmodulin do not cause lysosomal labilization whereas cGMP does but only at high concentration (10^-4 M). It is concluded that lysosomal hydrolases play no significant part in rapid muscle damage. It is suggested that rises in [Ca]_i activate two separate pathways causing (i) myofilament damage; (ii) sarcolemmal (and possibly lysosomal) membrane damage via phospholipase A₂ and lipoxygenase activity. Dinitrophenol triggers both pathways independently and thus may cause lysosome labilization. The possibility that the sarcoplasmic reticulum is the site generating myofilament damage is discussed.     

Chinese hamster ovary cell lysosomes rapidly exchange contents

We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid- phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC- conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.     

P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.     

Examining the retention of functional kleptoplasts and digestive activity in sacoglossan sea slugs

Solar-powered sea slugs (Sacoglossa: Gastropoda) have long captured the attention of laymen and scientists alike due to their remarkable ability to steal functional chloroplasts from their algal food, enslaving them to withstand long starvation periods. Recently, a wealth of data has shed insight into this remarkable relationship; however, the cellular mechanisms governing this process are still completely unknown. This study explores these mechanisms, providing insight into the chloroplast retention and delayed digestion, occurring within the slug’s digestive gland. We examine the relationships between functional chloroplast and lysosome abundances during starvation, in live material, for the long-term retaining species Elysia timida, the ambiguous long/short-term retaining Elysia viridis, and the short-term retaining Thuridilla hopei, to elucidate digestive differences that contribute to the development of functional kleptoplasty. Functional chloroplast and lysosome abundance are measured using chlorophyll a autofluorescence and the pH-dependent stain acridine orange. In each species, the number of chloroplasts and lysosomes is indirectly proportional, with the plastid density decreasing when starvation begins. We also present a new FIJI/Image J Plugin, the 3D—Accounting and Measuring Plugin, 3D-AMP, which enables the reliable analysis of large image sets.     

LYST Affects Lysosome Size and Quantity,but not Trafficking or Degradation Through Autophagy or Endocytosis

Mutations in the large BEACH domain‐containing protein LYST causes Chediak–Higashi syndrome. The diagnostic hallmark is enlarged lysosomes and lysosome‐related organelles in various cell types. Dysfunctional secretion of enlarged lysosome‐related organelles has been observed in cells with mutations in LYST, but the capacity of the enlarged lysosomes to degrade endogenous proteins has not been studied. Here, we show for the first time that small interfering RNA‐depletion of LYST in human cell lines recapitulates the LYST mutant phenotype of enlarged lysosomes. We found no evidence for an effect of LYST depletion on autophagy or endocytic degradation. Autophagosomes are formed in normal size and quantities and are able to fuse to the enlarged lysosomes, leading to normal rates of degradation. Degradation of the epidermal growth factor receptor (EGFR) was similarly not affected, indicating that the enlarged lysosomes are fully functional in degrading endogenous proteins. Retrograde trafficking of toxins as well as the localization of transporters of lysosomal proteins, adaptor protein‐3 (AP‐3) and cation‐independent mannose‐6‐phosphate receptor (CI‐MPR), were all found to be unaffected by LYST. Quantitative analysis of the enlarged lysosomes shows that LYST depletion causes a reduction in vesicle quantity per cell, while the total enzymatic content and vesicular pH are unaffected, supporting a role for LYST in lysosomal fission and/or fusion events.      

Ultrastructural Studies of the Visceral Muscles of Chaetognaths

The visceral musculature of Chaetognaths was studied with special attention given to the digestive apparatus muscle. In the head the digestive apparatus muscle is relatively thick; individual muscles are difficult to distinguish at the anatomical level; in the anterior part of the oesophagus discontinuous bundles and layers of cross-striated fibers are found. As a group however, the oesophageal musculature completely covers the oesophageal epithelium. The prominent muscle layers around the oesophagus probably help to force food into the intestine against the turgor pressure of the trunk cavity which tends to collapse the intestine. Around the intestine the musculature is largely circular and smooth. The intestinal epithelium is ciliated despite its muscular covering. Muscle fibers are not individually innervated. They form myoepithelial structures with various intercellular junction types. In the intestinal muscle the fibers show myoendothelial-like junctions. Sphincters composed of myoepidermal cells surround the anus and the female gonopores. The somatic side of the general cavity is lined with a polymorphic squamous epithelium. Sometimes myoepithelial cells are found, with the occasional presence of extracellular matrix basal to the layer of the squamous epithelium. The ontogenetic relations between the polymorphic epithelium and the composite ‘mesenteries’ remain to be established. We have now some idea about the architecture of the body of Chaetognaths in relation to contractile structures.     

Lysosome Transport as a Function of Lysosome Diameter

Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter.     

The biogenesis of lysosomes: Is it a kiss and run,continuous fusion and fission process?

Molecules are transferred to lysosomes, the major, acid pH, digestive compartment in eukaryotic cells, by a complex series of pathways that converge at a late endosome/prelysosomal compartment. Here, we discuss the relationship between this compartment and the lysosome. We propose that lysosomes are maintained within cells by a repeated series of kiss and run, transient fusion and fission processes with the late endosome/prelysosome compartment. Directionality to these processes may be conferred by pH gradients and retrieval mechanisms. The future challenge in testing this and any other proposed hypothesis for lysosomal biogenesis will be the establishment of molecular mechanisms.     

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells

Background

Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear.

Methodology/Principal Finding

In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion.

Conclusion/Significance

Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are available in the cell and that cholesterol depletion may modulate the fusion of pre-docked lysosomes at the cell cortex.     

Association of purified thyroid lysosomes to reconstituted microtubules

We report the characteristics of the interaction between reconstituted microtubules and purified thyroid lysosomes. Microtubules were extracted from pig brain by temperature-dependent assembly-disassembly and labelled with 125I by conjugation with the Bolton-Hunter reagent. Thyroid lysosomes were purified from pig thyroid by isopycnic centrifugation on Percoll gradients. The formation of microtubule-lysosome complexes has been studied by electron microscopy, using negative staining, and by differential centrifugation. The association of lysosomes to microtubules is time- and temperature-dependent (between 25 degrees C and 37 degrees C). The rate of microtubule-lysosome complex formation is related to the concentration of lysosomes. The higher the lysosome concentration is, the higher also is the rate of the interaction. Changes in microtubule concentration merely alter the amount of complex formed; there is a linear relationship between the amount of complexes and the microtubule concentration. However, lysosomes seem to possess a limited number of 'microtubule-binding sites', since a saturation of the complex formation can be obtained at high microtubule concentration. Two main types of complex have been observed by electron microscopy on negatively stained samples; simple complexes composed of a lysosome in close contact with a microtubule and complexes formed by a lysosome surrounded by several microtubules. The formation of microtubule-lysosome complexes was totally inhibited in the presence of 100 microM N-ethylmaleimide; the rate of the interaction was slightly increased in the presence of dithiothreitol (25-100 microM). The interaction we describe here in an acellular system might be relevant to the association of lysosomes to microtubules observed in intact cells (Collot, M., Louvard D. and Singer S.J. (1984) Proc. Natl. Acad. Sci. USA 81, 788-792) and will constitute a useful model to study the regulation mechanisms of microtubule-vesicle interaction.     

Lysosomal Ca2+ release channel TRPML1 regulates lysosome size by promoting mTORC1 activity

Lysosomal Ca²⁺ release channel TRPML1 has been suggested to regulate lysosome size by activating calmodulin (CaM). To further understand how TRPML1 and CaM regulate lysosome size, in this study, we report that inhibiting mTORC1 causes enlarged lysosomes, and the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. We also show that lysosome vacuolation induced by inhibiting TRPML1 is corrected by mTORC1 upregulation, and the facilitating effect of TRPML1 on the recovery of enlarged lysosomes is suppressed by inhibiting mTORC1. In the meantime, lysosome vacuolation induced by inhibiting CaM is corrected by mTORC1 upregulation, and mTORC1 overexpression corrects the inhibitory effect of CaM antagonist on the recovery of enlarged lysosomes. Conversely, the vacuolation induced by suppressing mTORC1 is not corrected by upregulating CaM. These data suggest that mTORC1 functions downstream of TRPML1 and CaM to regulate lysosome size. Together with our recent finding showing that TRPML1, CaM and mTORC1 form a macromolecular complex to control mTORC1 activity, we suggest that TRPML1 and CaM control lysosome fission through regulating mTORC1, identifying an mTORC1-dependent molecular mechanism for lysosomal membrane fission.     

Isolation of monkey aortic lysosomes using Percoll density gradient

A novel technique involving the Percoll density gradient and 0.01M phosphate buffer has been employed for the first time on aortic tissue for isolation of lysosomes. The purity of the lysosomes has been established by marker-enzymes, acid phosphatase and N-acetyl-beta-D-glucosaminidase and latent activities of lysosomal hydrolases. The heavier fraction (density 1.08) obtained after Percoll density gradient centrifugation showed high specific activities of lysosomal hydrolases and these enzymes were markedly latent. Moreover this heavier (lysosome rich) fraction has been noted to be free of other sub-cellular contaminants.     

内分泌细胞中溶酶体对激素分泌调节的作用

Enzyme cytochemistry and immunocytochemistry were utilized to study the morphological alterations of the lysosomes and associated crinophagic and autophagic structures in the hypo-secreting pituitary gonadotrophin and Leydig cells induced by exogenous androgen. The lysosomes and autophagic vacuoles in the electron micrographs were quantitatively analysed. The morphological and quantitative data led to the following conclusions: 1) The hypo-secreting gonadotrophin showed an increase in the number of lysosomes and an enhancement of crinophagy. It demonstrated once again that the lysosomes in the protein and polypeptide hormone secreting cells play a role in the regulation of secretion process by means of the crinophagy. 2) The hypo-secreting Leydig cells showed an increase in the number of lysosomes and an enhancement of autophagic activity. This indicated that the lysosomes in the steroid hormone secreting cells also function in the regulation of hormone secretion but by means of autophagy which scavenge a part of steroid-producing apparatus and hormone. The autophagy might have similar effect in regulation of steroid secretion to the crinophagy in regulation of protein secretion.     

Two patients walk into a clinic...a genomics perspective on the future of schizophrenia

Background

Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release.

Results

NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse.

Conclusions

These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells.     

The digestive tract of Helicoradomenia (Solenogastres, Mollusca), aplacophoran molluscs from the hydrothermal vents of the East Pacific Rise

Abstract. Species of Helicoradomenia are constantly found at hydrothermal vent sites of the eastern and western Pacific Ocean. The digestive tract of 2 species of the genus was investigated with special focus on the ultrastructure and histochemistry of epithelia and glandular organs. The preoral cavity and foregut epithelia are composed of microvillous main cells, secretory cells producing protein-rich substances, and sensory cells with specialized cilia. The foregut bears a pair of glands with 3 types of extremely long-necked glandular cells surrounded by musculature. Each glandular cell opens directly into the radula pocket without a gland duct. The large radula apparatus consists of pairs of denticulated bars resting on a flexible radular membrane without elaboration of a subradular membrane. The midgut has a narrow, mid-dorsal tract of ciliary cells, but most of the epithelium is composed of digestive cells with a highly developed lysosomal system. The hindgut is lined by ciliated cells and free of glands. The foregut and radula seem to be highly efficient in the capture of relatively large, motile prey. Food contents within the midgut lumen and within some of the large secondary lysosomes indicate a triploblastic metazoan prey of non-cnidarian origin. The digestive tract is not adapted to microvory and there is no indication of a symbiosis with chemoautotrophic bacteria.     

Secretory lysosome biogenesis in cytotoxic T lymphocytes from normal and Chediak Higashi syndrome patients

The lytic proteins mediating target cell killing are stored in the lysosomes of activated cytotoxic T lymphocytes (CTL) and are secreted upon recognition of a target cell. These secretory lysosomes cannot be detected in resting T lymphocytes. Interaction of a resting cell with a target cell activates de novo formation of secretory lysosomes. CTL clones in culture mimic this behaviour, and so provide an ideal system for studying secretory lysosome biogenesis and maturation. In the genetic disease, Chediak Higashi syndrome (CHS), all lysosomes in the cells are enlarged and reduced in number compared with wild-type (WT) cells. We have used CTL from this disease to study secretory lysosome biogenesis and maturation. We show that at early stages after activation the secretory lysosomes are identical in WT and mutant cells, and that delivery of proteins to the secretory lysosome along the biosynthetic and endocytic pathways is normal in the mutant cells. With time, the lysosomes in the mutant cells aggregate, become larger and fewer in number and eventually form giant structures. Our results show that the initial steps of secretory lysosome formation are normal in CHS, but that the organelles subsequently fuse together during cell maturation to form the giant secretory lysosomes.