What's special about secretory lysosomes?

Lysosomes are organelles specialised for their role in intracellular protein degradation. A small number of cell types also use their lysosomes as regulated secretory organelles. These secretory lysosomes package additional secretory products, respond to extracellular stimuli and fuse with the plasma membrane to release their contents. Recent research has identified unique components of the secretory machinery in these cells. However, studies on conventional lysosomes in non-secretory cells reveal that even their lysosomes can fuse with the plasma membrane in response to membrane damage. What then is special about secretory lysosomes?     

Endocytosis in secretory cells

Membranes of secretion granules inserted during exocytosis into the luminal plasma membranes of glandular cells are retrieved by endocytosis as revealed by electron dense tracers applied selectively to the apical cell surfaces. Two major pathways that endocytic vesicles may take are described: (1) a direct route to the Golgi complex (e.g. in parotid and exocrine pancreas) with later appearance of the tracer in the periphery of mature secretion granules; (2) an indirect route with lysosomes as a first station and the subsequent appearance of tracer in stacked Golgi cisternae. It is presumed that some of the retrieved membrane follows the same pathways and is reutilized in the secretory cycle.     

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.     

Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca²⁺, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca²⁺ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.

Here we demonstrate that elevation in the intracellular free Ca²⁺ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca²⁺ ionophore ionomycin or addition of Ca²⁺containing buffers to streptolysin O–permeabilized cells induced exocytosis of ~10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca²⁺ and to be temperature and ATP dependent, similar to Ca²⁺-regulated secretory mechanisms in specialized cells.

These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca²⁺-regulated exocytosis.

     

Widespread association of annexin II with intracellular membrane systems and involvement of annexin II in granule--granule fusion in addition to granule--plasma membrane fusion in anterior pituitary cells

The subcellular localization in anterior pituitary secretory cells of annexin II, one of the Ca2+-dependent phospholipid-binding proteins, was examined by immunohistochemistry and immunoelectron microscopy. Annexin II was associated with the plasma membrane, the membranes of secretory granules and cytoplasmic organelles, such as rough endoplasmic reticulum, mitochondria and vesicles, and with the nuclear envelope. Annexin II was frequently detected at the contact sites of secretory granules with other granules and with the plasma membrane. The anterior pituitary and adrenal medulla were treated with Clostridium perfringens enterotoxin, which induces Ca2+ influx, and examined under an electron microscope. The anterior pituitary cells showed multigranular exocytosis, i.e. multiple fusions of secretory granules with each other and with the plasma membrane, but adrenal chromaffin cells, which lack annexin II on the granule membranes, never showed granule--granule fusion and only single granule exocytosis. From these results, we conclude that, in anterior pituitary secretory cells, annexin II is involved in granule--granule fusion in addition to granule--plasma membrane fusion. © 1998 Chapman & Hall     

Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells

Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface.     

Effects of hypergravity environment of the ultrastructure of the golden hamster parathyroid gland

The fine structure of the parathyroid glands of golden hamsters exposed to 2, 5 or 10 g environment for 5 h was studied. In the centrifuged hamsters, many secretory granules are located in a peripheral position just beneath the plasma membrane of chief cells, and the Golgi complexes and cisternae of the granular endoplasmic reticulum are significantly increased compared with those of control animals. There are no significant differences between the control and centrifuged animals with regard to secretory granules, large secretory granules, lysosomes, vacuolar bodies and lipid droplets. These findings suggest that the secretory activity of the parathyroid gland may be stimulated in response to hypergravity environment.     

High- and low-uptake forms of β-hexosaminidase in human platelets Selective retention of the high-uptake form during stimulation with thrombin

Human platelets are rich in β-hexosaminidase and other acid hydrolases contained in organelles (lysosomes) distinct from α-granules and dense granules. Incubation of platelets with bovine or human thrombin (100 U/ml for 5 min at 37°C) induces the secretion of 100% of the contents of α- and dense granules, but only 40–60% of total β-hexosaminidase from lysosomes. Both isozymes Hex A and Hex B are secreted in the same proportion as found intracellulary. There is no selective recapture or plasma membrane binding by platelets of secreted β-hexosaminidase. The secreted enzyme is of the low-uptake type, i.e., it is poorly recognized by the phosphomannosyl receptor-mediated uptake mechanism of fibroblasts, while the retained enzyme is a 3-fold higher uptake form. Preincubation of platelets with NH₄Cl (10 mM, 2 h), followed by thrombin stimulation, results in secretion of all β-hexosaminidase as a low-uptake form. The data support the hypothesis that there are secretory and nonsecretory forms of lysosomes. The secretory lysosomes would contain low-uptake forms of hydrolases in addition to acid phosphatase, while the nonsecretory lysosomes would contain high-uptake hydrolases and be acid phosphatase-deficient. Conditions where the contents of both lysosomal populations were released together, i.e., amine treatment followed by thrombin induction, or extraction of unstimulated cells, would result in the exposure of high-uptake phosphomannosylated hydrolases released from one population of lysosomes to acid phosphatase released from the second population of lysosomes with their subsequent conversion to low-uptake forms.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.     

Protein kinase C-dependent supply of secretory granules to the plasma membrane

To elucidate the mechanism for supplying secretory granules to the cell membrane, chromaffin cells isolated from the bovine adrenal medulla were observed by the evanescent wave microscopy after staining their granules with acridine orange. The secretory granules showed only a very small fluctuation, indicating their docking to the plasma membrane. The rate and range of movement increased greatly by application of botulinum toxin A or C. The number of secretory granules docked to the plasma membrane significantly decreased by botulinum toxin C. Conversely, the number increased greatly by activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu). In the presence of an anti-actin reagent cytochalasin D, no increasing effect of PDBu on the number of docked granules was observed. While in the presence of an anti-mitotic reagent, colchicine, a clear increasing effect of PDBu was observed. The final step for supplying granules to the plasma membrane in endocrine cells is concluded to be mediated by a phosphorylation-dependent and actin-based transport system.     

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.     

Cholesterol regulates prostasome release from secretory lysosomes in PC-3 human prostate cancer cells

Prostasomes are vesicles secreted by epithelial cells of the prostate gland. However, little is known about the mechanism and the regulation of prostasome secretion. Since endocytic organelles may be involved in prostasome release, PC-3-derived prostasomes were investigated by Western blot analysis for the presence of marker proteins normally associated with these organelles. Prostasomes secreted by PC-3 cells contain clathrin, Tsg101, Hrs, Rab11, Rab5, LAMP-1, LAMP-2, LAMP-3/CD63, and annexin II. Moreover, electron microscopy of PC-3 cells revealed the presence of characteristic multivesicular body-like secretory lysosomes containing vesicles with the same size-distribution as released prostasomes. Ultrastructural immunogold labelling showed that LAMP-1, LAMP-2 and LAMP-3/CD63 were associated with these vesicles. In addition, we have investigated whether cholesterol plays a role in prostasome release by the human prostate cancer cell line PC-3. Interestingly, prostasome release was significantly increased when the cholesterol levels of PC-3 cells were reduced by the cholesterol-sequestering agent methyl-beta-cyclodextrin (MBCD), or by treatment with lovastatin and mevalonate. In conclusion, these studies indicate that cholesterol plays an important role in the release of prostasomes by the human prostate cancer PC-3 cells, and suggest that prostasomes may be released after fusion of secretory lysosomes with the plasma membrane.     

Regulated secretion of conventional lysosomes

Regulated secretion has been traditionally regarded as a specialized process present in only a few cell types. Similarly, the secretory lysosomes of hematopoietic cells have been viewed as 'modified' organelles that acquired the machinery for regulated exocytosis. However, there is evidence that conventional lysosomes can, in many cell types, respond to rises in the intracellular free Ca2+ concentration by fusing with the plasma membrane. These findings profoundly change the current view of lysosomes as a 'final' station of the endocytic pathway and suggest a previously unsuspected active role for this organelle.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

Cytochemistry of Ca(++)-dependent adenosine triphosphatase (Ca-ATPase) in rat anterior pituitary cells

In the present study, we demonstrate the localization of Ca(++)-ATPase in the anterior pituitary of the male rat. Ca(++)-ATPase was mainly distributed on the membrane system of the granular cells, which included the plasma membrane, the outer mitochondrial membrane, the enveloping membrane of secretory granules, the smooth endoplasmic reticulum and some components of the Golgi complex. No reaction product was detected on the membrane of the rough endoplasmic reticulum or that surrounding the lysosomes. A positive reaction was clearly observed on the membranes surrounding 'large' secretory granules, while that present on the membranes of the 'small' granules was comparatively weak. The cells which contained the 'large' granules were interpreted as growth hormone-secreting cells and those in which the 'small' granules were located as gonadotrophs. There were either no reaction or one that was barely detectable on the plasma membrane of the folliculo-stellate cells. These data along with our previous findings (Soji, 1982, 1984) suggest that the membranous enzymes are not uniformly distributed over all pituitary cells but rather are specific for a given cell population(s).     

Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.     

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.     

Effects of colchicine on DNA synthesis, endocytosis and fine structure of cultivated arterial smooth muscle cells

Confluent secondary cultures of rat arterial smooth muscle cells were exposed to cationic and anionic derivatives of ferritin and horseradish peroxidase and studied electron microscopically in order to clarify the influence of molecular net charge on surface binding and endocytosis of proteins. The cationic markers bound uniformly to the plasma membrane. They were then ingested by membrane invagination and via small vesicles transported to lysosomes and the Golgi complex. These organelles were both labelled already after 30 min of incubation. With longer exposure times (2-4 h), an increasing accumulation within the lysosomes was observed, whereas the labelling of the Golgi complex decreased. In spite of continued interiorization of plasma membrane carrying the cationic markers, the cells retained their ability to bind the latter to the surface. The anionic markers did not bind to the cell surface, were taken up in the fluid phase, and later observed only in lysosomes. If assuming that the cationic and anionic proteins serve as markers for the plasma membrane and fluid phase, respectively, but do not affect the intracellular path of interiorized membrane, these results indicate that the endocytic vesicles fuse with and empty their content into lysosomes and that part of the incoming membrane subsequently is transferred to the Golgi complex for possible recirculation back to the cell surface. If, on the other hand, the net charge of the exogenous marker influences the path of the vesicles, there may exist more than one recovery route for membrane interiorized by endocytosis.     

Intracellular pathways of receptor-bound GnRH agonist in pituitary gonadotropes

Summary Localization of GnRH receptors in rat pituitary gonadotropes was studied by use of ¹²⁵I-[azidobenzoyl-D-Lys⁶]GnRH which, upon photolysis, is covalently bound to the receptor molecule. Using high resolution autoradiography, it was found that, after a 90-min incubation of the analog with pituitary cells at 4° C, 93% of the silver grains were associated with the plasma membrane of the gonadotropes. After 45-min incubation of the cells at 37° C, clustering and internalization of the receptor-bound GnRH analog were evident. Silver grains were associated with coated pits, intracellular vesicles, Golgi complexes, lysosome-like structures and secretory granules. The data indicate that receptor-bound GnRH agonist is internalized, at least in part, via coated pits and is subsequently routed to lysosomes where degradation of the hormone-receptor complex may occur. The presence of a considerable amount of silver grains associated with secretory granules may suggest that some of the internalized receptor molecules can escape degradation and be recycled to the cell membrane.     

Secretory lysosomes

Regulated secretion of stored secretory products is important in many cell types. In contrast to professional secretory cells, which store their secretory products in specialized secretory granules, some secretory cells store their secretory proteins in a dual-function organelle, called a secretory lysosome. Functionally, secretory lysosomes are unusual in that they serve both as a degradative and as a secretory compartment. Recent work shows that cells with secretory lysosomes use new sorting and secretory pathways. The importance of these organelles is highlighted by several genetic diseases, in which immune function and pigmentation--two processes that normally involve secretory lysosomes--are impaired.