A New Look at Transudation: The Apocrine Connection

Entrance phase - endocytosis and early sorting

Transcytosis has been observed in connection with numerous transport and externalization processes of the cell. Several detailed studies of transcytosis-linked processes described in the literature are associated with engulfment or endocytosis and baso-apical recycling of the vacuolar ATPase proton pump (vATPase) required for luminal acidification of endosomes, characteristic for many transporting epithelia (Brown and Sabolić, 1993, Brown and Breton, 2000). Although under various circumstances transport from the basal and basolateral pole to the apical pole or vice versa does not fulfill the features of complete transcytosis, it does represent the bipolar transcellular transport of membrane constituents (Schneider et al. 1979, de Chastellier et al. 1987), therefore we still can learn important information from these studies. In experiments with artificial cargo such as native ferritin (NF) or horseradish peroxidase (HRP) to trace fluid-phase endocytosis, or with cationized ferritin (CF) to examine adsorptive endocytosis, it is possible to follow clathrin-mediated endocytosis when tracers first enter an apical tubulovesicular endosomal system, then larger apical endosomal vesicles and later multivesicular bodies (MVB).

It is widely accepted that the intracellular network of endosomes is a highly organized cluster of membrane-derived compartments that act as sorting sites for both, endosomal and biosynthetic cargo. The subsequent fate of the engulfed cargo depends upon its interaction with a variety of molecularly distinct processing complexes, which tightly regulate the close relationship between the sorting of their particular load and the native process of membrane re-carving, essential for the proper formation of transport carriers (McGough and Cullen 2011, Shen et al. 2018). One such complex, retromer, among others primarily mediates retrograde transport from endosomes to the trans-Golgi network (TGN) (Burd and Cullen 2014, Gallon and Cullen 2015, Abubakar et al. 2017, Kvainickas et al. 2017, Simonetti et al. 2017). Nonetheless, the bipolar transport of membrane components, such as vATPase, must be able to escape these degradative routes. Even though this membrane component is not necessarily produced by other distal tissues (and therefore not has undergone adsorptive endocytosis), it is still able to enter the same efficient intracellular transport and delivery mechanism as foreign cargo, which is either identical or similar to the transcytotic route.

Kumagai et al. (2011) described the binding of IgG to the neonatal Fc receptor (FcRn) followed by internalization of this complex, and suggested apical receptor-mediated transcytosis via the TGN. Subsequently, the FcRn located inside the apical plasma membrane binds maternal IgG, and becomes internalized by endocytosis into the absorptive cells. The FcRn plays a key role in providing the fetus or newborn with humoral immunity before its immune system becomes fully developed as it transports maternal IgG across various epithelial barriers. The authors concluded that in newborns, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. The work of He et al. (2008) showed that the efficient unidirectional transport of IgG within the intestinal epithelial cells is facilitated by a pH difference between the apical (pH 6.0–6.5) and basolateral (pH 7.4) sides, since FcRn binds IgG at pH 6.0–6.5 but not at pH 7 or more, the basolateral pH 7.4 facilitates release of the cargo. The maternal IgG is absorbed by FcRn-expressing cells in the proximal portion of the small intestine (the duodenum and jejunum) when milk passes through the neonatal digestion, the remaining proteins are absorbed or degraded by FcRn-negative cells in the distal portion of the small intestine (ileum). In this context, a more complex view in which Fc moves throughout networks of entangled tubular and irregular vesicles before it reaches the basolateral surface was investigated by a combination of electron tomography and non-perturbing endocytic labeling (He et al. 2008). Distinct markers for early, late and recycling endosomes, each labeling vesicles in different and overlapping morphological classes, have helped these authors to reveal the spatial complexity in endolysosomal trafficking. These observations elucidated important features of transcytosis, including transport involving multivesicular bodies, inner vesicles/tubules and exocytosis through clathrin-coated pits. They also indicate that MVBs, most likelly a specific subclass of them, are involved in processive transcytosis and act in a distinct non-lysosomal (non-degradative) pathway, possibly requiring a specific sorting mechanism.

Post-entrance phase - the central role of retromer

The endosomal system is composed of an interconnected network of diverse membrane-bound structures and subcellular compartments whose primary function is to receive, dissociate, and sort cargo that originates either at the plasma membrane or from biosynthetic pathways (McGough and Cullen 2011, Burd and Cullen 2014, Gallon and Cullen 2015). Several years ago, there was a large amount of interest in a system associated with transcytosis after endosomal uptake and trafficking of polymeric immunoglobulin receptor (pIgR) and the role of a retromer complex in this process (Kobayashi et al. 2002, Vergés et al. 2004, 2007). Retromer consists of two major components: (1) a heterotrimer encoded by the vacuolar protein sorting (Vps) genes Vps26, Vps29, and Vps35, the role of which is to select cargo, and (2) a dimer of phosphoinositide-binding sorting nexins (SNX), whose function is to deform the membrane (Bonifacino and Hurley 2008, Vergés 2016). In other words, retromer is a heteropentameric complex that associates with the cytosolic face of vesicles and mediates the retrograde transport of transmembrane cargo from endosomes to the TGN. It comprises a sorting nexin dimer composed of a combination of SNX1, SNX2, SNX5, or SNX6, and a cargo-recognition trimer containing Vps26, Vps29 and Vps35. The SNX subunits contribute by Bin/Amphiphysin/Rvs (BAR) and phosphoinositide-binding phox homology (PX) domains that allow binding to the PI₃P-enriched highly curved membranes of endosomal vesicles, whereas Vps26, Vps29 and Vps35 subunits functionally contribute by phosphoesterase, arrestin and α-solenoid folds, respectively (Bujny et al. 2007, Bonifacino and Hurley 2008, van Weering et al. 2010, Vergés 2008, 2016).

Following endocytosis, pIgR is trafficked from the basal to the apical membrane. This trafficking pathway may be attenuated by the suppression of Vps35, a key constituent of retromer (McGough et al. 2014). Similarly, cells expressing some pIgR mutations defective in trafficking can have their transcytosis phenotype rescued by the overexpression of wild-type Vps35. In a study using endosomal fractions prepared from liver, pIgR antibody co-immunoprecipitated Vps26, Vps29 and Vps35, showing the close interactions and overall biochemical relationship between the trimer components of retromer and pIgR (Vergés et al. 2004). Although only negligible amounts of sorting nexin SNX1 and SNX2 were pulled down in these experiments, it still remains possible that sorting nexins are also required for this process, as the rescue by Vps35 is PI₃P sensitive (Vergés et al. 2007). In connection with the above mentionedd MVBs capable of avoiding the degradative route, these findings suggest that retromer complex may play a central or crucial role in escaping pIgR, and potentially other cargoes, from the lysosomal pathway and directing it for transcytosis.

Game of multiple traffick and its fine tuning

Retromer is known as a critical regulator of multiple export and sorting pathways from endosomes. Other cargo proteins that rely on retromer for trafficking also exist, including Drosophila crumbs, a protein essential for maintaining apico-basolateral polarity (Pocha et al. 2011, Zhou et al. 2011), serpentine, a protein required for tracheal development in Drosophila (Dong et al. 2013) and auxin efflux carriers in plants (Kleine-Vehn et al. 2008). Originally, Vps35 was identified in a screen for genes that function in endocytosis in the D. melanogaster Schneider 2 cultured cell line (Korolchuk et al. 2007), and further characterized as a protein required for the endocytosis of mBSA, the scavenger receptor Croquemort, EGF receptor and various other proteins, but not for synaptic vesicle endocytosis. These and other authors speculate that the endocytosis defect is a result of the regulation of Rac by Vps35, by an undefined mechanism (Korolchuk et al. 2007, Dong et al. 2013). The proposed theory is based upon a genetic interaction between Rac and Vps35, as well as the increased levels of F-actin present in the Vps35 mutant background. As such, it is logical to assume that the list of proteins that are known to be carried by retromer will grow significantly. However, the specific mechanism that distinguishes whether a particular cargo should avoid the endo-lysosomal pathway and instead undergo transcyctosis remains to be elucidated.

When considering pIgR, one working hypothesis is that the retromer could function by directly promoting pIgR transcytosis by rescuing the receptor-ligand complex from the degradative pathway (Vergés et al. 2004, 2007). To test this hypothesis, the region of the pIgR cytoplasmic domain that interacts with Vps35 was mapped. Interaction between the cytoplasmic domain of pIgR and Vps35 is necessary for retromer to promote transcytosis. However, such an interaction is not necessary to avoid cargo degradation, as very little pIgA internalized by pIgR-725t is degraded, with or without altering Vps35 levels (Luton et al. 1998), suggesting that retromer does not function primarily by rescuing pIgR from degradation. At present, transcytosis and degradation are considered to be alternative pathways, and thus anything that directly affects one is likely to indirectly affect the other (Breitfeld et al. 1990, Vergés et al. 2004, 2007, Vergés 2008, 2016). Because of this tight connection, it is difficult to completely exclude a function for retromer in avoidance of, or retrieval from, the degradative pathway. However, a plausible mechanistic explanation may involve consideration of additional, but thus far unidentified players. This could be, for example, additional interacting partner(s) or a posttranslational modification of a retromer subunit or endosomal cargo vesicle component.

Retromer and sorting in concert

Biochemical and immunohistochemical studies have revealed the subcellular localization of retromer and retromer subunits in the post-endocytic pathway. Although subunits of the cargo-selective complex were enriched in early endosomes (EEs), levels of SNX2 were greater in sorting endosomes. An estimated 25 % of total Vps26 and SNX2 was localized in EEs, with negligible portions in recycling endosomes as well as in late endosomes and lysosomes. Although Vps26 was found in structures of more heterogeneous size and shape than SNX2, these markedly overlapped. As a consequence, the two retromer subcomplexes mostly colocalized. Remarkably, retromer was found preferentially as part of the transcytotic pathway. Other studies have shown that pharmacological inhibition of phosphoinositide 3-kinase affected the co-distribution of retromer with pIgA and the cation-independent mannose 6-phosphate receptor (CI-MPR), delaying pIgA progress to the apical surface (Mellado et al. 2014, Vergés 2016, Kvainickas et al. 2017).

Retromer as an evolutionarily conserved protein complex required for endosome-to-Golgi transport is necessary also for the retrieval of lysosomal hydrolases receptors (McGough and Cullen 2011, Gallon and Cullen 2015, Abubakar et al. 2017). A dimer of two sorting nexins, typically SNX1/SNX2 or SNX1/SNX5 or SNX1/SNX6, deforms the membrane and thus cooperates with retromer to ensure cargo sorting. Wassmer et al. (2009) established that retromer activity depends on a dynamic spatial organization of the endosomal network regulated via the association of SNX5/SNX6 with the “glued” p150 component of dynactin, which serves as an activator of the minus-end directed microtubule motor dynein. The spatial organization of the retromer activity is directed through the association of SNX1 with the proposed TGN-localized Rab6-interacting protein-1 (RIP-1). The stepwise sequence of these interactions is fundamental for retromer-mediated transport and strongly suggests that a critical element required for efficient retromer-mediated sorting is defined by the spatial organization of the retromer network. The role of retromer as well as SNXs in endosomal protein (re)cycling and protein targeting to specialized plasma membrane domains in polarized cells, including transcytosis, adds further complexity and has implications in growth control, the establishment of developmental patterns, cell adhesion, migration, etc.

However, it needs to be stressed here that some proteins may be targeted to their ultimate destination immediately after endocytosis via a less circuitous route that can involve direct movement between the basal/basolateral and apical domains (Kobayashi et al. 2002, Rojas and Apodaca 2002, Hoessli et al. 2004, Fuchs and Ellinger 2004). This type of simplified transcytosis is considered to be clathrin-independent uptake which occurs via caveolae or other membrane microdomains. Caveolae are small flask-shaped plasma membrane invaginations that contain the protein caveolin-1. They are relatively static structures, but a number of factors can stimulate caveolae uptake, creating caveosomes (Parton et al. 1994, Pelkmans and Helenius 2002, Thomsen et al. 2002, Mukherjee et al. 2006). The uptake of caveosomes is likely to be similar to clathrin-mediated endocytosis because caveolae are enriched in proteins that function in membrane docking and fusion events (Schnitzer et al. 1995, Oh et al. 1998). Caveolae may be a subgroup in a broader class of membrane microdomains that are involved in facilitating endocytic events (Johannes and Lamaze 2002, Laude and Prior 2004, Alfalah et al. 2005, Stan 2005, Cheng et al. 2006).

A link between transudation and apocrine

Historically, the transudation process has mainly been associated with the exudation of various serum components (e.g. immunoglobulins, albumins, numerous enzymes) into mucous body fluids including nasal mucus, saliva, tears, ascitic fluid, pulmonary fluids, etc. and often shown to be exacerbated during injury and inflammation (Auerswald et al. 1952, Schorn et al. 1975, Malmendier and Amerijckx 1976, Selinger et al. 1979, Gachon et al. 1982, Janssen and van Bijsterveld 1983, Kuhn et al. 1983, Bard et al. 2002, Bours et al. 2005). In addition to these body fluids, the process has also been observed in association with cervical fluid (or cervicovaginal secretions) (Schumacher 1970, Schwarz et al. 2010, Petäjä et al. 2011), middle ear fluid (MEF) or cerumen (Ishikawa et al. 1972, Howie et al. 1973, Jones et al. 1979, Bernstein 2002, Kaur et al. 2012), bronchoalveolar fluid (Osebold et al. 1975), seminal plasma (Tauber et al. 1975, Lizana et al. 1987), colonic mucosa (Prigent-Delecourt et al. 1995), sputum (Vogel et al. 1997), corporeal fluid (Bélec 2002), oviductal secretions (Buhi et al. 2000), and peritoneal fluid (as a result of endosalpingeal secretion) (Hunter et al. 2007).

In the great majority of cases the common feature of these transudates or exudates is the presence of the same or closely related proteins such as serum albumin, immunoglobulins (IgG1, IgG2, IgA, and IgM), lactoferrin, transferrin, alpha 1-antitrypsin (AAT), lysozyme, C′3-component of complement, ceruloplasmin, fibrinogen, eosinophil cationic protein (ECP), tumor necrosis factor α (TNF-α), transforming growth factor (TGF)-beta1 (TGF-β1), alpha 2-macroglobulin. Consequently, some of these proteins e.g. serum albumin, secretory immunoglobulin A (S-IgA), AAT, lysozyme, lactoferrin and transferrin were then frequently used as transudation markers (Dufour et al. 2005). The mucosal regions of the body, where these proteins are secreted, are responsible for defense against environmental pathogens. Particularly in the lumen of the vas deferens, intestinal and colonic lumens, airway lumen, oviductal and vaginal lumen the antibody- and defense proteins-mediated immune responses are critical for preventing invasion by pathogens (Gonnella and Neutra 1984, Hermo and de Melo, 1987, Oshikawa et al. 1996, Parr et al. 1998, Buhi et al. 2000, Hermo et al. 2000, MacPherson and Uhr 2004, Hamburger et al. 2006). Interestingly, all of the listed proteins are also markers of apocrine secretion, as previously compiled and defined (Farkaš 2015). This list of apocrine markers is now longer and continuously growing, however, there is no doubt that all listed tissues or organs displaying the capability of transudation are also polarized epithelia using apocrine secretion to deliver intracellular proteins to mucosal regions of the body where they serve as a barrier at the exterior interface.

Apocrine mechanism

The definition of apocrine secretion as a non-canonical and non-vesicular transport and secretory mechanism was possible due to its serendipitous discovery in prepupal salivary glands of Drosophila (Farkaš et al. 2014). This genetically well defined model organism provided us with an exceptional opportunity to elucidate the mechanistic aspects of this fundamental process through molecular, cellular, and developmental analyses. It was also a chance to undertake a critical reappraisal and comprehensive review of our knowledge on apocrine secretion in order to place new findings in a historical context and, at the same time, develop new concepts that encapsulate the basic features of apocrine secretion using insights from the most recent data. In addition to the already established phrases and definitions which have been used in the description of apocrine secretion for almost a century, the most intense phase of apocrine extrusion in the Drosophila salivary gland is accompanied by the release of large cellular fragments including entire organelles or their structurally compact nondisintegrated parts: mitochondria, microsomes, Golgi zone and areas of the ER (Farkaš et al. 2014, Farkaš 2015). Thus, apocrine secretion is composed of membranous, cytoskeletal, microsomal, mitochondrial, ribosomal, and most surprisingly, nuclear and even nucleolar proteins. Apocrine secretion is characterized by massive protein release, rather than being solely devoted to the secretion of oily substances, as was previously incorrectly defined (Purkyně 1833b, Schiefferdecker 1917, 1921, Richter 1932, Kuno 1938, Hurley and Shelley 1954, Gesase et al. 1996, Gesase and Satoh 2003).

Preliminary and current proteomic analyses of the apocrine secretory material have revealed that it is a highly complex mixture consisting of hundreds to thousands of proteins from different subcellular locations and with highly diverse functions. These proteins include enzymes, structural proteins, signaling molecules, membrane and intracellular receptors, chaperones, storage proteins, transcription factors, coactivators and corepressors, chromatin and nucleolar components (Farkaš 2015, 2016). Although numerous nuclear proteins are released, nuclear DNA and nuclei appear completely intact (Farkaš et al. 2014). By isolating freshly secreted material directly from the lumen of the Drosophila salivary glands and evaluating the presence of specific proteins using a broad panel of antibodies in western blotting, we demonstrated that proteins are released undegraded, and most probably fully functional (Farkaš et al. 2014). Even though many of the proteins that we analyzed are actively released to undetectable levels inside cells, they start to slowly reappear at their original subcellular locations within about an hour after the completion of apocrine secretion. In addition, the tissue, even though it has undergone massive apocrine secretion, continues to live and produce gene products as shown by the incorporation of radioactive precursors into newly transcribed RNA and newly synthesized proteins. When the fluorescent signal from secondary antibodies or GFP-/RFP-/YFP- fusion proteins that were released into the lumen was quantified, more than 90–95 % of these proteins were found to be secreted from the cells, suggesting that the remaining few percent of a cell’s proteome is sufficient to maintain tissue viability and allow for its rapid regenration (Farkaš et al. 2014).

Heavy endosomal traffick, vacuolation and resecretion in an apocrine organ

Equally unanticipated as apocrine secretion was the finding that Drosophila salivary glands during the early prepupal period are remarkably active in endocytosis, generating high numbers of small acidic vacuoles that continuously fuse to larger ones. Midway through the prepupal period, there is an abundance of late endosomal trafficking and vacuole growth, later followed by vacuole neutralization and disappearance via membrane consolidation (Farkaš et al. 2015). All this endosomal activity, in which a crucial role is played by dynamin, clathrin, Rab5, Rab11, syntaxins, vacuolar ATPases Vha36-1, Vha55, Vha68-2, etc., takes place a few hours prior to apocrine secretion. The final phases of this process, involving vacuole disappearance and their fusion with membranes of ER and TGN, are controlled by Drosophila retromer constituent, Vps35 (Farkaš et al. 2015). It has been determined that among apocrine secretory products which are released by salivary glands there are several proteins which are not produced by themselves, but must be taken up from the surrounding hemolymph by endocytosis (Farkaš et al. 2014).

It should be emphasized that apocrine secretion is characterized by massive protein release, which is in striking contrast to exocytosis when only a single product or a few proteins are released from vesicles, homotypically fusing with the cell membrane. Thus, in the majority if not all above mentioned cases, when immunoglobulins, albumins, various enzymes, lactoferrin, transferrin, AAT, lysozyme, alpha 2-macroglobulin, ceruloplasmin, fibrinogen, ECP, C′3-component of complement, TNF-α, and many others that are not synthesized by a secretory organ, are released into exocrine mucous regions and body fluids of mammals, apocrine secretion is involved. All these data suggest that early endocytosis as well as further cargo sorting and selection for the post-endocytic pathway using retromer (Vergés et al. 2004, 2007, Vergés, 2008, 2016, Bonifacino and Hurley 2008, Kleine-Vehn and Friml 2008, van Weering et al. 2010, Burd and Cullen 2014, Gallon and Cullen 2015) might be common to both the re-release of engulfed proteins by exocytosis (transcytosis) and apocrine secretion (transudation). However, at present we cannot conclude which of the retromer subcomplexes are preferentially used in transcytosis and which are used in transudation, or whether they are distinct at all.

Retromer or its vicinity - potenial intersection point for transcytosis and transudation

Due to the non-vesicular character of apocrine transport and the secretory mechanism, differences at the post-endocytic pathway and later stages at the level of cargo recognition, sorting and/or selection will exist which will allow bulky recruitment of the specified cargo to this massive externalization pathway. This conclusion opens up new vistas for investigation in the future. There is no exocytosis during or after apocrine secretion in Drosophila salivary glands (Farkaš et al. 2014, 2016) and thus proteins taken up from circulation via endocytosis during the early prepupal stages can be released or secreted only by the apocrine pathway. The involvement of retromer in this preceding endocytosis has been already evidenced (Farkaš et al. 2015). This finding poses an important question: in the case of apocrine secretion, does any preceding endocytosis for the purpose of re-secretion, require clathrin-dependent retromer-associated sorting machinery and automatically exclude caveolae-mediated endocytosis?

Transcytosis may be either unidirectional or bidirectional. Unidirectional transcytosis may occur selectively in the luminal to abluminal direction, or in the reverse direction (abluminal to luminal). However, apocrine secretion occurs exclusively on the apical pole and therefore this type of transport and re-secretion is only unidirectional providing grounds to consider these as two separate mechanisms, another reason to clearly differentiate transudation from trancytosis (see Figs 1 and 2). This differentiation opens up several new and interesting avenues of enquiry, such as the existence of crosstalk or the interconnection between an endosomal (vesicular) system and apocrine secretion which in principle is a non-vesicular mechanism, or in other words, how endocytosed material of foreign origin becomes integrated into apocrine machinery or whether this endocytosed material needs a specific sorting mechanism to escape the vesicular pathway on the route to being released. The above described system of Drosophila salivary glands can easily be used to explore these and similar questions by designing biochemical protein-protein interaction studies or screens for genetic interactions focused directly on key trafficking components and the apocrine machinery itself.