The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Regional differences in the ultrastructure of mucosal macrophages in the rat large intestine.

We previously clarified the histological characteristics of macrophages in the rat small intestine using serial block-face scanning electron microscopy (SBF-SEM). However, the regional differences in the characteristics of macrophages throughout the large intestine remain unknown. Here, we performed a pilot study to explore the regional differences in the ultrastructure of mucosal macrophages in the large intestine by using SBF-SEM analysis. SBF-SEM analysis conducted on the luminal side of the cecum and descending colon revealed macrophages as amorphous cells possessing abundant lysosomes and vacuoles. Macrophages in the cecum exhibited a higher abundance of lysosomes and a lower abundance of vacuoles than those in the descending colon. Macrophages with many intraepithelial cellular processes were observed beneath the intestinal superficial epithelium in the descending colon. Moreover, macrophages in contact with nerve fibers were more prevalent in the cecum than in the descending colon, and a subset of them surrounded a nerve bundle only in the cecum. In conclusion, the present pilot study suggested that the quantity of some organelles (lysosomes and vacuoles) in macrophages differed between the cecum and the descending colon and that there were some region-specific subsets of macrophages like nerve-associated macrophages in the cecum.

Systematic Transmission Electron Microscopy-Based Identification and 3D Reconstruction of Cellular Degradation Machinery.

Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer-scale micrographs of cellular degradation components in a fixed sample. Serial block face-scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

Neurodegenerative Lysosomal Storage Disorders: TPC2 Comes to the Rescue!

Lysosomal storage diseases (LSDs) resulting from inherited gene mutations constitute a family of disorders that disturb lysosomal degradative function leading to abnormal storage of macromolecular substrates. In most LSDs, central nervous system (CNS) involvement is common and leads to the progressive appearance of neurodegeneration and early death. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of neurodegenerative LSDs. One of the main basic mechanisms through which the endolysosomal ion channels regulate the function of the endolysosomal system is Ca2+ release, which is thought to be essential for intracellular compartment fusion, fission, trafficking and lysosomal exocytosis. The intracellular TRPML (transient receptor potential mucolipin) and TPC (two-pore channel) ion channel families constitute the main essential Ca2+-permeable channels expressed on endolysosomal membranes, and they are considered potential drug targets for the prevention and treatment of LSDs. Although TRPML1 activation has shown rescue effects on LSD phenotypes, its activity is pH dependent, and it is blocked by sphingomyelin accumulation, which is characteristic of some LSDs. In contrast, TPC2 activation is pH-independent and not blocked by sphingomyelin, potentially representing an advantage over TRPML1. Here, we discuss the rescue of cellular phenotypes associated with LSDs such as cholesterol and lactosylceramide (LacCer) accumulation or ultrastructural changes seen by electron microscopy, mediated by the small molecule agonist of TPC2, TPC2-A1-P, which promotes lysosomal exocytosis and autophagy. In summary, new data suggest that TPC2 is a promising target for the treatment of different types of LSDs such as MLIV, NPC1, and Batten disease, both in vitro and in vivo.

The perinuclear region concentrates disordered proteins with predicted phase separation distributed in a 3D network of cytoskeletal filaments and organelles.

Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins.

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural-functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.

Amyloid-like aggregating proteins cause lysosomal defects in neurons via gain-of-function toxicity.

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like ß-sheet proteins (ß proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, ß proteins interact with and sequester AP-3 µ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3µ1 expression ameliorates neurotoxicity caused by ß proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.

Prefused lysosomes cluster on autophagosomes regulated by VAMP8.

Lysosome-autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes-vesicle-associated membrane protein 8 (VAMP8)-plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

Sulforaphane downregulated fatty acid synthase and inhibited microtubule-mediated mitophagy leading to apoptosis.

We previously demonstrated that sulforaphane (SFN) inhibited autophagy leading to apoptosis in human non-small cell lung cancer (NSCLC) cells, but the underlying subcellular mechanisms were unknown. Hereby, high-performance liquid chromatography-tandem mass spectrometry uncovered that SFN regulated the production of lipoproteins, and microtubule- and autophagy-associated proteins. Further, highly expressed fatty acid synthase (FASN) contributed to cancer malignancy and poor prognosis. Results showed that SFN depolymerized microtubules, downregulated FASN, and decreased its binding to α-tubulin; SFN downregulated FASN, acetyl CoA carboxylase (ACACA), and ATP citrate lyase (ACLY) via activating proteasomes and downregulating transcriptional factor SREBP1; SFN inhibited the interactions among α-tubulin and FASN, ACACA, and ACLY; SFN decreased the amount of intracellular fatty acid (FA) and mitochondrial phospholipids; and knockdown of FASN decreased mitochondrial membrane potential (ΔΨm) and increased reactive oxygen species, mitochondrial abnormality, and apoptosis. Further, SFN downregulated mitophagy-associated proteins Bnip3 and NIX, and upregulated mitochondrial LC3 II/I. Transmission electron microscopy showed mitochondrial abnormality and accumulation of mitophagosomes in response to SFN. Combined with mitophagy inducer CCCP or autophagosome-lysosome fusion inhibitor Bafilomycin A1, we found that SFN inhibited mitophagosome-lysosome fusion leading to mitophagosome accumulation. SFN reduced the interaction between NIX and LC3 II/I, and reversed CCCP-caused FA increase. Furthermore, knockdown of α-tubulin downregulated NIX and BNIP3 production, and upregulated LC3 II/I. Besides, SFN reduced the interaction and colocalization between α-tubulin and NIX. Thus, SFN might cause apoptosis via inhibiting microtubule-mediated mitophagy. These results might give us a new insight into the mechanisms of SFN-caused apoptosis in the subcellular level.

Implications of exosomes derived from cholesterol-accumulated astrocytes in Alzheimer's disease pathology.

Amyloid ß (Aß) peptides generated from the amyloid precursor protein (APP) play a critical role in the development of Alzheimer's disease (AD) pathology. Aß-containing neuronal exosomes, which represent a novel form of intercellular communication, have been shown to influence the function/vulnerability of neurons in AD. Unlike neurons, the significance of exosomes derived from astrocytes remains unclear. In this study, we evaluated the significance of exosomes derived from U18666A-induced cholesterol-accumulated astrocytes in the development of AD pathology. Our results show that cholesterol accumulation decreases exosome secretion, whereas lowering cholesterol increases exosome secretion, from cultured astrocytes. Interestingly, exosomes secreted from U18666A-treated astrocytes contain higher levels of APP, APP-C-terminal fragments, soluble APP, APP secretases and Aß1-40 than exosomes secreted from control astrocytes. Furthermore, we show that exosomes derived from U18666A-treated astrocytes can lead to neurodegeneration, which is attenuated by decreasing Aß production or by neutralizing exosomal Aß peptide with an anti-Aß antibody. These results, taken together, suggest that exosomes derived from cholesterol-accumulated astrocytes can play an important role in trafficking APP/Aß peptides and influencing neuronal viability in the affected regions of the AD brain.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

Current Knowledge of Endolysosomal and Autophagy Defects in Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) refers to a group of neurological disorders involving the degeneration of motor neurons. Due to their clinical and genetic heterogeneity, finding common effective therapeutics is difficult. Therefore, a better understanding of the common pathological mechanisms is necessary. The role of several HSP genes/proteins is linked to the endolysosomal and autophagic pathways, suggesting a functional convergence. Furthermore, impairment of these pathways is particularly interesting since it has been linked to other neurodegenerative diseases, which would suggest that the nervous system is particularly sensitive to the disruption of the endolysosomal and autophagic systems. In this review, we will summarize the involvement of HSP proteins in the endolysosomal and autophagic pathways in order to clarify their functioning and decipher some of the pathological mechanisms leading to HSP.

Physcion Induces Potential Anticancer Effects in Cervical Cancer Cells.

BACKGROUND: The extent of morphological and ultrastructural changes in HeLa cells was assessed by optical, fluorescence and electron microscopy after exposure to various concentrations of physcion, taking into account the biological properties of the test compound. METHODS: Cell viability was assessed by MTT assay, while the cell cycle, LC3 expression, apoptosis, change of mitochondrial potential, Bcl-2 protein expression level and the level of reactive oxygen species were analyzed by flow cytometry. RESULTS: As a result of physcion encumbrance, concentration-dependent inhibition of HeLa cell viability and the G0/G1 phase of the cell cycle was observed. Activation of the lysosomal system was also revealed, which was expressed by an increased number of lysosomes, autophage vacuoles and increased expression of the LC3 protein, a marker of the autophagy process. Transmission electron microscopy and fluorescence microscopy showed that physcion induced clear changes in cervical cancer cells, especially in the structure of the nucleus and mitochondria, which correlated with the production of reactive oxygen species by the test compound and indicated the induction of the oxidative process. At the same time, the pro-apoptotic effect of physcion was demonstrated, and this mechanism was dependent on the activation of caspases 3/7 and the reduction in Bcl-2 protein expression. CONCLUSION: The obtained results indicate an antitumor mechanism of action of physcion, based on the induction of oxidative stress, autophagy and apoptosis.

Kainate-Induced Degeneration of Hippocampal Neurons. Protective Effect of Activation of the Endocannabinoid System.

We studied the prolonged action of kainic acid on glutamatergic neurons in the dorsal hippocampus and the endocannabinoid-dependent protection against neurodegeneration. The pyramidal neurons of the CA3 field of the hippocampus, as well as granular and mossy cells of the dentate gyrus were examined. Light and electron microscopy revealed substantial damage to the components of the protein-synthesizing (rough endoplasmic reticulum, Golgi apparatus, and polyribosomes) and catabolic (lysosomes, autophagosomes, multivesicular structures, and lipofuscin formations) systems in all cells. Pyramidal and mossy neurons die mainly by the necrotic pathway. The death of granular cells occurred through both apoptosis and necrosis. The most vulnerable cells are mossy neurons located in the hilus. Activation of the endocannabinoid system induced by intracerebral injection of URB597, an inhibitor of degradation of endocannabinoid anandamide, protected the normal structure of the hippocampus and prevented neuronal damage and death induced by KA.

TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism.

Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.

SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum.

The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin.

α-Synuclein fibrils subvert lysosome structure and function for the propagation of protein misfolding between cells through tunneling nanotubes.

The accumulation of α-synuclein (α-syn) aggregates in specific brain regions is a hallmark of synucleinopathies including Parkinson disease (PD). α-Syn aggregates propagate in a "prion-like" manner and can be transferred inside lysosomes to recipient cells through tunneling nanotubes (TNTs). However, how lysosomes participate in the spreading of α-syn aggregates is unclear. Here, by using super-resolution (SR) and electron microscopy (EM), we find that α-syn fibrils affect the morphology of lysosomes and impair their function in neuronal cells. In addition, we demonstrate that α-syn fibrils induce peripheral redistribution of lysosomes, likely mediated by transcription factor EB (TFEB), increasing the efficiency of α-syn fibrils' transfer to neighboring cells. We also show that lysosomal membrane permeabilization (LMP) allows the seeding of soluble α-syn in cells that have taken up α-syn fibrils from the culture medium, and, more importantly, in healthy cells in coculture, following lysosome-mediated transfer of the fibrils. Moreover, we demonstrate that seeding occurs mainly at lysosomes in both donor and acceptor cells, after uptake of α-syn fibrils from the medium and following their transfer, respectively. Finally, by using a heterotypic coculture system, we determine the origin and nature of the lysosomes transferred between cells, and we show that donor cells bearing α-syn fibrils transfer damaged lysosomes to acceptor cells, while also receiving healthy lysosomes from them. These findings thus contribute to the elucidation of the mechanism by which α-syn fibrils spread through TNTs, while also revealing the crucial role of lysosomes, working as a Trojan horse for both seeding and propagation of disease pathology.

Autophagy of mucin granules contributes to resolution of airway mucous metaplasia.

Exacerbations of muco-obstructive airway diseases such as COPD and asthma are associated with epithelial changes termed mucous metaplasia (MM). Many molecular pathways triggering MM have been identified; however, the factors that regulate resolution are less well understood. We hypothesized that the autophagy pathway is required for resolution of MM by eliminating excess non-secreted intracellular mucin granules. We found increased intracellular levels of mucins Muc5ac and Muc5b in mice deficient in autophagy regulatory protein, Atg16L1, and that this difference was not due to defects in the known baseline or stimulated mucin secretion pathways. Instead, we found that, in mucous secretory cells, Lc3/Lamp1 vesicles colocalized with mucin granules particularly adjacent to the nucleus, suggesting that some granules were being eliminated in the autophagy pathway rather than secreted. Using a mouse model of MM resolution, we found increased lysosomal proteolytic activity that peaked in the days after mucin production began to decline. In purified lysosomal fractions, Atg16L1-deficient mice had reduced proteolytic degradation of Lc3 and Sqstm1 and persistent accumulation of mucin granules associated with impaired resolution of mucous metaplasia. In normal and COPD derived human airway epithelial cells (AECs), activation of autophagy by mTOR inhibition led to a reduction of intracellular mucin granules in AECs. Our findings indicate that during peak and resolution phases of MM, autophagy activity rather than secretion is required for elimination of some remaining mucin granules. Manipulation of autophagy activation offers a therapeutic target to speed resolution of MM in airway disease exacerbations.

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes.

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as "phagosome resolution." Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

Distinct antigen uptake receptors route to the same storage compartments for cross-presentation in dendritic cells.

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.