Enhanced translation expands the endo-lysosome size and promotes antigen presentation during phagocyte activation.

The mechanisms that govern organelle adaptation and remodelling remain poorly defined. The endo-lysosomal system degrades cargo from various routes, including endocytosis, phagocytosis, and autophagy. For phagocytes, endosomes and lysosomes (endo-lysosomes) are kingpin organelles because they are essential to kill pathogens and process and present antigens. During phagocyte activation, endo-lysosomes undergo a morphological transformation, going from a collection of dozens of globular structures to a tubular network in a process that requires the phosphatidylinositol-3-kinase-AKT-mechanistic target of rapamycin (mTOR) signalling pathway. Here, we show that the endo-lysosomal system undergoes an expansion in volume and holding capacity during phagocyte activation within 2 h of lipopolysaccharides (LPS) stimulation. Endo-lysosomal expansion was paralleled by an increase in lysosomal protein levels, but this was unexpectedly largely independent of the transcription factor EB (TFEB) and transcription factor E3 (TFE3), which are known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of acute organelle expansion via mTOR Complex 1 (mTORC1)-dependent increase in translation, which appears to be mediated by both S6Ks and 4E-BPs. Moreover, we show that stimulation of RAW 264.7 macrophage cell line with LPS alters translation of a subset but not all of mRNAs encoding endo-lysosomal proteins, thereby suggesting that endo-lysosome expansion is accompanied by functional remodelling. Importantly, mTORC1-dependent increase in translation activity was necessary for efficient and rapid antigen presentation by dendritic cells. Collectively, we identified a previously unknown and functionally relevant mechanism for endo-lysosome expansion that relies on mTORC1-dependent translation to stimulate endo-lysosome biogenesis in response to an infection signal.

Biogenesis of lysosome-related organelles complex-1 (BORC) regulates late endosomal/lysosomal size through PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate.

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.

Phagocytosis: what's on the menu? 1.

Phagocytosis is an evolutionarily conserved process. In Protozoa, phagocytosis fulfills a feeding mechanism, while in Metazoa, phagocytosis diversified to play multiple organismal roles, including immune defence, tissue homeostasis, and remodeling. Accordingly, phagocytes display a high level of plasticity in their capacity to recognize, engulf, and process targets that differ in composition and morphology. Here, we review how phagocytosis adapts to its multiple roles and discuss in particular the effect of target morphology in phagocytic uptake and phagosome maturation.

Lysophosphatidic acid represses autophagy in prostate carcinoma cells.

Lysophosphatidic acid (LPA) is a small signaling phospholipid that mediates diverse functions including cell proliferation, migration, and survival by engaging LPA-agonized G-protein coupled receptors. Autophagy is a survival mechanism in response to nutrient depletion or organellar damage that encloses idle or damaged organelles within autophagosomes that are then delivered to lysosomes for degradation. However, the relationship between LPA and autophagy is largely unknown. The purpose of this study is to elucidate whether LPA affects autophagy through the ERK1/2 and (or) the Akt-mTOR signaling pathways. In this study, we investigated the effect of LPA on autophagy-regulating pathways in various prostate-derived cancer cells including PC3, LNCaP, and Du145 cells grown in complete medium and exposed to serum-free medium. Using Western blotting and ELISA, we determined that LPA stimulates the ERK and mTOR pathways in complete and serum-free medium. The mTOR pathway led to phosphorylation of S6K and ULK, which respectively stimulates protein synthesis and arrests autophagy. Consistent with this, LPA exposure suppressed autophagy as measured by LC3 maturation and formation of GFP-LC3 puncta. Altogether, these results suggest that LPA suffices to activate mTORC1 and suppress autophagy in prostate cancer cells.

Lysosome remodelling and adaptation during phagocyte activation.

Lysosomes are acidic and hydrolytic organelles responsible for receiving and digesting cargo acquired during endocytosis, phagocytosis, and autophagy. For macrophages and dendritic cells, the lysosome is kingpin, playing a direct role in microbe killing and antigen processing for presentation. Strikingly, the historic view that lysosomes are homogeneous and static organelles is being replaced with a more elegant paradigm, in which lysosomes are heterogeneous, dynamic, and respond to cellular needs. For example, lysosomes are signalling platforms that integrate stress detection and molecular decision hubs such as the mTOR complex 1 and AMPK to modulate cellular activity. These signals can even adjust lysosome activity by modulating transcription factors such as transcription factor EB (TFEB) and TFE3 that govern lysosome gene expression. Here, we review lysosome remodelling and adaptation during macrophage and dendritic cell stimulation. First, we assess the functional outcomes and regulatory mechanisms driving the dramatic restructuring of lysosomes from globular organelles into a tubular network during phagocyte activation. Second, we discuss lysosome adaptation and scaling in macrophages driven by TFEB and TFE3 stimulation in response to phagocytosis and microbe challenges. Collectively, we are beginning to appreciate that lysosomes are dynamic and adapt to serve phagocyte differentiation in response to microbes and immune stress.

pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels.

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3)P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes.

mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells.

Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase-Akt-mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes.