A novel live-cell imaging assay reveals regulation of endosome maturation

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

TBC1D18, a novel Rab5-GAP, coordinates endosome maturation together with Mon1

Endosome maturation is essential for efficient degradation of internalized extracellular molecules and plasma membrane proteins. Two Rab GTPases, Rab5 and Rab7, are known to regulate endosome maturation, and a Rab5-to-Rab7 conversion mediated by a Rab7 activator, Mon1-Ccz1, is essential for progression of the maturation process. However, the importance and mechanism of Rab5 inactivation during endosome maturation is poorly understood. Here we report a novel Rab5 inactivator (Rab5-GTPase activating protein [Rab5-GAP]), TBC1D18, which is associated with Mon1 and mediates endosome maturation. We found that Rab5 hyperactivation in addition to Rab7 inactivation occurs in the absence of Mon1. We present evidence showing that the severe defects in endosome maturation observed in Mon1-KO cells are attributable to Rab5 hyperactivation rather than to Rab7 inactivation. We then identified TBC1D18 as a Rab5-GAP by comprehensive screening of TBC-domain-containing Rab-GAPs. Expression of TBC1D18 in Mon1-KO cells rescued the defects in endosome maturation, whereas its depletion attenuated endosome formation and degradation of endocytosed cargos. Moreover, TBC1D18 was found to be able to interact with Mon1, and it localized in close proximity to lysosomes in a Mon1-dependent manner. Thus, TBC1D18 is a crucial regulator of endosome maturation that functions together with Mon1.

Strategies of Mycoplasma ovipneumoniae to evade immune clearance by alveolar macrophages

Evidences showed that M. ovipneumoniae might associate with the development and duration of chronic pneumonia. Moreover, sheep infected with M. ovipneumoniae are easily infected by other organisms, suggesting that M. ovipneumoniae may play an immunosuppressive role during infection. However, the mechanism is still poorly understood. The infection occurs in the airway, where resident alveolar macrophages first encounter M. ovipneumoniae. Therefore, primary alveolar macrophages (AMs) were collected from the lungs of healthy adult sheep, and the (iTRAQ) protein assay was used to investigate the immunosuppressive effects of M. ovipneumoniae on sheep AMs. The RAW264.7 cells were used to confirm the findings. The results showed that M. ovipneumoniae promoted higher expression of anti-apoptotic proteins and lower expression of apoptosis-related proteins in the infected AMs. Moreover, the number of infected AMs increased. However, M. ovipneumoniae reduced ATP levels in AMs and impaired late endosome maturation and phagolysosome fusion. Furthermore, M. ovipneumoniae inhibited the autophagy pathway via the Akt-mTOR axis in AMs. These findings indicated that M. ovipneumoniae had distinctive strategies to evade elimination caused by the AMs. The findings might explain the chronic infection and co-infection in sheep infected by M. ovipneumoniae.

A novel live cell imaging assay reveals regulation of endosome maturation

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1- and Rab11-dependent endosomal recycling occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

The Rab GTPase activating protein TBC-2 extends longevity by facilitating release of FOXO/DAF-16 from endosomes

FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is also increased by disrupting the Rab GTPase activating protein TBC-2, or decreased by inhibiting the RAB-5 or RAB-7 GTPases, key regulators of early to late endosome maturation. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 decreased the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Finally, we show that the ability of TBC-2 proteins, TBC1D2 and TBC1D2B, to regulate FOXO protein localization is conserved in human cells. Overall, this work identifies endosomal localization as a mechanism regulating FOXO/DAF-16, which is important for its functions in metabolism and aging.

Fusion and fission events regulate endosome maturation and viral escape

Endosomes are intracellular vesicles that mediate the communication of the cell with its extracellular environment. They are an essential part of the cell’s machinery regulating intracellular trafficking via the endocytic pathway. Many viruses, which in order to replicate require a host cell, attach themselves to the cellular membrane; an event which usually initiates uptake of a viral particle through the endocytic pathway. In this way viruses hijack endosomes for their journey towards intracellular sites of replication and avoid degradation without host detection by escaping the endosomal compartment. Recent experimental techniques have defined the role of endosomal maturation in the ability of enveloped viruses to release their genetic material into the cytoplasm. Endosome maturation depends on a family of small hydrolase enzymes (or GTPases) called Rab proteins, arranged on the cytoplasmic surface of its membrane. Here, we model endosomes as intracellular compartments described by two variables (its levels of active Rab5 and Rab7 proteins) and which can undergo coagulation (or fusion) and fragmentation (or fission). The key element in our approach is the “per-cell endosomal distribution” and its dynamical (Boltzmann) equation. The Boltzmann equation allows us to derive the dynamics of the total number of endosomes in a cell, as well as the mean and the standard deviation of its active Rab5 and Rab7 levels. We compare our mathematical results with experiments of Dengue viral escape from endosomes. The relationship between endosomal active Rab levels and pH suggests a mechanism that can account for the observed variability in viral escape times, which in turn regulate the viability of a viral intracellular infection.

Non-canonical ago loading of EV-derived exogenous single stranded miRNA in recipient cells

MicroRNAs, the tiny regulators of gene expression, can be transferred between neighbouring cells via Extracellular Vesicles (EV) to control the expression of genes in both donor and recipient cells. How the EV-derived miRNAs get internalized and become functional in target cells is an unresolved question. We have expressed liver specific microRNA, miR-122, in non-hepatic cells for packaging in the released EVs. With these EVs, we have followed the trafficking of miR-122 to recipient HeLa cells that otherwise don't express this miRNA. We found that EV-associated miR-122 are primarily single stranded and, to become functional, get loaded onto the recipient cell Ago proteins without requiring host Dicer1. Following endocytosis, EV-associated miR-122 get loaded onto the host cell Ago on the endosomal membrane where the release of internalized miRNAs occurs in a pH-dependent manner facilitating the formation of the exogenous miRNP pool in the recipient cells. Endosome maturation defect affects EV-mediated entry of exogeneous miRNAs in mammalian cells.

Interaction Between Phosphoinositide 3-Kinase and the VDAC2 Channel Tethers Endosomes to Mitochondria and Promotes Endosome Maturation

SUMMARYIntracellular organelles of mammalian cells communicate with each other during various cellular processes. The functions and molecular mechanisms of such interorganelle association remain largely unclear, however. We here identified voltage-dependent anion channel 2 (VDAC2), a mitochondrial outer membrane protein, as a binding partner of phosphoinositide 3-kinase (PI3K), a regulator of clathrin-independent endocytosis downstream of the small GTPase Ras. VDAC2 was found to tether endosomes positive for the Ras-PI3K complex to mitochondria in response to cell stimulation with epidermal growth factor and to promote clathrin-independent endocytosis as well as endosome maturation at membrane contact sites. With a newly developed optogenetics system to induce mitochondrion-endosome association, we found that, in addition to its structural role in such association, the pore function of VDAC2 is also required for the promotion of endosome maturation. Our findings thus uncover a previously unappreciated role of mitochondrion-endosome association in the regulation of endocytosis and endosome maturation.HighlightsThe mitochondrial protein VDAC2 binds PI3K and tethers endosomes to mitochondriaVDAC2 promotes clathrin-independent endocytosisVDAC2-PI3K interaction induces acidification of endosomes associated with mitochondriaThe pore function of VDAC2 also contributes to endosome maturation at contact sites