Phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate regulate phagolysosome biogenesis

Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome–lysosome fusion. Phagosome–early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.

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Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.

Journal of Experimental Medicine ◽

10.1084/jem.177.3.583 ◽

1993 ◽

Vol 177 (3) ◽

pp. 583-596 ◽

Cited By ~ 95

Author(s):

P Romagnoli ◽

C Layet ◽

J Yewdell ◽

O Bakke ◽

R N Germain

Keyword(s):

Major Histocompatibility Complex ◽

Mhc Class Ii ◽

Class Ii ◽

Endocytic Pathway ◽

Invariant Chain ◽

Major Histocompatibility ◽

Histocompatibility Complex ◽

Late Endosomes ◽

Early Endosomes ◽

Endocytic Compartments

Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.

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Internalization and endosomal degradation of receptor-bound antigens regulate the efficiency of cross presentation by human dendritic cells

Blood ◽

10.1182/blood-2012-01-402370 ◽

2012 ◽

Vol 120 (10) ◽

pp. 2011-2020 ◽

Cited By ~ 119

Author(s):

Bithi Chatterjee ◽

Anna Smed-Sörensen ◽

Lillian Cohn ◽

Cécile Chalouni ◽

Richard Vandlen ◽

...

Keyword(s):

Dendritic Cells ◽

Vaccine Development ◽

Mannose Receptor ◽

Antigen Delivery ◽

Cross Presentation ◽

Late Endosomes ◽

Early Endosomes ◽

Antibody Conjugates ◽

Human Dendritic Cells ◽

Endocytic Compartments

Abstract Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.

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Rabenosyn-5, a Novel Rab5 Effector, Is Complexed with Hvps45 and Recruited to Endosomes through a Fyve Finger Domain

The Journal of Cell Biology ◽

10.1083/jcb.151.3.601 ◽

2000 ◽

Vol 151 (3) ◽

pp. 601-612 ◽

Cited By ~ 222

Author(s):

Erik Nielsen ◽

Savvas Christoforidis ◽

Sandrine Uttenweiler-Joseph ◽

Marta Miaczynska ◽

Frederique Dewitte ◽

...

Keyword(s):

Early Endosome ◽

Effector Proteins ◽

Endosomal Trafficking ◽

Membrane Domains ◽

Phosphatidylinositol 3 Kinase ◽

Early Endosomes ◽

Site Of Action ◽

Endosomal Membranes ◽

Novel Protein ◽

Phosphatidylinositol 3

Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase–dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

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Endosomal trafficking regulates receptor-mediated transcytosis of antibodies across the blood brain barrier

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x17740031 ◽

2017 ◽

Vol 38 (4) ◽

pp. 727-740 ◽

Cited By ~ 19

Author(s):

Arsalan S Haqqani ◽

Christie E Delaney ◽

Eric Brunette ◽

Ewa Baumann ◽

Graham K Farrington ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Early Endosome ◽

Brain Barrier ◽

Endosomal Trafficking ◽

Brain Endothelial Cells ◽

Density Fractions ◽

Blood Brain ◽

Late Endosomes ◽

Early Endosomes

Current methods for examining antibody trafficking are either non-quantitative such as immunocytochemistry or require antibody labeling with tracers. We have developed a multiplexed quantitative method for antibody ‘tracking’ in endosomal compartments of brain endothelial cells. Rat brain endothelial cells were co-incubated with blood-brain barrier (BBB)-crossing FC5, monovalent FC5Fc or bivalent FC5Fc fusion antibodies and control antibodies. Endosomes were separated using sucrose-density gradient ultracentrifugation and analyzed using multiplexed mass spectrometry to simultaneously quantify endosomal markers, receptor-mediated transcytosis (RMT) receptors and the co-incubated antibodies in each fraction. The quantitation showed that markers of early endosomes were enriched in high-density fractions (HDF), whereas markers of late endosomes and lysosomes were enriched in low-density fractions (LDF). RMT receptors, including transferrin receptor, showed a profile similar to that of early endosome markers. The in vitro BBB transcytosis rates of antibodies were directly proportional to their partition into early endosome fractions of brain endothelial cells. Addition of the Fc domain resulted in facilitated antibody ‘redistribution’ from LDF into HDF and additionally into multivesicular bodies (MVB). Sorting of various FC5 antibody formats away from late endosomes and lysosomes and into early endosomes and a subset of MVB results in increased antibody transcytosis at the abluminal side of the BBB.

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De novo formation of an early endosome during Rab5 to Rab7 transition

10.1101/2020.07.06.189050 ◽

2020 ◽

Author(s):

Frode Miltzow Skjeldal ◽

Linda Hofstad Haugen ◽

Duarte Mateus ◽

Dominik Frei ◽

Oddmund Bakke

Keyword(s):

De Novo ◽

Early Endosome ◽

Late Endosome ◽

Second Phase ◽

Specific Domain ◽

Endosomal Membrane ◽

Late Endosomes ◽

Early Endosomes ◽

Main Determinants ◽

Insight Into

AbstractRab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch where Rab5 is exchanged with Rab7a on the same endosome. Here we provide new insight into the mechanism of endosomal maturation where we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrate a diffusion-like exchange between cytosol and endosomal membrane, and the second phase is slower where Rab5 converges into a specific domain that specifically detaches as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5 to Rab7a switch induce the formation of a new fully functional early endosome. Progression through a stepwise early endosomal maturation regulates the direction of the transport and concomitantly regulates the homeostasis of early endosomes.

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Rab5-mediated endosome formation is regulated at the trans-Golgi network

Communications Biology ◽

10.1038/s42003-019-0670-5 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 3

Author(s):

Makoto Nagano ◽

Junko Y. Toshima ◽

Daria Elisabeth Siekhaus ◽

Jiro Toshima

Keyword(s):

Plasma Membrane ◽

Early Endosome ◽

Vesicle Transport ◽

Nucleotide Exchange ◽

Trafficking Pathway ◽

Transport Vesicles ◽

Late Endosomes ◽

Early Endosomes ◽

Critical Location ◽

Golgi Network

AbstractEarly endosomes, also called sorting endosomes, are known to mature into late endosomes via the Rab5-mediated endolysosomal trafficking pathway. Thus, early endosome existence is thought to be maintained by the continual fusion of transport vesicles from the plasma membrane and the trans-Golgi network (TGN). Here we show instead that endocytosis is dispensable and post-Golgi vesicle transport is crucial for the formation of endosomes and the subsequent endolysosomal traffic regulated by yeast Rab5 Vps21p. Fittingly, all three proteins required for endosomal nucleotide exchange on Vps21p are first recruited to the TGN before transport to the endosome, namely the GEF Vps9p and the epsin-related adaptors Ent3/5p. The TGN recruitment of these components is distinctly controlled, with Vps9p appearing to require the Arf1p GTPase, and the Rab11s, Ypt31p/32p. These results provide a different view of endosome formation and identify the TGN as a critical location for regulating progress through the endolysosomal trafficking pathway.

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The acyltransferase LYCAT controls specific phosphoinositides and related membrane traffic

Molecular Biology of the Cell ◽

10.1091/mbc.e16-09-0668 ◽

2017 ◽

Vol 28 (1) ◽

pp. 161-172 ◽

Cited By ~ 22

Author(s):

Leslie N. Bone ◽

Roya M. Dayam ◽

Minhyoung Lee ◽

Nozomu Kono ◽

Gregory D. Fairn ◽

...

Keyword(s):

Acyl Chain ◽

Membrane Traffic ◽

Cell Physiology ◽

Phosphatidylinositol Synthase ◽

Lipid Kinases ◽

Acyl Chains ◽

And Function ◽

Phosphatidylinositol 4 ◽

Phosphatidylinositol 3

Phosphoinositides (PIPs) are key regulators of membrane traffic and signaling. The interconversion of PIPs by lipid kinases and phosphatases regulates their functionality. Phosphatidylinositol (PI) and PIPs have a unique enrichment of 1-stearoyl-2-arachidonyl acyl species; however, the regulation and function of this specific acyl profile remains poorly understood. We examined the role of the PI acyltransferase LYCAT in control of PIPs and PIP-dependent membrane traffic. LYCAT silencing selectively perturbed the levels and localization of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and phosphatidylinositol-3-phosphate and the membrane traffic dependent on these specific PIPs but was without effect on phosphatidylinositol-4-phosphate or biosynthetic membrane traffic. The acyl profile of PI(4,5)P2 was selectively altered in LYCAT-deficient cells, whereas LYCAT localized with phosphatidylinositol synthase. We propose that LYCAT remodels the acyl chains of PI, which is then channeled into PI(4,5)P2. Our observations suggest that the PIP acyl chain profile may exert broad control of cell physiology.

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Phosphoinositides in Constitutive Membrane Traffic

Physiological Reviews ◽

10.1152/physrev.00033.2003 ◽

2004 ◽

Vol 84 (3) ◽

pp. 699-730 ◽

Cited By ~ 208

Author(s):

Michael G. Roth

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Membrane Traffic ◽

Spatial Segregation ◽

Peripheral Membrane Proteins ◽

Transport Vesicles ◽

Early Endosomes ◽

Lipid Kinases ◽

Phosphatidylinositol 4 ◽

Different Parts

Proteins that make, consume, and bind to phosphoinositides are important for constitutive membrane traffic. Different phosphoinositides are concentrated in different parts of the central vacuolar pathway, with phosphatidylinositol 4-phosphate predominate on Golgi, phosphatidylinositol 4,5-bisphosphate predominate at the plasma membrane, phosphatidylinositol 3-phosphate the major phosphoinositide on early endosomes, and phosphatidylinositol 3,5-bisphosphate found on late endocytic organelles. This spatial segregation may be the mechanism by which the direction of membrane traffic is controlled. Phosphoinositides increase the affinity of membranes for peripheral membrane proteins that function for sorting protein cargo or for the docking and fusion of transport vesicles. This implies that constitutive membrane traffic may be regulated by the mechanisms that control the activity of the enzymes that produce and consume phosphoinositides. Although the lipid kinases and phosphatases that function in constitutive membrane traffic are beginning to be identified, their regulation is poorly understood.

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Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex

The Journal of Cell Biology ◽

10.1083/jcb.202002075 ◽

2021 ◽

Vol 220 (2) ◽

Author(s):

Jennifer Hirst ◽

Geoffrey G. Hesketh ◽

Anne-Claude Gingras ◽

Margaret S. Robinson

Keyword(s):

Protein Complex ◽

Adaptor Protein ◽

Coincidence Detection ◽

Rag Gtpases ◽

Fyve Domain ◽

Late Endosomes ◽

Early Endosomes ◽

Mtorc1 Pathway ◽

Phosphatidylinositol 3

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.

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Degradation of phagosomal components in late endocytic organelles

Journal of Cell Science ◽

10.1242/jcs.111.1.141 ◽

1998 ◽

Vol 111 (1) ◽

pp. 141-148

Author(s):

T.E. Tjelle ◽

B. Saigal ◽

M. Froystad ◽

T. Berg

Keyword(s):

Subcellular Fractionation ◽

Endocytic Pathway ◽

Phagocytic Cells ◽

Fusion Event ◽

Late Endosomes ◽

Early Endosomes ◽

Efficient Communication ◽

J774 Cells ◽

Shift Technique

Phagosomes are formed when phagocytic cells ingest particles such as bacteria, viruses or synthetic beads of different kinds. The environment within the phagosome gradually changes to generate degradative conditions. These changes require multiple interactions between the maturing phagosomes and the endocytic and the biosynthetic pathway. The phagosomes probably communicate with endocytic organelles by a transient fusion event, often referred to as the ‘kiss-and-run’ hypothesis. We have studied the role of endocytic organelles in the phagocytic pathway of J774 cells, a mouse macrophage cell line. We have used magnetic Dynabeads coated with 125ITC-IgG and 125ITC-OVA as phagocytic probes and were able to isolate the phagosomal fraction by means of a magnet. To separate lysosomes from other organelles in the endocytic pathway we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three endocytic fractions corresponding to early endosomes (the light Percoll fraction), late endosomes (the dense Percoll fraction) and lysosomes (the gold fraction). We observed that the proteins linked to the ingested beads are initially cleaved in the phagosomes. This cleavage is inhibited by leupeptin, a thiol-protease inhibitor, and requires an acidic environment. However, efficient communication between the phagosomes and the endocytic pathway leads to the transfer of dissociated phagocytosed peptides of different sizes to late endosomes and lysosomes for further processing. Consequently, the late endosomes and the lysosomes may be involved in the degradation of phagocytosed compounds.

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