Synaptic Vesicles Form by Budding from Tubular Extensions of Sorting Endosomes in PC12 Cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15°C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37°C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.

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Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes

Journal of Cell Science ◽

10.1242/jcs.103.4.1139 ◽

1992 ◽

Vol 103 (4) ◽

pp. 1139-1152

Author(s):

J.W. Kok ◽

K. Hoekstra ◽

S. Eskelinen ◽

D. Hoekstra

Keyword(s):

Plasma Membrane ◽

Intracellular Ph ◽

Brefeldin A ◽

Fluid Phase ◽

Endocytic Pathway ◽

Late Endosomes ◽

Early Endosomes ◽

Recycling Pathway ◽

Golgi Network ◽

Extensive Involvement

Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6(-)[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosyl sphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membrane occurs, as could be shown after treating the cells with the microtubule-disrupting agent nocodazole, which causes inhibition of the glycolipid's trafficking from peripheral early endosomes to centrally located late endosomes. When the microtubuli are intact, at least part of the glucosylceramide is transported from early to late endosomes together with ricin. Interestingly, also N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a membrane marker of the fluid-phase endocytic pathway, is transported to this endosomal compartment. However, in contrast to both ricin and N-Rh-PE, the glucosylceramide can escape from this organelle and recycle to the plasma membrane. Monensin and brefeldin A have little effect on this recycling pathway, which would exclude extensive involvement of early Golgi compartments in recycling. Hence, the small fraction of the glycolipid that colocalizes with transferrin (Tf) in the Golgi area might directly recycle via the trans-Golgi network. When the intracellular pH was lowered to 5.5, recycling was drastically reduced, in accordance with the impeding effect of low intracellular pH on vesicular transport during endocytosis and in the biosynthetic pathway. Our results thus demonstrate the existence of at least two recycling pathways for glucosylceramide and indicate the relevance of early endosomes in recycling of both proteins and lipids.

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The Inhibitory Role of Rab11b in Osteoclastogenesis through Triggering Lysosome-Induced Degradation of c-Fms and RANK Surface Receptors

International Journal of Molecular Sciences ◽

10.3390/ijms21249352 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9352

Author(s):

Manh Tien Tran ◽

Yuka Okusha ◽

Yunxia Feng ◽

Masatoshi Morimatsu ◽

Penggong Wei ◽

...

Keyword(s):

Osteoclast Differentiation ◽

Activated T Cells ◽

Surface Receptors ◽

Late Endosomes ◽

Early Endosomes ◽

D Cell ◽

D Cells ◽

Vitro Model

Rab11b, abundantly enriched in endocytic recycling compartments, is required for the establishment of the machinery of vesicle trafficking. Yet, no report has so far characterized the biological function of Rab11b in osteoclastogenesis. Using in vitro model of osteoclasts differentiated from murine macrophages like RAW-D cells or bone marrow-derived macrophages, we elucidated that Rab11b served as an inhibitory regulator of osteoclast differentiation sequentially via (i) abolishing surface abundance of RANK and c-Fms receptors; and (ii) attenuating nuclear factor of activated T-cells c1 (NFATc-1) upstream signaling cascades, following RANKL stimulation. Rab11b was localized in early and late endosomes, Golgi complex, and endoplasmic reticulum; moreover, its overexpression enlarged early and late endosomes. Upon inhibition of lysosomal function by a specific blocker, chloroquine (CLQ), we comprehensively clarified a novel function of lysosomes on mediating proteolytic degradation of c-Fms and RANK surface receptors, drastically ameliorated by Rab11b overexpression in RAW-D cell-derived osteoclasts. These findings highlight the key role of Rab11b as an inhibitor of osteoclastogenesis by directing the transport of c-Fms and RANK surface receptors to lysosomes for degradation via the axis of early endosomes-late endosomes-lysosomes, thereby contributing towards the systemic equilibrium of the bone resorption phase.

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Sorting to Synaptic-like Microvesicles from Early and Late Endosomes Requires Overlapping but Not Identical Targeting Signals

Molecular Biology of the Cell ◽

10.1091/mbc.11.5.1801 ◽

2000 ◽

Vol 11 (5) ◽

pp. 1801-1814 ◽

Cited By ~ 20

Author(s):

Anastasiya D. Blagoveshchenskaya ◽

Daniel F. Cutler

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Pc12 Cells ◽

Protein Complex ◽

Adaptor Protein ◽

Neuroendocrine Cells ◽

Dependent Manner ◽

Late Endosomes ◽

Early Endosomes ◽

Epidermal Growth

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.

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Role of the RAB7 Protein in Tumor Progression and Cisplatin Chemoresistance

Cancers ◽

10.3390/cancers11081096 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1096 ◽

Cited By ~ 7

Author(s):

Guerra ◽

Bucci

Keyword(s):

Cancer Progression ◽

Extracellular Vesicle ◽

Endocytic Pathway ◽

Cellular Processes ◽

Molecular Events ◽

Late Endosomes ◽

Early Endosomes ◽

Cellular Context ◽

Guanosine Triphosphatase

RAB7 is a small guanosine triphosphatase (GTPase) extensively studied as regulator of vesicular trafficking. Indeed, its role is fundamental in several steps of the late endocytic pathway, including endosome maturation, transport from early endosomes to late endosomes and lysosomes, clustering and fusion of late endosomes and lysosomes in the perinuclear region and lysosomal biogenesis. Besides endocytosis, RAB7 is important for a number of other cellular processes among which, autophagy, apoptosis, signaling, and cell migration. Given the importance of RAB7 in these cellular processes, the interest to study the role of RAB7 in cancer progression is widely grown. Here, we describe the current understanding of oncogenic and oncosuppressor functions of RAB7 analyzing cellular context and other environmental factors in which it elicits pro and/or antitumorigenic effects. We also discuss the role of RAB7 in cisplatin resistance associated with its ability to regulate the late endosomal pathway, lysosomal biogenesis and extracellular vesicle secretion. Finally, we examined the potential cancer therapeutic strategies targeting the different molecular events in which RAB7 is involved.

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Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

Viruses ◽

10.3390/v11020126 ◽

2019 ◽

Vol 11 (2) ◽

pp. 126 ◽

Cited By ~ 10

Author(s):

Cheng Li ◽

Hongqing Zheng ◽

Yifan Wang ◽

Wang Dong ◽

Yaru Liu ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Mammalian Cells ◽

Classical Swine Fever ◽

Fever Virus ◽

Late Endosomes ◽

Early Endosomes ◽

Infected Cells ◽

The Relationship ◽

Antiviral Role

The proteins IFITM1, IFITM2, and IFITM3 are host effectors against a broad range of RNA viruses whose roles in classical swine fever virus (CSFV) infection had not yet been reported. We investigated the effect of these proteins on CSFV replication in mammalian cells. The proteins were overexpressed and silenced using lentiviruses. Confocal microscopy was used to determine the distribution of these proteins in the cells, and immunofluorescence colocalization analysis was used to evaluate the relationship between IFITMs and the CSFV endosomal pathway, including early endosomes, late endosomes, and lysosomes. IFITM1, IFITM2, or IFITM3 overexpression significantly inhibited CSFV replication, whereas protein knockdown enhanced CSFV replication. In porcine alveolar macrophages (PAMs), IFITM1 was mainly located at the cell surface, whereas IFITM2 and IFITM3 were mainly located in the cytoplasm. Following CSFV infection, the distribution of IFITM1 changed. IFITM1, IFITM2, and IFITM3 colocalization with Lamp1, IFITM2 with Rab5 and Rab7, and IFITM3 with Rab7 were observed in CSFV-infected cells. Collectively, these results provide insights into the possible mechanisms associated with the anti-CSFV action of the IFITM family.

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Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

The Journal of Cell Biology ◽

10.1083/jcb.202106044 ◽

2021 ◽

Vol 221 (1) ◽

Author(s):

Jan van der Beek ◽

Cecilia de Heus ◽

Nalan Liv ◽

Judith Klumperman

Keyword(s):

Electron Microscopy ◽

Fluorescence Microscopy ◽

Functional Organization ◽

Light And Electron Microscopy ◽

Correlative Microscopy ◽

Late Endosomes ◽

Early Endosomes ◽

Endocytic Vesicles ◽

Endosomal System

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.

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The giant organelles in beige and Chediak-Higashi fibroblasts are derived from late endosomes and mature lysosomes.

Journal of Experimental Medicine ◽

10.1084/jem.178.6.1845 ◽

1993 ◽

Vol 178 (6) ◽

pp. 1845-1856 ◽

Cited By ~ 76

Author(s):

J K Burkhardt ◽

F A Wiebel ◽

S Hester ◽

Y Argon

Keyword(s):

Cell Lines ◽

Secretory Granules ◽

Mutant Cell ◽

Endocytic Pathway ◽

Wild Type ◽

Marker Proteins ◽

Late Endosomes ◽

Early Endosomes ◽

Acidic Organelles ◽

Respective Wild Type

Chediak-Higashi Syndrome (CHS) is an autosomal recessive disease affecting secretory granules and lysosomes-like organelles. In CHS fibroblasts, acidic organelles are abnormally large and clustered in the perinuclear area. We have analyzed fibroblast cell lines from a CHS patient and from the murine model for CHS, the beige mouse, to determine which lysosome-like compartments are affected. Uptake of neutral red showed that in both beige and CHS cell lines, the acidic organelles were markedly clustered in the perinuclear region of the cells. Giant organelles (> 4 microns) were observed in a fraction of the cells, and these were more dramatic in the beige fibroblasts than in the CHS fibroblasts. The total dye uptake of both mutant cell lines was similar to their respective wild type fibroblasts, suggesting that the overall volume of acidic compartments is unaffected by the disorder. Histochemistry and immunofluorescence showed that the giant organelles in both beige and CHS fibroblasts were positive for cathepsin D, lysosome-associated membrane protein (LAMP) 1, LAMP 2, and a 120-kD lysosomal glycoprotein, all marker proteins for late endosomes and lysosomes. The giant organelles were also negative for transferrin receptor and mannose-6-phosphate receptor, and most of them were also negative for rab 7. This distribution of marker proteins shows that the giant organelles in both beige and CHS are derived from late compartments of the endocytic pathway. This conclusion was confirmed using endocytic tracers. BSA was transported to the giant organelles, but only after long incubation times, and only at 37 degrees C. alpha 2-Macroglobulin was taken up and degraded at similar rates by CHS or beige cells and their respective wild type control cells. Taken together, our results indicate that the mutation in CHS specifically affects late endosomes and lysosomes, with little or no effect on early endosomes. Although the mutation clearly causes mislocalization of these organelles, it appears to have little effect on their endocytic and degradative functions.

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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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Bilayered Clathrin Coats on Endosomal Vacuoles Are Involved in Protein Sorting toward Lysosomes

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0525 ◽

2002 ◽

Vol 13 (4) ◽

pp. 1313-1328 ◽

Cited By ~ 237

Author(s):

Martin Sachse ◽

Sylvie Urbé ◽

Viola Oorschot ◽

Ger J. Strous ◽

Judith Klumperman

Keyword(s):

Kinase Inhibitor ◽

Brefeldin A ◽

Phosphatidylinositol 3 Kinase ◽

Recycling Endosomes ◽

Early Endosomes ◽

Epidermal Growth ◽

Quantitative Immunoelectron Microscopy ◽

Endocytic Vesicles ◽

Coated Membrane ◽

Proteasomal Inhibitor

In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron miscroscopy we show that the coat contains clathrin heavy as well as light chain, but lacks the adaptor complexes AP1, AP2, and AP3, by which it differs from clathrin coats on endocytic vesicles and recycling endosomes. The coat is insensitive to short incubations with brefeldin A, but disappears in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin. No association of endosomal coated areas with tracks of tubulin or actin was found. By quantitative immunoelectron microscopy, we found that the lysosomal-targeted receptors for growth hormone (GHR) and epidermal growth factor are concentrated in the coated membrane areas, whereas the recycling transferrin receptor is not. In addition, we found that the proteasomal inhibitor MG 132 induces a redistribution of a truncated GHR (GHR-369) toward recycling vesicles, which coincided with a redistribution of endosomal vacuole-associated GHR-369 to the noncoated areas of the limiting membrane. Together, these data suggest a role for the bilayered clathrin coat on vacuolar endosomes in targeting of proteins to lysosomes.

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Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170

Molecular Biology of the Cell ◽

10.1091/mbc.10.12.4107 ◽

1999 ◽

Vol 10 (12) ◽

pp. 4107-4120 ◽

Cited By ~ 202

Author(s):

Caterina Valetti ◽

Dawn M. Wetzel ◽

Michael Schrader ◽

M. Josh Hasbani ◽

Steven R. Gill ◽

...

Keyword(s):

Cytoplasmic Dynein ◽

Endocytic Pathway ◽

Wild Type ◽

Microtubule Binding ◽

Late Endosomes ◽

Early Endosomes ◽

Endocytic Traffic ◽

Binding Subunit

The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150Glued, the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway.

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