Trafficking of prion proteins through a caveolae-mediated endosomal pathway

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A–gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

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Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

Molecular Biology of the Cell ◽

10.1091/mbc.8.1.13 ◽

1997 ◽

Vol 8 (1) ◽

pp. 13-31 ◽

Cited By ~ 76

Author(s):

L Hicke ◽

B Zanolari ◽

M Pypaert ◽

J Rohrer ◽

H Riezman

Keyword(s):

Fusion Protein ◽

Secretory Pathway ◽

Protein A ◽

Brefeldin A ◽

Early Endosome ◽

Late Endosome ◽

Endocytic Pathway ◽

Early Secretory Pathway ◽

Late Endosomes

Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.

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Nav1.5 sodium channels in macrophages in multiple sclerosis lesions

Multiple Sclerosis Journal ◽

10.1177/1352458512460417 ◽

2012 ◽

Vol 19 (5) ◽

pp. 532-542 ◽

Cited By ~ 20

Author(s):

Joel A Black ◽

Jia Newcombe ◽

Stephen G Waxman

Keyword(s):

Multiple Sclerosis ◽

Protein A ◽

Proteolipid Protein ◽

Endocytic Pathway ◽

Late Endosomes ◽

Myelin Degradation ◽

Active Multiple Sclerosis

Background: Macrophages are dynamic participants in destruction of white matter in active multiple sclerosis (MS) plaques. Regulation of phagocytosis and myelin degradation along endosomal pathways in macrophages is highly-orchestrated and critically-dependent upon acidification of endosomal lumena. Evidence from in vitro studies with macrophages and THP-1 cells suggests that sodium channel Nav1.5 is present in the limiting membrane of maturing endosomes where it plays a prominent role in the accumulation of protons. However, a contribution of the Nav1.5 channel to macrophage-mediated events in vivo has not been demonstrated. Method: We examined macrophages within active MS lesions by immunohistochemistry to determine whether Nav1.5 is expressed in these cells in situ and, if expressed, whether it is localized to specific compartments along the endocytic pathway. Results: Our results demonstrate that Nav1.5 is expressed within macrophages in active MS lesions, and that it is preferentially expressed in late endosomes and phagolysosomes (Rab7+, LAMP-1+), and sparsely expressed in early (EEA-1+) endosomes. Triple-immunolabeling studies showed localization of Nav1.5 within Rab7+ endosomes containing proteolipid protein, a myelin marker, in macrophages within active MS plaques. Conclusions: These observations support the suggestion that Nav1.5 contributes to the phagocytic pathway of myelin degradation in macrophages in vivo within MS lesions.

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Identification of a new, Rab14-dependent, endo-lysosomal pathway

10.1101/2021.03.25.436964 ◽

2021 ◽

Author(s):

Evgeniya Trofimenko ◽

Yuta Homma ◽

Mitsunori Fukuda ◽

Christian Widmann

Keyword(s):

Common Ancestor ◽

Cell Penetrating Peptides ◽

Small Gtpase ◽

Endocytic Pathway ◽

Last Eukaryotic Common Ancestor ◽

Surrounding Environment ◽

Late Endosomes ◽

Early Endosomes ◽

Endosomal Pathway ◽

Dependent Pathway

Cells can endocytose material from the surrounding environment. Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Several endocytosis pathways have been described (e.g. clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway). Besides possible recycling routes to the plasma membrane and various organelles, these pathways all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs) used in research and clinic, we have discovered a second endocytic pathway that moves material to late endosomes/lysosomes and that is fully independent of Rab5 and Rab7 but requires the Rab14 protein. This newly identified pathway differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already and is not affected by a series of compounds that inhibit the Rab5-dependent pathway. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins. Rab14 is expressed by the last eukaryotic common ancestor. The Rab14-dependent pathway may therefore correspond to a primordial endosomal pathway taken by cationic cargos.

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Rapid Quantification of Prion Proteins

10.26434/chemrxiv.11299097 ◽

2019 ◽

Author(s):

Matthew Healey ◽

Muttuswamy Sivakumaran ◽

Mark Platt

Keyword(s):

Prion Proteins ◽

Prion Diseases ◽

Sample Volume ◽

Cellular Prion Protein ◽

Dna Aptamer ◽

Superparamagnetic Beads ◽

Resistive Pulse ◽

Large Sample Volume ◽

Complex Sample ◽

Neurological Conditions

Prion diseases are a group of fatal transmissible neurological conditions caused by the change in conformation of the normal intrinsic cellular prion protein (PrPC) in to the highly ordered insoluble amyloid state conformer (PrPSC). We present a rapid assay using Aptamers and Resistive Pulse Sensing, RPS, to extract and quantify proteins from complex sample matrices, demonstrate with the quantification of PrPc. We functionalise the surface of superparamagnetic beads, SPBs, with a DNA aptamer. First SPB’s termed P-Beads, are used to pre-concentrate the analyte from a large sample volume. The PrPc protein is then eluted from the P-Beads before aptamer modified sensing beads, S-Beads, are added. The velocity of the S-Beads through the nanopore reveals the concentration of the PrPc protein. The process is done in under an hour and allows the detection of picomol’s of protein. The technique could be easily adopted to the mutated version of the protein and integrated into clinical workflows for the screening of blood donations and transfusions.

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Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol

Acta Neuropathologica ◽

10.1007/s00401-020-02254-3 ◽

2021 ◽

Author(s):

Juan Carlos Polanco ◽

Gabriel Rhys Hand ◽

Adam Briner ◽

Chuanzhou Li ◽

Jürgen Götz

Keyword(s):

Critical Role ◽

Membrane Integrity ◽

Lysosomal Degradation ◽

Escape Route ◽

Cellular Invasion ◽

Endosomal Membrane ◽

Late Endosomes ◽

Endosomal Membranes ◽

Endosomal Pathway ◽

Tau Aggregation

AbstractThe microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

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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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Trafficking of interleukin 2 and transferrin in endosomal fractions of T lymphocytes

Journal of Cell Science ◽

10.1242/jcs.107.5.1289 ◽

1994 ◽

Vol 107 (5) ◽

pp. 1289-1295 ◽

Cited By ~ 1

Author(s):

V. Duprez ◽

M. Smoljanovic ◽

M. Lieb ◽

A. Dautry-Varsat

Keyword(s):

Horseradish Peroxidase ◽

Interleukin 2 ◽

Fluid Phase ◽

Endocytic Pathway ◽

Recycling Endosomes ◽

High Affinity ◽

Human T Cell ◽

Late Endosomes ◽

Discontinuous Sucrose Gradient ◽

Acidic Organelles

The T lymphocyte growth factor interleukin 2 binds to surface high-affinity receptors and is rapidly internalized and degraded in acidic organelles. The alpha and beta chains of high-affinity interleukin 2 receptors are internalized together with interleukin 2. To identify the intracellular pathway followed by interleukin 2, we have compared the subcellular distribution of interleukin 2, transferrin and a fluid-phase marker, horseradish peroxidase, in the human T cell line IARC 301.5. Transferrin was used as a marker of early and recycling endosomes, and horseradish peroxidase to probe for the whole endocytic pathway. Fractionation of intracellular organelles on a discontinuous sucrose gradient showed that internalized interleukin 2 is initially mostly found in compartments with similar densities to transferrin, e.g. early and recycling endosomes. The kinetics of entry and exit of interleukin 2 from such organelles was much slower than that of transferrin. Later on, interleukin 2 is predominantly found in dense lysosome-containing fractions. Very little, if any, interleukin 2 was found in fractions corresponding to late endosomes containing horseradish peroxidase. These results suggest that, after endocytosis, interleukin 2 enters early or recycling endosomes before it reaches dense lysosomes.

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Rab22a affects the morphology and function of the endocytic pathway

Journal of Cell Science ◽

10.1242/jcs.114.22.4041 ◽

2001 ◽

Vol 114 (22) ◽

pp. 4041-4049 ◽

Cited By ~ 2

Author(s):

Rosana Mesa ◽

Cristina Salomón ◽

Marcelo Roggero ◽

Philip D. Stahl ◽

Luis S. Mayorga

Keyword(s):

Fluorescent Protein ◽

Fluid Phase ◽

Small Gtpases ◽

Endocytic Pathway ◽

Site Directed Mutagenesis ◽

Rab Proteins ◽

Rab Protein ◽

Late Endosomes ◽

And Function ◽

Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

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Subcellular location of serum- and glucocorticoid-induced kinase-1 in renal and mammary epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00399.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. C1971-C1981 ◽

Cited By ~ 13

Author(s):

Emily Cordas ◽

Anikó Náray-Fejes-Tóth ◽

Géza Fejes-Tóth

Keyword(s):

Subcellular Localization ◽

Mammary Epithelial Cells ◽

Collecting Duct ◽

Subcellular Location ◽

Mammary Epithelial ◽

Live Cells ◽

Epithelial Cell Line ◽

Cortical Collecting Duct ◽

Mitochondrial Marker ◽

Amino Acid Region

Serum- and glucocorticoid-induced kinase-1 (SGK1) is involved in aldosterone-induced Na+ reabsorption by increasing epithelial Na+ channel (ENaC) activity in cortical collecting duct (CCD) cells, but its exact mechanisms of action are unknown. Although several potential targets such as Nedd4-2 have been described in expression systems, endogenous substrates mediating SGK1's physiological effects remain to be identified. In addition, subcellular localization studies of SGK1 have provided controversial results. We determined the subcellular location of SGK1 using SGK1-autofluorescent protein (AFP) fusion proteins. Rabbit CCD (RCCT-28A) cells were transiently transfected with a construct encoding for SGK1-AFP and were stained or cotransfected with markers for various subcellular compartments. In live cells, transiently expressed SGK1-AFP clearly colocalized with the mitochondrial marker rhodamine 123. Similarly, SGK1-AFP colocalized with the mitochondrial marker MitoTracker when stably expressed using a retroviral system in either RCCT-28A cells or the mammary epithelial cell line MCF10A. To determine which region of SGK1 is responsible for this subcellular localization, we generated RCCT-28A cell lines stably expressing SGK1 mutants. The results indicate that the NH2-terminal 60-amino acid region of SGK1 is necessary and sufficient for its subcellular localization. Localization of SGK1 to the mitochondria raises the possibility that SGK1 may play a role in regulating energy metabolism.

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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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