Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments: evidence for early endosome-to-TGN trafficking of MPR300

It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans-Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes.     

Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system

The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. In conclusion: (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles.     

Recycling of apoprotein E is associated with cholesterol efflux and high density lipoprotein internalization

After receptor-mediated endocytosis of triglyceride-rich lipoproteins (TRL) into the liver, TRL particles are immediately disintegrated in peripheral endosomal compartments. Whereas core lipids and apoprotein B are delivered for degradation into lysosomes, TRL-derived apoE is efficiently recycled back to the plasma membrane. This is followed by apoE re-secretion and association of apoE with high density lipoproteins (HDL). Because HDL and apoE can independently promote cholesterol efflux, we investigated whether recycling of TRL-derived apoE in human hepatoma cells and fibroblasts could be linked to intracellular cholesterol transport. In this study we demonstrate that HDL(3) does not only act as an extracellular acceptor for recycled apoE but also stimulates the recycling of internalized TRL-derived apoE. Furthermore, radioactive pulse-chase experiments indicate that apoE recycling is accompanied by cholesterol efflux. Confocal imaging reveals co-localization of apoE and cholesterol in early endosome antigen 1-positive endosomes. During apoE re-secretion, HDL(3)-derived apoA-I is found in these early endosome antigen 1, cholesterol-containing endosomes. As shown by time-lapse fluorescence microscopy, apoE recycling involves the intracellular trafficking of apoA-I to pre-existing and TRL-derived apoE/cholesterol-containing endosomes in the periphery. Thus, these studies provide evidence for a new intracellular link between TRL-derived apoE, cellular cholesterol transport, and HDL metabolism.     

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins

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.     

Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells

In mammalian cells, macromolecules internalized by endocytosis are transported via endosomes for digestion by lysosomal acid hydrolases . The mechanism by which endosomes and lysosomes exchange content remains equivocal . However, lysosomes are reusable organelles because they remain accessible to endocytic enzyme replacement therapies and undergo content mixing with late endosomes . The maturation model, which proposes that endosomes mature into lysosomes , cannot explain these observations. Three mechanisms for content mixing have been proposed. The first is vesicular transport, best supported by a yeast cell-free assay . The second suggests that endosomes and lysosomes engage in repeated transient fusions termed "kiss-and-run" . The third is that endosomes and lysosomes fuse completely, yielding hybrid compartments from which lysosomes reform , termed "fusion-fission" . We utilized time-lapse confocal microscopy to test these hypotheses in living cells. Lysosomes were loaded with rhodamine dextran by pulse-chase, and subsequently late endosomes were loaded with Oregon green 488 dextran. Direct fusions were observed between endosomes and lysosomes, and one such event was captured by correlative electron microscopy. Fluorescence intensity analyses of endosomes that encountered lysosomes revealed a gradual accumulation of lysosomal content. Our data are compatible with a requirement for direct contact between organelles before content is exchanged.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1(-/-) fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1(-/-) fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2(-/-) cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1(-/-) and NPC2(-/-) fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1(-/-) fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2(-/-) fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1(-/-) cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from receptor-mediated endocytosis. We conclude that the spectral properties of C5-DMB-SM can be used to distinguish unique populations of early endosomes from one another and to record dynamic changes in their number and distribution within living cells.     

Occurrence of an anomalous endocytic compartment in fibroblasts from Sandhoff disease patients

Sandhoff disease (SD) is a lysosomal storage disorder due to mutations in the gene encoding for the β-subunit of β-hexosaminidase, that result in β-hexosaminidase A (αβ) and β-hexosaminidase B (ββ) deficiency. This leads to the storage of GM2 ganglioside in endosomes and lysosomes, which ends in a progressive neurodegeneration. Currently, very little is known about the biochemical pathways leading from GM2 ganglioside accumulation to pathogenesis. Defects in transport and sorting by the endosomal–lysosomal system have been described for several lysosomal storage disorders. Here, we have investigated the endosomal–lysosomal compartment in fibroblasts from SD patients and observed that both late endosomes and lysosomes, but not early endosomes, have a higher density in comparison with normal fibroblasts. Moreover, Sandhoff fibroblasts have an intracellular distribution of terminal endocytic organelles that differs from the characteristic perinuclear punctate pattern observed in normal fibroblasts and endocytic vesicles also appear larger. These findings reveal the occurrence of an alteration in the terminal endocytic organelles of Sandhoff fibroblasts, suggesting an involvement of this compartment in the disruption of cell metabolic and signalling pathways and in the onset of the pathological state.     

Accumulation of sialic acid in endocytic compartments interferes with the formation of mature lysosomes. Impaired proteolytic processing of cathepsin B in fibroblasts of patients with lysosomal sialic acid storage disease.

The impact of an altered endocytic environment on the biogenesis of lysosomes was studied in fibroblasts of patients suffering from sialic acid storage disease (SASD). This inherited disorder is characterized by the accumulation of acidic monosaccharides in lysosomal compartments and a concomitant decrease of their buoyant density. We demonstrate that C-terminal trimming of the lysosomal cysteine proteinase cathepsin B is inhibited in SASD fibroblasts. This late event in the biosynthesis of cathepsin B normally takes place in mature lysosomes, suggesting an impaired biogenesis of these organelles in SASD cells. When normal fibroblasts are loaded with sucrose, which inhibits transport from late endosomes to lysosomes, C-terminal cathepsin B processing is prevented to the same extent. Further characterization of the terminal endocytic compartments of SASD cells revealed properties usually associated with late endosomes/prelysosomes. In addition to a decreased buoyant density, SASD "lysosomes" show a reduced acidification capacity and appear smaller than their normal counterparts. We conclude that the accumulation of small non-diffusible compounds within endocytic compartments interferes with the formation of mature lysosomes and that the acidic environment of the latter organelles is a prerequisite for C-terminal processing of lysosomal hydrolases.     

The Niemann-Pick C1 gene is downregulated by feedback inhibition of the SREBP pathway in human fibroblasts

The Niemann-Pick C1 (NPC1) protein regulates the transport of cholesterol from late endosomes/lysosomes to other compartments responsible for maintaining intracellular cholesterol homeostasis. The present study examined the expression of the NPC1 gene and the distribution of the NPC1 protein that resulted from the transport of LDL-derived cholesterol through normal human fibroblasts. A key finding was that the transport of cholesterol from late endosomes/lysosomes to the sterol-regulatory pool at the endoplasmic reticulum, as determined by feedback inhibition of the sterol-regulatory element binding protein (SREBP) pathway, was associated with the downregulation of the NPC1 gene. Consistent with these results, fibroblasts incubated with LDL had decreased amounts of SREBP protein that interacted with sterol-regulatory element (SRE) sequences positioned within the NPC1 gene promoter region. Finally, partial colocalization of the NPC1 protein with late endosomes/lysosomes and distinct regions of the endoplasmic reticulum suggested that the NPC1 protein may facilitate the transport of cholesterol directly between these two compartments. Together, these results indicate that the transport of LDL-derived cholesterol from late endosomes/lysosomes to the sterol-regulatory pool, known to be regulated by the NPC1 protein, is responsible for promoting feedback inhibition of the SREBP pathway and downregulation of the NPC1 gene.     

Reconstitution of lysosomal NAADP-TRP-ML1 signaling pathway and its function in TRP-ML1(-/-) cells

It is well known that the mutation of TRP-ML1 (transient receptor potential-mucolipin-1) causes mucolipidosis IV, a lysosomal storage disease. Given that lysosomal nicotinic acid adenine dinucleotide phosphate (NAADP)-Ca(2+) release channel activity is associated with TRP-ML1, the present study was designed to test the hypothesis that NAADP regulates lysosome function via activation of TRP-ML1 channel activity. Using lysosomal preparations from wild-type (TRP-ML1(+/+)) human fibroblasts, channel reconstitution experiments demonstrated that NAADP (0.01-1.0 μM) produced a concentration-dependent increase in TRP-ML1 channel activity. This NAADP-induced activation of TRP-ML1 channels could not be observed in lysosomes from TRP-ML1(-/-) cells, but was restored by introducing a TRP-ML1 transgene into these cells. Microscopic Ca(2+) fluorescence imaging showed that NAADP significantly increased intracellular Ca(2+) concentration to 302.4 ± 74.28 nM (vs. 180 ± 44.13 nM of the basal) in TRP-ML1(+/+) cells, but it had no effect in TRP-ML1(-/-) cells. If a TRP-ML1 gene was transfected into TRP-ML1(-/-) cells, the Ca(2+) response to NAADP was restored to the level comparable to TRP-ML1(+/+) cells. Functionally, confocal microscopy revealed that NAADP significantly enhanced the dynamic interaction of endosomes and lysosomes and the lipid delivery to lysosomes in TRP-ML1(+/+) cells. This functional action of NAADP was abolished in TRP-ML1(-/-) cells, but restored after TRP-ML1 gene was rescued in these cells. Our results suggest that NAADP increases lysosomal TRP-ML1 channel activity to release Ca(2+), which promotes the interaction of endosomes and lysosomes and thereby regulates lipid transport to lysosomes. Failure of NAADP-TRP-ML1 signaling may be one of the important mechanisms resulting in intracellular lipid trafficking disorder and consequent mucolipidosis.     

Gangliosides are Transported from the Plasma Membrane to Intralysosomal Membranes as Revealed by Immuno-Electron Microscopy

A biotin-labeled derivative of the ganglioside GM1 (biotin-GM1) was used to study its transport along the endocytic pathway of cultured fibroblasts by immuno-electron microscopy. Using electron dense endocytic tracers we could demonstrate that late endosomes and lysosomes of these cells are long living organelles with a high content of internal membranes. Our studies show that during endocytosis the biotin-GM1 was transported to these intraendosomal and intralysosomal membranes. These observations support the hypothesis that glycosphingolipids (GSL) are preferentially degraded in intralysosomal vesicles.     

pH-induced microtubule-dependent redistribution of late endosomes in neuronal and epithelial cells

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to approximately 6.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell.     

The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.     

Intra-endosomal membrane traffic

Following endocytosis, ubiquitinated signaling receptors are incorporated within intraluminal vesicles of forming multivesicular endosomes. These vesicles then follow the pathway from early to late endosomes, remaining within the endosomal lumen, and are eventually delivered to lysosomes, where they are degraded together with their protein cargo. However, intraluminal vesicles do not always end up in lysosomes for degradation; they can also fuse back with the limiting membrane of late endosomes. This route, which might be regulated by lyso-bisphosphatidic acid and its putative effector Alix, can be hijacked by the anthrax toxin and vesicular stomatitis virus and is presumably exploited by proteins and lipids that transit through intraluminal vesicles. Alternatively, these vesicles can be released extracellularly, like HIV in macrophages, upon fusion of endosomes or lysosomes with the plasma membrane.     

Membrane traffic to and from lysosomes

In the late endocytic pathway, it has been proposed that endocytosed macromolecules are delivered to a proteolytic environment by 'kiss-and-run' events or direct fusion between late endosomes and lysosomes. To test whether the fusion hypothesis accounts for delivery to lysosomes in living cells, we have used confocal microscopy to examine content mixing between lysosomes loaded with rhodamine-dextran and endosomes subsequently loaded with Oregon-Green-dextran. Both kissing and explosive fusion events were recorded. Data from cell-free content-mixing assays have suggested that fusion is initiated by tethering, which leads to formation of a trans-SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein complex and then release of lumenal Ca(2+), followed by membrane bilayer fusion. We have shown that the R-SNARE (arginine-containing SNARE) protein VAMP (vesicle-associated membrane protein) 7 is necessary for heterotypic fusion between late endosomes and lysosomes, whereas a different R-SNARE, VAMP 8 is required for homotypic fusion of late endosomes. After fusion of lysosomes with late endosomes, lysosomes are re-formed from the resultant hybrid organelles, a process requiring condensation of content and the removal/recycling of some membrane proteins.     

Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents

In this study, we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular glands were chosen as model organs because they can be exposed easily, imaged without compromising their function and, furthermore, they are amenable to pharmacological and genetic manipulations. We show that the fibroblasts within the stroma of the glands readily internalize systemically injected molecules such as fluorescently conjugated dextran and BSA, providing a robust model to study endocytosis. We dynamically image the trafficking of these probes from the early endosomes to the late endosomes and lysosomes while also visualizing homotypic fusion events between early endosomes. Finally, we demonstrate that pharmacological agents can be delivered specifically to the submandibular salivary glands, thus providing a powerful tool to study the molecular machinery regulating endocytosis in a physiological context.     

Molecular mechanisms of late endosome morphology, identity and sorting

Recent studies using electron microscopy, protein crystallography, classic biochemistry and novel live-cell imaging have provided numerous insights into the endocytic pathway, describing a dynamic system in which compartment morphology, molecular identity and the mechanics of cargo sorting are intimately connected. Current evidence supports a model of maturation in which the lipids, cargo proteins and Rab population at the endosome determine its competence to perform the functions of late endosomes, including the sorting of cargoes into lumenal vesicles and fusion with lysosomes.     

A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments

Rab5 is a small GTPase that plays roles in the homotypic fusion of early endosomes and regulation of intracellular vesicle transport. We show here that expression of GFP-tagged GTPase-deficient form of Rab5b (Rab5bQ79L) in NRK cells results in the sequential formation of three morphologically and functionally distinct types of endosomes. Expression of GFP-Rab5bQ79L initially caused a homotypic fusion of early endosomes accompanying a redistribution of the TGN-resident cargo molecules, and subsequent fusion with late endosomes/lysosomes, leading to the formation of giant hybrid organelles with features of early endosomes and late endosomes/lysosomes. Surprisingly, the giant endosomes gradually fragmented and shrunk, leading to the accumulation of early endosome clusters and concurrent reformation of late endosomes/lysosomes, a process accelerated by treatment with a phosphatidylinositol-3-kinase (PI(3)K) inhibitor, wortmannin. We postulate that such sequential processes reflect the biogenesis and maintenance of late endosomes/lysosomes, presumably via direct fusion with early endosomes and subsequent fission from hybrid organelles. Thus, our findings suggest a regulatory role for Rab5 in not only the early endocytic pathway, but also the late endocytic pathway, of membrane trafficking in coordination with PI(3)K activity.     

Endosomal ricin transport: involvement of Rab4- and Rab5-positive compartments

Transport of the ribosome-inactivating protein ricin through endosomes was studied in A431 cells expressing Rab5-, Rab4-, and Rab11-GFP. It was shown that Rab5- and Rab4-positive functional domains of early endosomes are involved in ricin transport. Ricin enters cells by both clathrin-dependent and clathrin-independent mechanisms. The main pool of internalized toxin accumulates in early endosomes and remains associated with them for a long time. In contrast to earlier observations, current observations indicate that the majority of ricin avoids transport to lysosomes. The low level of ricin association with Rab11 as well as with transferrin accumulated in the pericentriolar recycling compartment shows that the compartment is not responsible for keeping ricin away from degradation in lysosomes. Escape from degradation in lysosomes is assumed to result from the potentiality of ricin to form assemblies within compartments.