Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells

A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1 microns 2 domain. A signal-to-noise ratio better than 30 was obtained for approximately 30,000 fluorescein-labeled band 3 molecules in a 2 microns 2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues.     

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.     

Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes

After endocytosis, lysosomally targeted ligands pass through a series of endosomal compartments. The endocytic apparatus that accomplishes this passage may be considered to take one of two forms: (a) a system in which lysosomally targeted ligands pass through preexisting, long-lived early sorting endosomes and are then selectively transported to long-lived late endosomes in carrier vesicles, or (b) a system in which lysosomally targeted ligands are delivered to early sorting endosomes which themselves mature into late endosomes. We have previously shown that sorting endosomes in CHO cells fuse with newly formed endocytic vesicles (Dunn, K. W., T. E. McGraw, and F. R. Maxfield. 1989. J. Cell Biol. 109:3303-3314) and that previously endocytosed ligands lose their accessibility to fusion with a half-time of approximately 8 min (Salzman, N. H., and F. R. Maxfield. 1989. J. Cell Biol. 109:2097-2104). Here we have studied the properties of individual endosomes by digital image analysis to distinguish between the two mechanisms for entry of ligands into late endosomes. We incubated TRVb-1 cells (derived from CHO cells) with diO-LDL followed, after a variable chase, by diI-LDL, and measured the diO content of diI-containing endosomes. As the chase period was lengthened, an increasing percentage of the endosomes containing diO-LDL from the initial incubation had no detectable diI-LDL from the second incubation, but those endosomes that contained both probes showed no decrease in the amount of diO-LDL per endosomes. These results indicate that (a) a pulse of fluorescent LDL is retained by individual sorting endosomes, and (b) with time sorting endosomes lose the ability to fuse with primary endocytic vesicles. These data are inconsistent with a preexisting compartment model which predicts that the concentration of ligand in sorting endosomes will decline during a chase interval, but that the ability of the stable sorting endosome to receive newly endocytosed ligands will remain high. These data are consistent with a maturation mechanism in which the sorting endosome retains and accumulates lysosomally directed ligands until it loses its ability to fuse with newly formed endocytic vesicles and matures into a late endosome. We also find that, as expected according to the maturation model, new sorting endosomes are increasingly labeled during the chase period indicating that new sorting endosomes are continuously formed to replace those that have matured into late endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Inhibitory effect of intracellular lipid load on macroautophagy

Degradation of intracellular components via macroautophagy is a complex multi-step process that starts with the sequestration of cytosolic cargo in a de novo formed double-membrane vesicle or autophagosome. This compartment acquires the hydrolases required for cargo digestion by fusion with lysosomes. In contrast to the detailed molecular dissection of the components that participate in the induction, regulation and execution of the early steps in macroautophagy, through the engulfment of cargo in autophagosomes, the mechanisms involved in the lysosomal clearance of autophagosomes have been poorly characterized in mammals. One of the major limitations in this respect has been the fact that autophagosome-lysosome fusion in intact cells involves several independent steps, namely binding of the molecular motors associated to the surface of the vesicles with the cytoskeletal network, directional vesicular trafficking and fusion between the two vesicular compartments. Furthermore, both lysosomes and autophagosomes are very dynamic organelles that can fuse with different vesicular structures involved in macroautophagy, but also along the endocytic and phagocytic pathways. To resolve these limitations and directly analyze the fusion step between autophagosomes and different compartments of the endocytic-lysosomal pathway, we have recently developed an in vitro fusion assay with autophagosomes, lysosomes and endosomes isolated from cells or tissues. Fluorescent labeling of these compartments allows for the tracking of fusion events by fluorescence microscopy or by fluorescence activated cell sorting (FACS). Labeling of either membrane proteins on the surface of the organelles or dye-loading of the vesicles permits the monitoring of hemi-membrane fusion and complete vesicular fusion (cargo mixing).     

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 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees 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.     

APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway.     

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.     

Ferromagnetic isolation of endosomes: a novel method for subcellular fractionation of Xenopus oocytes

A novel method has been developed using ferric particles to label endosomes, and to achieve magnetic sorting of the various endocytic compartments involved in lipoprotein uptake into cells. Ferric particles conjugated to a receptor-recognized ligand are bound to coated membrane pits and become internalized into the cytoplasm inside coated vesicles. After apparent fusion of the vesicles to tubular endosomes, the conjugates accumulate and finally discharge into multivesicular endosomes. Pulse-chase experiments elucidate the pathway of internalized conjugates and allow both early compartments (pinosomes and tubular endosomes) and late compartments (multivesicular endosomes and storage organelles) to be selectively labelled. After ferroloading of the various transport compartments, the cells are homogenized and subcellularly fractionated. Sorting of labelled endosomes is performed by a specially designed "free-flow" magnetic chamber. Prophase I-arrested oocytes of the toad Xenopus laevis are used as a model system for studying the transport pathway and the conversion of the yolk precursor vitellogenin. It is possible to follow the route of internalization of vitellogenin-iron conjugates via coated pits, coated vesicles, uncoated vesicles, tubular endosomes, multivesicular endosomes, and light primordial yolk platelets. These endosomes shuttle the ferric particles together with the vitellogenin from oolemma to performed heavy yolk organelles which are still growing. In addition, these various compartments can be isolated according to their function and subjected to electron microscopy and to gel electrophoresis for detailed characterization of their limiting membranes as well as their contents.     

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.     

A myosin I is involved in membrane recycling from early endosomes

Geometry-based mechanisms have been proposed to account for the sorting of membranes and fluid phase in the endocytic pathway, yet little is known about the involvement of the actin-myosin cytoskeleton. Here, we demonstrate that Dictyostelium discoideum myosin IB functions in the recycling of plasma membrane components from endosomes back to the cell surface. Cells lacking MyoB (myoA(-)/B(-), and myoB(-) cells) and wild-type cells treated with the myosin inhibitor butanedione monoxime accumulated a plasma membrane marker and biotinylated surface proteins on intracellular endocytic vacuoles. An assay based on reversible biotinylation of plasma membrane proteins demonstrated that recycling of membrane components is severely impaired in myoA/B null cells. In addition, MyoB was specifically found on magnetically purified early pinosomes. Using a rapid-freezing cryoelectron microscopy method, we observed an increased number of small vesicles tethered to relatively early endocytic vacuoles in myoA(-)/B(-) cells, but not to later endosomes and lysosomes. This accumulation of vesicles suggests that the defects in membrane recycling result from a disordered morphology of the sorting compartment.     

Myo6 facilitates the translocation of endocytic vesicles from cell peripheries

Immunolocalization studies in epithelial cells revealed myo6 was associated with peripherally located vesicles that contained the transferrin receptor. Pulse-chase experiments after transferrin uptake showed that these vesicles were newly uncoated endocytic vesicles and that myo6 was recruited to these vesicles immediately after uncoating. GIPC, a putative myo6 tail binding protein, was also present. Myo6 was not present on early endosomes, suggesting that myo6 has a transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome.     

Receptor-mediated endocytosis and cytoskeleton

Receptor-mediated endocytosis is the most specific pathway for macromolecules and macromolecular complexes generally designated as ligands to enter cells. Upon binding to their transmembrane receptors, the ligands enter endocytic vesicles that fuse with each other giving rise to the so-called early endosomes. The sorting of ligand-receptor complexes internalized in these endosomes depends on their nature: metabolic receptors are recycled back to the plasma membrane, while signaling receptors and their ligands (e.g. receptor tyrosine kinases or receptors associated with tyrosine kinase) are delivered to internal vesicles of the multivesicular late endosomes and finally are degraded after interaction with lysosomes. During these processes, endosomes undergo translocation from the cell periphery to the juxtanuclear region, which is accompanied by multiple fusion, invagination, tabulation, and membrane fission events. This review considers modern concepts of the sorting mechanisms of ligand-receptor complexes, the crosstalk between endosomes, microtubules, and actin, and the role of this crosstalk in endosome maturation.     

Exocytosis of late endosomes does not directly contribute membrane to the formation of phagocytic cups or pseudopods in Dictyostelium

Exocytosis of late endocytic compartments in Dictyostelium has mostly been studied by live microscopy. Here we show that this exocytosis is accompanied by a complete fusion of late endosomes with the plasma membrane resulting in the transient formation of membrane microdomains that can be visualized by immunofluorescence in fixed cells. This permitted to demonstrate that fusion of late endocytic compartments with the cell surface does not occur in regions of the plasma membrane engaged in the formation of pseudopods, macropinosomes or phagosomes. Our results propose that exocytosis of late endosomes and actin-driven membrane remodeling are mutually exclusive processes.     

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.     

Acidification of morphologically distinct endosomes in mutant and wild- type Chinese hamster ovary cells

In the preceding paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2713-2721), we have shown that there is rapid acidification of endosomal compartments to pH 6.3 by 3 min in wild-type Chinese hamster ovary (CHO) cells. In contrast, early acidification of endosomes is markedly reduced in the CHO mutants, DTF 1-5-4 and DTF 1-5- 1. Since these CHO mutants are pleiotropically defective in endocytosis (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308), our results are consistent with a requirement for proper acidification of early endocytic compartments in many pH-regulated endocytic processes. In this paper, by measuring the pH of morphologically distinct endosomes using fluorescence microscopy and digital image analysis, we have determined in which of the endocytic compartments the defective acidification occurs. We found that the acidification of both the para- Golgi recycling endosomes and lysosomes was normal in the CHO mutants DTG 1-5-4 and DTF 1-5-1. The mean pH of large endosomes containing either fluorescein-labeled alpha 2-macroglobulin or fluorescein- isothiocyanate dextran was only slightly less acidic in the mutant cells than in wild-type cells. However, when we examined the pH of individual large (150-250 nm) endosomes, we found that there was an increased number of endosomes with a pH greater than 6.5 in the CHO mutants when compared with wild-type cells. Heterogeneity in the acidification of large endosomes was also seen in DTF 1-5-1 by a combined null point pH method and digital image analysis technique. In addition, both CHO mutants showed a marked decrease in the acidification of the earliest endosomal compartment, a diffusely fluorescent compartment comprised of small vesicles and tubules. We suggest that the defect in endosome acidification is most pronounced in the early, small vesicular, and tubular endosomes and that this defect partially carries over to the large endosomes that are involved in the sorting and processing of ligands. The proper step-wise acidification of the different endosomes along the endocytic pathway may have an important role in the regulation of endocytic processes.     

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.     

Internalization of the TGF-β type I receptor into caveolin-1 and EEA1 double-positive early endosomes

Endocytosis and intracellular sorting of transforming growth factor-β (TGF-β) receptors play an important regulatory role in TGF-β signaling. Two major endocytic pathways, clathrin- and caveolae-mediated endocytosis, have been reported to independently mediate the internalization of TGF-β receptors. In this study, we demonstrate that the clathrin- and caveolae-mediated endocytic pathways can converge during TGF-β receptor endocytic trafficking. By tracking the intracellular dynamics of fluorescently-labeled TGF-β type I receptor (TβRI), we found that after mediating TβRI internalization, certain clathrin-coated vesicles and caveolar vesicles are fused underneath the plasma membrane, forming a novel type of caveolin-1 and clathrin double-positive vesicles. Under the regulation of Rab5, the fused vesicles are targeted to early endosomes and thus deliver the internalized TβRI to the caveolin-1 and EEA1 double-positive early endosomes (caveolin-1-positive early endosomes). We further showed that the caveolin-1-positive early endosomes are positive for Smad3/SARA, Rab11 and Smad7/Smurf2, and may act as a multifunctional device for TGF-β signaling and TGF-β receptor recycling and degradation. Therefore, these findings uncover a novel scenario of endocytosis, the direct fusion of clathrin-coated and caveolae vesicles during TGF-β receptor endocytic trafficking, which leads to the formation of the multifunctional sorting device, caveolin-1-positive early endosomes, for TGF-β receptors.     

Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway

To study the intracellular sorting of internalized ligands and receptors, we examined the pathways of two ligands: transferrin, which is recycled, and alpha 2-macroglobulin (alpha 2M), which is degraded. In CHO cells the two ligands rapidly segregate into different intracellular compartments. Within 5 min fluorescein-labeled transferrin (F-Tf) is found in a large round juxtanuclear structure. Rhodamine-labeled alpha 2M is found in a punctate pattern. Ultra-structural localization studies demonstrate that colloidal gold-alpha 2M is found predominantly in endocytic vesicles, while ferritin-transferrin is found in small vesicles and tubular structures in a region adjacent to the Golgi complex. Using image intensified fluorescence microscopy and digital image analysis, we determined that the F-Tf containing structure has a pH of 6.4 +/- 0.2, while endocytic vesicles containing F-alpha 2M have a pH of 5.4 +/- 0.1. Our study defines a mildly acidic compartment, distinct from endocytic vesicles, that is involved in the recycling of internalized components back to the cell surface.     

Fluid phase endocytosis investigated by fluorescence with trimethylamino-diphenylhexatriene in L929 cells; the influence of temperature and of cytoskeleton depolymerizing drugs.

The temperature-dependence of fluid phase endocytosis was investigated in L929 cells, using a recently described fluorescence approach with trimethylamino-diphenylhexatriene (TMA-DPH). In interaction with cells, this probe is rapidly incorporated into the plasma membrane and follows its intracellular traffic of internalization-recycling, thus behaving as a suitable marker for fluid phase endocytosis. The kinetics of the process may be followed accurately by simple fluorescence intensity measurements, while complementary fluorescence anisotropy and micrographic data may be obtained in parallel with the same probe. It was shown that the formation of endocytic vesicles was not inhibited by cooling the cells, even down to 4 degrees C, but only reduced in a quasi-linear way with temperature. Conversely the further fusion events between the vesicles and large vacuolar bodies (endosomes, lysosomes) were strongly and discontinuously influenced: they were almost totally suppressed below 15 degrees C. The evolution of the membrane fluidity during endocytosis, which was monitored by fluorescence anisotropy measurements, indicated that the fusion inhibition was probably correlated with the inability of the endocytic vesicles to shed their initial clathrin coat at low temperature. Moreover, microscopic observations showed that at low temperature the endocytic vesicles hardly moved from the place of their formation. Pretreatment of the cells with microtubule and microfilament depolymerizing drugs (cytochalasin B, vinblastine) led to the conclusion that the cytoskeleton played little role in the vesicle movements. Altogether, the results suggested that the progression of the vesicles towards the cell core resulted from successive fusion events, which explained why they were considerably slowed down by cooling.