Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway.

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered lysosome-associated membrane protein (LAMP)-carrier vesicles around multivesicular bodies, as well as the appearance of 'hourglass' profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of charged multi-vesicular body protein 6 (CHMP6), and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

Juvenile mucopolysaccharidosis plus disease caused by a missense mutation in VPS33A.

A rare and fatal disease resembling mucopolysaccharidosis in infants, is caused by impaired intracellular endocytic trafficking due to deficiency of core components of the intracellular membrane-tethering protein complexes, HOPS, and CORVET. Whole exome sequencing identified a novel VPS33A mutation in a patient suffering from a variant form of mucopolysaccharidosis. Electron and confocal microscopy, immunoblotting, and glycosphingolipid trafficking experiments were undertaken to investigate the effects of the mutant VPS33A in patient-derived skin fibroblasts. We describe an attenuated juvenile form of VPS33A-related syndrome-mucopolysaccharidosis plus in a man who is homozygous for a hitherto unknown missense mutation (NM_022916.4: c.599 G>C; NP_075067.2:p. Arg200Pro) in a conserved region of the VPS33A gene. Urinary glycosaminoglycan (GAG) analysis revealed increased heparan, dermatan sulphates, and hyaluronic acid. We showed decreased abundance of VPS33A in patient derived fibroblasts and provided evidence that the p.Arg200Pro mutation leads to destablization of the protein and proteasomal degradation. As in the infantile form of mucopolysaccharidosis plus, the endocytic compartment in the fibroblasts also expanded-a phenomenon accompanied by increased endolysosomal acidification and impaired intracellular glycosphingolipid trafficking. Experimental treatment of the patient's cultured fibroblasts with the proteasome inhibitor, bortezomib, or exposure to an inhibitor of glucosylceramide synthesis, eliglustat, improved glycosphingolipid trafficking. To our knowledge this is the first report of an attenuated juvenile form of VPS33A insufficiency characterized by appreciable residual endosomal-lysosomal trafficking and a milder mucopolysaccharidosis plus than the disease in infants. Our findings expand the proof of concept of redeploying clinically approved drugs for therapeutic exploitation in patients with juvenile as well as infantile forms of mucopolysaccharidosis plus disease.

The lysosomal disease caused by mutant VPS33A.

A rare lysosomal disease resembling a mucopolysaccharidosis with unusual systemic features, including renal disease and platelet dysfunction, caused by the defect in a conserved region of the VPS33A gene on human chromosome 12q24.31, occurs in Yakuts-a nomadic Turkic ethnic group of Southern Siberia. VPS33A is a core component of the class C core vacuole/endosome tethering (CORVET) and the homotypic fusion and protein sorting (HOPS) complexes, which have essential functions in the endocytic pathway. Here we show that cultured fibroblasts from patients with this disorder have morphological changes: vacuolation with disordered endosomal/lysosomal compartments and-common to sphingolipid diseases-abnormal endocytic trafficking of lactosylceramide. Urine glycosaminoglycan studies revealed a pathological excess of sialylated conjugates as well as dermatan and heparan sulphate. Lipidomic screening showed elevated ß-D-galactosylsphingosine with unimpaired activity of cognate lysosomal hydrolases. The 3D crystal structure of human VPS33A predicts that replacement of arginine 498 by tryptophan will de-stabilize VPS33A folding. We observed that the missense mutation reduced the abundance of full-length VPS33A and other components of the HOPS and CORVET complexes. Treatment of HeLa cells stably expressing the mutant VPS33A with a proteasome inhibitor rescued the mutant protein from degradation. We propose that the disease is due to diminished intracellular abundance of intact VPS33A. Exposure of patient-derived fibroblasts to the clinically approved proteasome inhibitor, bortezomib, or inhibition of glucosylceramide synthesis with eliglustat, partially corrected the impaired lactosylceramide trafficking defect and immediately suggest therapeutic avenues to explore in this fatal orphan disease.

The Lysosome and Intracellular Signalling.

In addition to being the terminal degradative compartment of the cell's endocytic and autophagic pathways, the lysosome is a multifunctional signalling hub integrating the cell's response to nutrient status and growth factor/hormone signalling. The cytosolic surface of the limiting membrane of the lysosome is the site of activation of the multiprotein complex mammalian target of rapamycin complex 1 (mTORC1), which phosphorylates numerous cell growth-related substrates, including transcription factor EB (TFEB). Under conditions in which mTORC1 is inhibited including starvation, TFEB becomes dephosphorylated and translocates to the nucleus where it functions as a master regulator of lysosome biogenesis. The signalling role of lysosomes is not limited to this pathway. They act as an intracellular Ca2+ store, which can release Ca2+ into the cytosol for both local effects on membrane fusion and pleiotropic effects within the cell. The relationship and crosstalk between the lysosomal and endoplasmic reticulum (ER) Ca2+ stores play a role in shaping intracellular Ca2+ signalling. Lysosomes also perform other signalling functions, which are discussed. Current views of the lysosomal compartment recognize its dynamic nature. It includes endolysosomes, autolysosome and storage lysosomes that are constantly engaged in fusion/fission events and lysosome regeneration. How signalling is affected by individual lysosomal organelles being at different stages of these processes and/or at different sites within the cell is poorly understood, but is discussed.

Exosomes bind to autotaxin and act as a physiological delivery mechanism to stimulate LPA receptor signalling in cells.

Autotaxin (ATX; also known as ENPP2), the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA), is a secreted enzyme. Here we show that, once secreted, ATX can bind to the surface of cell-secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interactions, ATX releases the LPA to activate cell surface G-protein-coupled receptors of LPA; inhibition of signalling by the receptor antagonist Ki1642 suggests that these receptors are LPAR1 and LPAR3. The binding stimulates downstream signalling, including phosphorylation of AKT and mitogen-activated protein kinases, the release of intracellular stored Ca2+ and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means by which ATX-LPA signalling operates physiologically.

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I.

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.

Reversible assembly and disassembly of V-ATPase during the lysosome regeneration cycle.

Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.

VAMP2 regulates phase separation of α-synuclein.

α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson's disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells.

Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae, and is not inhibited by overexpression of GTPase-null dynamin mutants. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2). Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.

Mistargeting of SH3TC2 away from the recycling endosome causes Charcot-Marie-Tooth disease type 4C.

Mutations in the functionally uncharacterized protein SH3TC2 are associated with the severe hereditary peripheral neuropathy, Charcot-Marie-Tooth disease type 4C (CMT4C). Similarly, to other proteins mutated in CMT, a role for SH3TC2 in endocytic membrane traffic has been previously proposed. However, recent descriptions of the intracellular localization of SH3TC2 are conflicting. Furthermore, no clear functional pathogenic mechanisms have so far been proposed to explain why both nonsense and missense mutations in SH3TC2 lead to similar clinical phenotypes. Here, we describe our intracellular localization studies, supported by biochemical and functional data, using wild-type and mutant SH3TC2. We show that wild-type SH3TC2 targets to the intracellular recycling endosome by associating with the small GTPase, Rab11, which is known to regulate the recycling of internalized membrane and receptors back to the plasma membrane. Furthermore, we demonstrate that SH3TC2 interacts preferentially with the GTP-bound form of Rab11, identifying SH3TC2 as a novel Rab11 effector. Of clinical pathological relevance, all SH3TC2 constructs harbouring disease-causing mutations are shown to be unable to associate with Rab11 with consequent loss of recycling endosome localization. Moreover, we show that wild-type SH3TC2, but not mutant SH3TC2, influences transferrin receptor dynamics, consistent with a functional role on the endocytic recycling pathway. Our data therefore implicate mistargeting of SH3TC2 away from the recycling endosome as the fundamental molecular defect that leads to CMT4C.

Clathrin is required for the function of the mitotic spindle.

Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells, but is shut down during mitosis, when clathrin concentrates at the spindle apparatus. Here, we show that clathrin stabilizes fibres of the mitotic spindle to aid congression of chromosomes. Clathrin bound to the spindle directly by the amino-terminal domain of clathrin heavy chain. Depletion of clathrin heavy chain using RNA interference prolonged mitosis; kinetochore fibres were destabilized, leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of clathrin heavy chain, indicating that stabilization of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.

Clathrin light chains function in mannose phosphate receptor trafficking via regulation of actin assembly.

Clathrin-coated vesicles (CCVs) are major carriers for endocytic cargo and mediate important intracellular trafficking events at the trans-Golgi network (TGN) and endosomes. Whereas clathrin heavy chain provides the structural backbone of the clathrin coat, the role of clathrin light chains (CLCs) is poorly understood. We now demonstrate that CLCs are not required for clathrin-mediated endocytosis but are critical for clathrin-mediated trafficking between the TGN and the endosomal system. Specifically, CLC knockdown (KD) causes the cation-independent mannose-6 phosphate receptor (CI-MPR) to cluster near the TGN leading to a delay in processing of the lysosomal hydrolase cathepsin D. A recently identified binding partner for CLCs is huntingtin-interacting protein 1-related (HIP1R), which is required for productive interactions of CCVs with the actin cytoskeleton. CLC KD causes mislocalization of HIP1R and overassembly of actin, which accumulates in patches around the clustered CI-MPR. A dominant-negative CLC construct that disrupts HIP1R/CLC interactions causes similar alterations in CI-MPR trafficking and actin assembly. Thus, in mammalian cells CLCs function in intracellular membrane trafficking by acting as recruitment proteins for HIP1R, enabling HIP1R to regulate actin assembly on clathrin-coated structures.

Role of furin in granular acidification in the endocrine pancreas: identification of the V-ATPase subunit Ac45 as a candidate substrate.

Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient beta cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line betaTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.

Coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding.

Endocytosis has a crucial role in many cellular processes. The best-characterized mechanism for endocytosis involves clathrin-coated pits [1], but evidence has accumulated for additional endocytic pathways in mammalian cells [2]. One such pathway involves caveolae, plasma-membrane invaginations defined by caveolin proteins. Plasma-membrane microdomains referred to as lipid rafts have also been associated with clathrin-independent endocytosis by biochemical and pharmacological criteria [3]. The mechanisms, however, of nonclathrin, noncaveolin endocytosis are not clear [4, 5]. Here we show that coassembly of two similar membrane proteins, flotillin1 and flotillin2 [6-8], is sufficient to generate de novo membrane microdomains with some of the predicted properties of lipid rafts [9]. These microdomains are distinct from caveolin1-positive caveolae, are dynamic, and bud into the cell. Coassembly of flotillin1 and flotillin2 into microdomains induces membrane curvature, the formation of plasma-membrane invaginations morphologically similar to caveolae, and the accumulation of intracellular vesicles. We propose that flotillin proteins are defining structural components of the machinery that mediates a clathrin-independent endocytic pathway. Key attributes of this machinery are the dependence on coassembly of both flotillins and the inference that flotillin microdomains can exist in either flat or invaginated states.

Endosome-lysosome fusion.

The delivery of endocytosed cargo to lysosomes occurs through kissing and direct fusion of late endosomes/MVBs (multivesicular bodies) and lysosomes. Live-cell and electron microscopy experiments together with cell-free assays have allowed us to describe the characteristics of the delivery process and determine the core protein machinery required for fusion. The ESCRT (endosomal sorting complex required for transport) machinery is required for MVB biogenesis. The HOPS (homotypic fusion and vacuole protein sorting) complex is required for endosome-lysosome tethering and a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex including the R-SNARE VAMP7 (vesicle-associated membrane protein 7) mediates endosome-lysosome membrane fusion. Protein-binding partners of VAMP7 including the clathrin adaptors AP-3 (adaptor protein 3) and Hrb (HIV Rev-binding protein) are required for its correct intracellular localization and function. Overall, co-ordination of the activities of ESCRT, HOPS and SNARE complexes are required for efficient delivery of endocytosed macromolecules to lysosomes. Endosome-lysosome fusion results in a hybrid organelle from which lysosomes are re-formed. Defects in fusion and/or lysosome reformation occur in a number of lysosome storage diseases.

Mechanism and evolution of the Zn-fingernail required for interaction of VARP with VPS29.

VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.

The delivery of endocytosed cargo to lysosomes.

In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome-lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome-lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to lysosomes.

ESCRT proteins and the regulation of endocytic delivery to lysosomes.

In mammalian cells, there is evidence of cargo specificity in the requirement for particular ESCRT (endosomal sorting complex required for transport) proteins to sort cargo into the luminal vesicles of MVBs (multivesicular bodies). We have focussed on studying the ESCRT requirements for delivery of MHC class I to lysosomes following polyubiquitination by the Kaposi's sarcoma-associated herpesvirus protein K3. Down-regulation of polyubiquitinated cell-surface MHC class I in HeLa cells stably expressing K3 is achieved via clathrin-mediated endocytosis, followed by sorting into the luminal vesicles of MVBs and eventual delivery to lysosomes. Depletion of ESCRT-I and some ESCRT-III components interferes with this sorting and allows recycling of MHC class I to the cell surface. Depletion of ESCRT-II components has no effect on K3-mediated down-regulation of MHC class I and no gross morphological effect on endocytic compartments. Thus virally polyubiquitinated MHC class I does not require all of the ESCRT proteins in order to be sorted into the luminal vesicles of MVBs. However, there may be a further requirement for ESCRT-III proteins to ensure the efficient fusion of MVBs with lysosomes.

Clathrin-mediated endocytosis in AP-2-depleted cells.

We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

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.