Endocytic Organelles Research Articles

Spatial distribution of phagolysosomes is independent of the regulation of lysosome position by Rab34

Within a cell, the regulation of organelle positioning is considered to be critical in spatio-temporal responses. The position of late endocytic organelles (named here lysosomes for simplicity) is tightly controlled and has a functional impact on processes like endocytosis, phagocytosis and autophagocytosis. The cytoplasmic distribution profile of lysosomes can be easily determined in cells where the cytoplasm/nuclear ratio in a cross-section area is high. However, determining lysosomal position in cells with lower cytoplasm/nuclear ratio, such as macrophages is more challenging. Here, we describe a method that can be efficiently and accurately used to determine the position of organelles in macrophages using confocal microscopy in two-dimensional (2D) images. Using this approach in macrophages, we confirmed previous observations in epithelial cells that both changes in cytoplasmic pH and the levels of active Rab34 induced a re-distribution of lysosomes to the cell centre or periphery. Noteworthy is that this Rab34-dependent re-distribution of lysosomes did not significantly affect the spatial distribution profile of phagolysosomes in the cytoplasm. We conclude that although Rab34 regulates both lysosomal positioning and lysosome to phagosome fusion, the latter effect is not due to the regulation of the cytoplasmic accessibility of lysosomes to phagosomes by Rab34.

An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters

Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions.

Intracellular Trafficking of an Antibody Bipolar Bridged Complex of HSV‐1 gE‐gI, IgG, and a Viral Antigen

Herpes Simplex Virus 1 (HSV‐1) glycoproteins, gE and gI, mimic a human Fc‐receptor to hinder host immune responses. gE‐gI binds the Fc region of human IgG (hIgG), which can be internalized and released into endocytic organelles. gE‐gI also mediates antibody bipolar bridging (ABB), a process whereby IgG Fabs bind an antigen while its Fc region binds to gE‐gI. The cellular fate of the ABB complex is unknown. Here, we engineered an in vitro system to determine whether ABB complexes are internalized and degraded in host cells. Human cell lines transiently expressing gE‐gI and an HSV‐1 antigen, gD, normally localized at the cell surface of HSV‐1 virions and infected cells, were incubated with two forms of a monoclonal IgG anti‐gD antibody: one with human Fc to allow ABB and one with mouse Fc, a control which binds gD but not gE‐gI and cannot enable ABB. A hIgG against an irrelevant antigen was used as another non ABB control. 4D time‐lapse confocal fluorescence data indicate that gD is internalized in an ABB specific fashion and internalization is dependent upon gE‐gI. Internalized hIgGs bound to a surface viral antigen were targeted to lysosomes and biochemical analysis of enriched lysosomal compartments revealed degraded gD. These results suggest that gE‐gI plays an active role in clearing the infected cell surface of both host IgG and viral antigens, providing HSV‐1 with a mechanism to evade immune responses.

A novel co-infection model withToxoplasmaandChlamydia trachomatishighlights the importance of host cell manipulation for nutrient scavenging

Toxoplasma and Chlamydia trachomatis are obligate intracellular pathogens that have evolved analogous strategies to replicate within mammalian cells. Both pathogens are known to extensively remodel the cytoskeleton, and to recruit endocytic and exocytic organelles to their respective vacuoles. However, how important these activities are for infectivity by either pathogen remains elusive. Here, we have developed a novel co-infection system to gain insights into the developmental cycles of Toxoplasma and C. trachomatis by infecting human cells with both pathogens, and examining their respective ability to replicate and scavenge nutrients. We hypothesize that the common strategies used by Toxoplasma and Chlamydia to achieve development results in direct competition of the two pathogens for the same pool of nutrients. We show that a single human cell can harbour Chlamydia and Toxoplasma. In co-infected cells, Toxoplasma is able to divert the content of host organelles, such as cholesterol. Consequently, the infectious cycle of Toxoplasma progresses unimpeded. In contrast, Chlamydia's ability to scavenge selected nutrients is diminished, and the bacterium shifts to a stress-induced persistent growth. Parasite killing engenders an ordered return to normal chlamydial development. We demonstrate that C. trachomatis enters a stress-induced persistence phenotype as a direct result from being barred from its normal nutrient supplies as addition of excess nutrients, e.g. amino acids, leads to substantial recovery of Chlamydia growth and infectivity. Co-infection of C. trachomatis with slow growing strains of Toxoplasma or a mutant impaired in nutrient acquisition does not restrict chlamydial development. Conversely, Toxoplasma growth is halted in cells infected with the highly virulent Chlamydia psittaci. This study illustrates the key role that cellular remodelling plays in the exploitation of host intracellular resources by Toxoplasma and Chlamydia. It further highlights the delicate balance between success and failure of infection by intracellular pathogens in a co-infection system at the cellular level.

Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges

Cell penetrating peptides (CPPs) can deliver cell-impermeable therapeutic cargos into cells. In particular, CPP-cargo conjugates tend to accumulate inside cells by endocytosis. However, they often remain trapped inside endocytic organelles and fail to reach the cytosolic space of cells efficiently. In this review, the evidence for CPP-mediated endosomal escape is discussed. In addition, several strategies that have been utilized to enhance the endosomal escape of CPP-cargos are described. The recent development of branched systems that display multiple copies of a CPP is presented. The use of viral or synthetic peptides that can disrupt the endosomal membrane upon activation by the low pH of endosomes is also discussed. Finally, we survey how CPPs labeled with chromophores can be used in combination with light to stimulate endosomal lysis. The mechanisms and challenges associated with these intracellular delivery methodologies are discussed.

Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P2and PI(5)P

Normal steady-state levels of the signalling lipids PI(3,5)P(2) and PI(5)P require the lipid kinase FAB1/PIKfyve and its regulators, VAC14 and FIG4. Mutations in the PIKfyve/VAC14/FIG4 pathway are associated with Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis in humans, and profound neurodegeneration in mice. Hence, tight regulation of this pathway is critical for neural function. Here, we examine the localization and physiological role of VAC14 in neurons. We report that endogenous VAC14 localizes to endocytic organelles in fibroblasts and neurons. Unexpectedly, VAC14 exhibits a pronounced synaptic localization in hippocampal neurons, suggesting a role in regulating synaptic function. Indeed, the amplitude of miniature excitatory postsynaptic currents is enhanced in both Vac14(-/-) and Fig4(-/-) neurons. Re-introduction of VAC14 in postsynaptic Vac14(-/-) cells reverses this effect. These changes in synaptic strength in Vac14(-/-) neurons are associated with enhanced surface levels of the AMPA-type glutamate receptor subunit GluA2, an effect that is due to diminished regulated endocytosis of AMPA receptors. Thus, VAC14, PI(3,5)P(2) and/or PI(5)P play a role in controlling postsynaptic function via regulation of endocytic cycling of AMPA receptors.

Phagocytosis Here and Now

At the time of writing, a PubMed search using ‘endosome’ as the search term yielded 12 675 hits. A comparable search for ‘phagosome’ produced only 4646 hits. This is due, in part, to the fact that while all cells continuously perform endocytosis, only a few types of specialized cells – e.g. neutrophils, macrophages and dendritic cells, the professional phagocytes – are adept at phagocytosis. However, the comparative paucity of studies is also attributable to the misconception that phagocytosis is merely an overgrown version of endocytosis: that phagosomes are endosomes on steroids. In fact, unlike endocytic organelles (clathrin-coated vesicles, caveolae, clathrin-independent carriers/GPI-anchored-protein-enriched endosomal compartment (CLIC/GEEC)), whose size is dictated by the internalization machinery of the cells and hence remarkably constant, phagosome size is determined by the dimensions of the target particle, and can range from a fraction of 1 µm to upward of 10 µm. Thus, size is not the distinguishing characteristic of phagosomes. Instead, phagocytosis is unique in that it is triggered by a particulate, multivalent ligand, is strictly dependent on actin polymerization and remodeling, and often (but not always) requires activation of class I phosphatidylinositol 3-kinase. A caveat must be made: discussing phagocytosis as a single phenomenon is overly simplistic and misleading. There are dozens of receptors capable of inducing particle ingestion, and the signals they elicit and the mechanisms whereby they trigger particle engulfment are most likely to differ not only in detail, but in substance. Few of them have been studied systematically, but substantive differences have been already revealed between Fc receptors, dectin-1 and the integrins that serve as complement receptors. Thus, ‘phagocytosis’ should be used as an umbrella term that identifies a family of phenotypically related, but molecularly distinct phenomena. Phagocytosis mediates the clearance of apoptotic bodies and, as such, is central to embryogenesis, tissue remodeling and homeostasis. Just as importantly, the professional phagocytes are intimately involved in innate immunity: the forming phagosome is often the first event in host-pathogen confrontation. Antigens derived from the engulfment and destruction of pathogens are then presented by phagocytes to lymphoid cells for the development of adaptive immunity. Considering its unquestionable importance to the survival and well-being of metazoans (and even of some protozoans), the dearth of studies on phagocytosis – at least by comparison with endocytosis – is striking. It is in the spirit of correcting this trend that the Editors of Traffic decided to publish a series of reviews on various aspects of phagocytosis. The decision was spurred by the fact that remarkable advances have been made recently in the field, which warrant wide exposure. To this end, five teams of experts were convened to write manuscripts with emphasis on different aspects of phagocytosis. Three of these, those written by Drs Kagan and Iwasaki, Drs Goodridge, Underhill and Touret, and by Drs Soldati and Neyrolles are included in this issue. Two others will be forthcoming later this year. Each one of these analyses separate aspects of the phagocytic response. In this regard, it is worthwhile remembering that phagocytosis is a veritable compendium of many aspects of cell physiology: from receptor engagement and signal transduction, to cytoskeletal remodeling, membrane traffic and gene regulation. While those of us that work in the field and have made phagocytosis our province may not welcome the added competition, it is important for younger scientists and for experts in other areas to become aware of the recent developments and, in the process perhaps become interested in the fascinating biology of phagosome formation and maturation, and the challenges phagocytes face when confronted with wily pathogens.

TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells

BackgroundLysis of endocytic organelles is a necessary step in many cellular delivery methodologies. This is achieved efficiently in the photochemical internalization approach but the cell death that accompanies this process remains a problem. MethodsWe investigate the mechanisms of cell death that accompanies photochemical internalization of the fluorescent peptide TMR-TAT. ResultsTMR-TAT kills cells after endocytosis and light irradiation. The lysis of endocytic organelles by TMR-TAT causes a rapid increase in the concentration of calcium in the cytosol. TMR-TAT co-localizes with endocytic organelles containing calcium prior to irradiation and photochemical internalization leads to the release of the lumenal content of these organelles. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore, inhibit cell death. ConclusionsTMR-TAT mediated photochemical internalization leads to a disruption of calcium homeostasis. The subsequent import of calcium in mitochondria is a causative factor of the cell death that accompanies photochemical internalization.General significanceUnderstanding how the lysis of endocytic organelles affects cellular physiology and causes cell death is crucial to the development of optimal delivery methodologies.

Impaired retrograde membrane traffic through endosomes in a mutant CHO cell defective in phosphatidylserine synthesis

Phosphatidylserine (PS), a relatively minor constituent in the plasma membrane (PM), participates in various cellular processes such as clearance of apoptotic cells and recruitment of signaling molecules. PS also localizes in the membranes of endocytic organelles, such as recycling endosomes (REs). We recently showed that in REs, PS binds to the pleckstrin homology (PH) domain of evectin-2, thereby regulating retrograde traffic from REs to the Golgi. However, direct evidence that PS has a role in retrograde traffic is lacking. Here, we examined the contribution of PS to endosomal membrane traffic by exploiting a mutant CHO cell line (PSA-3) that is defective in PS synthesis. In PSA-3 cells, the Golgi localization of TGN38, a protein that circulates between the Golgi and the PM through endosomes by retrograde traffic, was abolished, whereas the localizations of other organelle markers remained unchanged. Increasing the cellular PS level by adding ethanolamine to the culture medium restored the Golgi localization of TGN38. Tracking the endocytic fate of cell surface TGN38 that was labeled by anti-TGN38 antibody showed that retrograde transport of TGN38 was impaired at endosomes, not at the PM. These findings provide direct evidence that intracellular PS is required for retrograde traffic through endosomes.

Spatial Restriction of Bone Morphogenetic Protein Signaling in Mouse Gastrula through the mVam2-Dependent Endocytic Pathway

The embryonic body plan is established through positive and negative control of various signaling cascades. Late endosomes and lysosomes are thought to terminate signal transduction by compartmentalizing the signaling molecules; however, their roles in embryogenesis remain poorly understood. We showed here that the endocytic pathway participates in the developmental program by regulating the signaling activity. We modified the mouse Vam2 (mVam2) locus encoding a regulator of membrane trafficking. mVam2-deficient cells exhibited abnormally fragmented late endosomal compartments. The mutant cells could terminate signaling after theremoval of the growth factors including TGF-β and EGF, except BMP-Smad1/Smad5 signaling. mVam2-deficient embryos exhibited ectopic activation of BMP signaling and disorganization of embryo patterning. We found that mVam2, which interacts with BMP type I receptor, is required for the spatiotemporal modulation of BMP signaling, via sequestration of the receptor complex in the late stages of the endocytic pathway.

Polyplexes Traffic through Caveolae to the Golgi and Endoplasmic Reticulum en Route to the Nucleus

The cellular machinery involved in the internalization of nonviral gene carriers and their subsequent trafficking to the nucleus directly impacts their therapeutic efficiency. Hence, identifying key endocytic pathways and organelles that contribute to the successful transfer of polyplexes to the nucleus generates new opportunities for improving carrier design. Previously, we showed that histone H3 tail peptides encoding a sequence known to participate in chromatin activation exhibit synergistic gene delivery activity with poly(ethylenimine) (PEI). Polyplexes containing H3 and PEI exhibited a reduced dependence on endocytic pathways that trafficked to lysosomes, and had enhanced sensitivity to an inhibitor associated with retrograde trafficking through the Golgi apparatus. Thus, we sought to determine whether caveolar uptake and transport through the Golgi and/or endoplasmic reticulum (ER) preceded nuclear delivery. By the use of a panel of chemical endocytic inhibitors, we determined that H3 polyplexes utilized caveolar pathways to a greater degree than PEI polyplexes. Caveolae-mediated endocytosis was found to be a productive route for gene expression by the H3/PEI-pDNA polyplexes, consistent with previous studies of polymer-mediated gene delivery. Additionally, the polyplexes substantially colocalized within the ER after only 5 min of incubation, and utilized retrograde Golgi-to-ER pathways at levels similar to pathogens known to traffic by these routes during infection. The results of this study have expanded our understanding of how caveolar polyplexes are trafficked to cell nuclei, and provide new evidence for the role of Golgi-ER pathways in transfection. These findings suggest new design criteria and opportunities to stragetically target nonviral gene delivery vehicles.

Endosomal Actin Remodeling by Coronin-1A Controls Lipoprotein Uptake and Degradation in Macrophages

The actin cytoskeleton has been implicated in the processing of atherogenic lipoproteins in macrophages. However, the functional role of actin and the regulatory proteins involved are unknown. Coronin-1A (Coro1A) was identified as a differentially expressed transcript in wild-type versus Niemann-Pick type C1 deficient macrophages exposed to acetylated low-density lipoproteins (AcLDL). We investigated whether Coro1A plays a role in the uptake or processing of modified lipoproteins in macrophages and if this is related to its actin regulatory functions. In wild-type primary macrophages, filamentous actin transiently decorated AcLDL containing endosomes that also recruited Coro1A. This dynamic association of F-actin with endosomes was disturbed in Coro1A deficient macrophages. In Coro1A knockout macrophages the uptake of AcLDL was increased, rate of AcLDL delivery to lysosomes enhanced, and lipoprotein-derived cholesteryl ester hydrolysis accelerated. Overexpression of wild-type Coro1A normalized AcLDL uptake in Coro1A knockout macrophages while a Coro1A actin binding mutant did not. Furthermore, the effects of macrophage Coro1A silencing on endosomal actin association and AcLDL delivery to lysosomes resembled those of cofilin silencing. Coro1A controls actin association with endocytic organelles, thereby negatively regulating endo-lysosomal delivery, degradation of modified lipoproteins and cholesterol deposition in macrophages.

Overcoming Endosomal Barrier by Amphotericin B-Loaded Dual pH-Responsive PDMA-b-PDPA Micelleplexes for siRNA Delivery

The endosomal barrier is a major bottleneck for the effective intracellular delivery of siRNA by nonviral nanocarriers. Here, we report a novel amphotericin B (AmB)-loaded, dual pH-responsive micelleplex platform for siRNA delivery. Micelles were self-assembled from poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-b-PDPA) diblock copolymers. At pH 7.4, AmB was loaded into the hydrophobic PDPA core, and siRNA was complexed with a positively charged PDMA shell to form the micelleplexes. After cellular uptake, the PDMA-b-PDPA/siRNA micelleplexes dissociated in early endosomes to release AmB. Live cell imaging studies demonstrated that released AmB significantly increased the ability of siRNA to overcome the endosomal barrier. Transfection studies showed that AmB-loaded micelleplexes resulted in significant increase in luciferase (Luc) knockdown efficiency over the AmB-free control. The enhanced Luc knockdown efficiency was abolished by bafilomycin A1, a vacuolar ATPase inhibitor that inhibits the acidification of the endocytic organelles. These data support the central hypothesis that membrane poration by AmB and increased endosomal swelling and membrane tension by a "proton sponge" polymer provided a synergistic strategy to disrupt endosomes for improved intracellular delivery of siRNA.

Individual organelle pH determinations of magnetically enriched endocytic organelles via laser-induced fluorescence detection.

The analysis of biotransformations that occur in lysosomes and other endocytic organelles is critical to studies on intracellular degradation, nutrient recycling, and lysosomal storage disorders. Such analyses require bioactive organelle preparations that are devoid of other contaminating organelles. Commonly used differential centrifugation techniques produce impure fractions and may not be compatible with microscale separation platforms. Density gradient centrifugation procedures reduce the level of impurities but may compromise bioactivity. Here we report on simple magnetic setup and a procedure that produce highly enriched bioactive organelles based on their magnetic capture as they traveled through open tubes. Following capture, in-line laser-induced fluorecence detection (LIF) determined for the first time the pH of each magnetically retained individual endocytic organelle. Unlike bulk measurements, this method was suitable to describe the distributions of pH values in endocytic organelles from L6 rat myoblasts treated with dextran-coated iron oxide nanoparticles (for magnetic retention) and fluorescein/TMRM-conjugated dextran (for pH measurements by LIF). Their individual pH values ranged from 4 to 6, which is typical of bioactive endocytic organelles. These analytical procedures are of high relevance to evaluate lysosomal-related degradation pathways in aging, storage disorders, and drug development.

Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

Actin regulation during abscission: unexpected roles of Rab35 and endocytic transport

Actin regulation during abscission: unexpected roles of Rab35 and endocytic transport

High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine

Phosphatidylserine (PS) plays a central role in cell signaling and in the biosynthesis of other lipids. To date, however, the subcellular distribution and transmembrane topology of this crucial phospholipid remain ill-defined. We transfected cells with a GFP-tagged C2 domain of lactadherin to detect by light and electron microscopy PS exposed on the cytosolic leaflet of the plasmalemma and organellar membranes. Cytoplasmically exposed PS was found to be clustered on the plasma membrane, and to be associated with caveolae, the trans-Golgi network, and endocytic organelles including intraluminal vesicles of multivesicular endosomes. This labeling pattern was compared with the total cellular distribution of PS as visualized using a novel on-section technique. These complementary methods revealed PS in the interior of the ER, Golgi complex, and mitochondria. These results indicate that PS in the lumenal monolayer of the ER and Golgi complex becomes exposed cytosolically at the trans-Golgi network. Transmembrane flipping of PS may contribute to the exit of cargo from the Golgi complex.

Smart Multifunctional Hollow Microspheres for the Quick Release of Drugs in Intracellular Lysosomal Compartments

Prepared to self-destruct: when poly(D, L-lactic-co-glycolic acid) (PLGA) hollow microspheres containing NaHCO(3) entered the endocytic organelles of a live cell, the NaHCO(3) in the aqueous core reacted with protons that infiltrated from the compartment to generate CO(2) gas. The evolution of CO(2) bubbles led to the formation of small holes in the PLGA shell and thus rapid release of the encapsulated drug doxorubicin.

Loss of Cleavage at β′-Site Contributes to Apparent Increase in β-Amyloid Peptide (Aβ) Secretion by β-Secretase (BACE1)-Glycosylphosphatidylinositol (GPI) Processing of Amyloid Precursor Protein

Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated β-amyloid peptide (Aβ) production. Notably, targeting β-secretase (β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aβ secretion. Paradoxically, Aβ secretion is not reduced by the expression of non-raft resident S-palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)). We addressed this apparent discrepancy in raft microdomain-associated BACE1 processing of APP in this study. As previously reported, we found that expression of BACE1-GPI elevated Aβ secretion as compared with wild-type BACE1 (WTBACE1) or BACE1-4C/A. However, this increase occurred without any difference in the levels of APP ectodomain released following BACE1 cleavage (soluble APPβ), arguing against an overall increase in BACE1 processing of APP per se. Further analysis revealed that WTBACE1 cleaves APP at β- and β'-sites, generating +1 and +11 β-C-terminal fragments and secreting intact as well as N-terminally truncated Aβ. In contrast, three different BACE1-GPI chimeras preferentially cleaved APP at the β-site, mainly generating +1 β-C-terminal fragment and secreting intact Aβ. As a consequence, cells expressing BACE1-GPI secreted relatively higher levels of intact Aβ without an increase in BACE1 processing of APP. Markedly reduced cleavage at β'-site exhibited by BACE1-GPI was cell type-independent and insensitive to subcellular localization of APP or the pathogenic KM/NL mutant. We conclude that the apparent elevation in Aβ secretion by BACE1-GPI is mainly attributed to preferential cleavage at the β-site and failure to detect +11 Aβ species secreted by cells expressing WTBACE1.

Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems

BackgroundCytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release.ResultsNK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse.ConclusionsThese results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells.