Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).

The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1⋅s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1⋅s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2 addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233 and Cys222 is possible and likely to participate in the catalytic cycle. Recognizing the ability of TcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). TcAPx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.

Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy.

Severe thrombocytopenia (≤50×109 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca2+ flux, integrin a ß activation and P-selectin expression when stimulated with a panelIIbof3 agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca2+ store content was unaffected, the patients' platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (P<0.001) and low oxygen consumption (P<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, P=0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients.

SLC35D3 delivery from megakaryocyte early endosomes is required for platelet dense granule biogenesis and is differentially defective in Hermansky-Pudlak syndrome models.

Platelet dense granules are members of a family of tissue-specific, lysosome-related organelles that also includes melanosomes in melanocytes. Contents released from dense granules after platelet activation promote coagulation and hemostasis, and dense granule defects such as those seen in Hermansky-Pudlak syndrome (HPS) cause excessive bleeding, but little is known about how dense granules form in megakaryocytes (MKs). In the present study, we used SLC35D3, mutation of which causes a dense granule defect in mice, to show that early endosomes play a direct role in dense granule biogenesis. We show that SLC35D3 expression is up-regulated during mouse MK differentiation and is enriched in platelets. Using immunofluorescence and immunoelectron microscopy and subcellular fractionation in megakaryocytoid cells, we show that epitope-tagged and endogenous SLC35D3 localize predominantly to early endosomes but not to dense granule precursors. Nevertheless, SLC35D3 is depleted in mouse platelets from 2 of 3 HPS models and, when expressed ectopically in melanocytes, SLC35D3 localizes to melanosomes in a manner requiring a HPS-associated protein complex that functions from early endosomal transport intermediates. We conclude that SLC35D3 is either delivered to nascent dense granules from contiguous early endosomes as MKs mature or functions in dense granule biogenesis directly from early endosomes, suggesting that dense granules originate from early endosomes in MKs.

Human platelets contain a pool of free zinc in dense granules.

Background: Activated platelets release procoagulant factors that include Ca2+ and Zn2+. Releasable Ca2+ stores have been identified in platelet dense granules and the dense tubular system, but similar stores of free Zn2+ have not been identified. Objectives: Guided by studies of platelet Ca2+, we employed minimally disruptive methods to identify and localize concentrated free Zn2+ in human platelets. Methods: Resting platelets from normal donors (NDs), patients with gray platelet syndrome (GPS) lacking α-granules, and patients with Hermansky-Pudlak syndrome (HPS) deficient in dense granules were loaded with cell-permeant fluorescent probes specific to free Ca2+ or Zn2+. Ion concentrations were detected in fixed cells as bright puncta via high-resolution confocal microscopy. Ions were also directly detected via transmission electron microscopy and energy dispersive X-ray analysis. Levels of total platelet Ca, Zn, and Mg were measured via inductively coupled plasma optical emission spectroscopy. Results: Fluorescent Zn2+ puncta counts were similar in ND and GPS platelets and markedly lower in HPS platelets, pointing to dense granules as likely reservoirs of free Zn2+. This localization was supported by direct detection of Ca2+, Zn2+, and Na+ in platelet dense granules via transmission electron microscopy and energy dispersive X-ray analysis. Measurements of total platelet Ca, Zn, and Mg via inductively coupled plasma optical emission spectroscopy indicated that free Zn2+ represents a small proportion of total platelet zinc, consistent with the strong affinity of Zn2+ for binding proteins, including several abundant in platelet α-granules. Conclusion: We conclude that normal human platelets contain a pool of free Zn2+ concentrated in dense granules that is available for secretion upon platelet activation and potentially contributes to hemostasis.

Pleiotropic platelet defects in mice with disrupted FOG1-NuRD interaction.

Understanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)-like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule-like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.

The platelet interior revisited: electron tomography reveals tubular alpha-granule subtypes.

We have used (cryo) electron tomography to provide a 3-dimensional (3D) map of the intracellular membrane organization of human platelets at high spatial resolution. Our study shows that the open canalicular system and dense tubular system are highly intertwined and form close associations in specialized membrane regions. 3D reconstructions of individual alpha-granules revealed large heterogeneity in their membrane organization. On the basis of their divergent morphology, we categorized alpha-granules into the following subtypes: spherical granules with electron-dense and electron-lucent zone containing 12-nm von Willebrand factor tubules, subtypes containing a multitude of luminal vesicles, 50-nm-wide tubular organelles, and a population with 18.4-nm crystalline cross-striations. Low-dose (cryo) electron tomography and 3D reconstruction of whole vitrified platelets confirmed the existence of long tubular granules with a remarkably curved architecture. Immunoelectron microscopy confirmed that these extended structures represent alpha-granule subtypes. Tubular alpha-granules represent approximately 16% of the total alpha-granule population and are detected in approximately half of the platelet population. They express membrane-bound proteins GLUT3 and alphaIIb-beta3 integrin and contain abundant fibrinogen and albumin but low levels of beta-thromboglobulin and no von Willebrand factor. Our 3D study demonstrates that, besides the existence of morphologically different alpha-granule subtypes, high spatial segregation of cargo exists within individual alpha-granules.

Platelet protein disulfide isomerase is localized in the dense tubular system and does not become surface expressed after activation.

Evidence is accumulating that circulating tissue factor (TF) contributes to the initiation of coagulation and the formation of fibrin. The majority of circulating TF is cryptic, and it has been suggested that close vicinity with anionic phospholipids on the cell surface increases the active conformation of TF. Two recent papers have shown that encryption of TF and initiation of coagulation are facilitated by the enzyme protein disulfide isomerase (PDI), possibly on the surface of activated platelets or endothelial cells. In this brief report, we demonstrate that the majority of PDI in platelets is intracellular where it is exclusively located in the dense tubular system. On activation, PDI remains confined to the intracellular stores of the dense tubular system and is neither released nor targeted to the cell surface. Similar results were obtained in endothelium where PDI remains exclusively localized in the endoplasmic reticulum, both at steady state and after thrombin stimulation.

Spatial organization of the transforming MHC class II compartment.

BACKGROUND INFORMATION: DC (dendritic cells) continuously capture pathogens and process them into small peptides within the endolysosomal compartment, the MIIC (MHC class II-containing compartment). In MIICs peptides are loaded on to MHC class II and rapidly redistributed to the cell surface. This redistribution is accompanied by profound changes of the MIICs into tubular structures. An emerging concept is that MIIC tubulation provides a means to transport MHC class II-peptide complexes to the cell surface, either directly or through vesicular intermediates. To obtain spatial information on the reorganization of the MIICs during DC maturation, we performed electron tomography on cryo-immobilized and freeze-substituted mouse DCs after stimulation with LPS (lipopolysaccharide). RESULTS: In non-stimulated DCs, MIICs are mostly spherical. After 3 h of LPS stimulation, individual MIICs transform into tubular structures. Three-dimensional reconstruction showed that the MIICs frequently display fusion profiles and after 6 h of LPS stimulation, MIICs become more interconnected, thereby creating large MIIC reticula. Microtubules and microfilaments align these MIICs and reveal physical connections. In our tomograms we also identified a separate population of MIIC-like intermediates, particularly at extended ends of MIIC tubules and in close proximity to the trans-Golgi network. No fusion events were captured between reticular MIICs and the plasma membrane. CONCLUSIONS: Our results indicate that MIICs have the capacity to fuse together, whereby the cytoskeleton possibly provides a scaffold for the MIIC shape change and directionality. MIIC-like intermediates may represent MHC class II carriers.

The acute effect of increased laser energy during the excimer laser-assisted non-occlusive anastomosis procedure on the vessel wall of the recipient artery: a histopathological study.

BACKGROUND AND OBJECTIVE: The excimer laser-assisted non-occlusive anastomosis (ELANA) technique is a way of making an anastomosis of vessels without temporal occlusion that is used for cerebral revascularization. Currently, 10 mJ of laser energy is used during the ELANA procedure. We have recently demonstrated that increasing the laser energy may increase flap retrieval rate. The aim of the present study was to study the acute effect of increased laser energy during the ELANA procedure on the recipient vessel wall. MATERIALS AND METHODS: The ELANA technique was performed on the abdominal aortas of rabbits under anesthesia using three categories of laser energy (two laser episodes of 10, 13, and 15 mJ, respectively). The rabbits were subsequently sacrificed and the anastomoses were removed. A non-lased rabbit aorta was used as control. Recipient arteries were studied using histopathology and transmission electron microscopy. RESULTS: In all three categories of laser energy and in the control group, the tunica media and adventitia adjacent to the anastomosis were intact, apart from damage caused by sutures. In the control group, the endothelium was fully intact. In the 10 and 13 mJ subgroups, the endothelium was mostly intact [92% (range 85-98) and 87% (range 80-90) for 10 and 13 mJ, respectively]. In the 15 mJ subgroup, most of the endothelium was absent [32% (range 20-40) of endothelium intact], predominantly at the side opposed to the anastomosis. CONCLUSION: Increasing the laser energy during the ELANA procedure from 10 to 13 mJ does not cause additional acute damage to the vessel wall. Increasing the laser energy from 13 to 15 mJ results in increased acute damage of the endothelium, whereas tunica media and adventitia remain unaffected. Further studies are required to assess the long-term effects of increased laser energy during the ELANA technique.

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency.

BACKGROUND: δ-storage pool disease (δ-SPD) is a bleeding disorder characterized by a reduced number of platelet-dense granules. The diagnosis of δ-SPD depends on the measurement of platelet ADP content, but this test is time consuming and requires a relatively large blood volume. Flow cytometric analysis of platelet mepacrine uptake is a potential alternative, but this approach lacks validation, which precludes its use in a diagnostic setting. OBJECTIVES: To evaluate the performance of platelet mepacrine uptake as a diagnostic test for δ-SPD. PATIENTS/METHODS: Mepacrine fluorescence was determined with flow cytometry before and after platelet activation in 156 patients with a suspected platelet function disorder and compared with platelet ADP content as a reference test. Performance was analyzed with a receiver operating characteristic (ROC) curve. RESULTS: Eleven of 156 patients had δ-SPD based on platelet ADP content. Mepacrine fluorescence was inferior to platelet ADP content in identifying patients with δ-SPD, but both mepacrine uptake (area under the ROC curve [AUC] 0.87) and mepacrine release after platelet activation (AUC 0.80) had good discriminative ability. In our tertiary reference center, mepacrine uptake showed high negative predicitive value (97%) with low positive predictive value (35%). Combined with a negative likelihood ratio of 0.1, these data indicate that mepacrine uptake can be used to exclude δ-SPD in patients with a bleeding tendency. CONCLUSION: Mepacrine fluorescence can be used as a screening tool to exclude δ-SPD in a large number of patients with a suspected platelet function disorder.

Increased protein nitration burden in the atherosclerotic lesions and plasma of apolipoprotein A-I deficient mice.

Apolipoprotein A-I (apoA-I), the major protein constituent within high-density lipoprotein (HDL), has been associated with antiatherogenic protection by mechanisms that include reverse cholesterol transport and antiinflammatory functions. To evaluate the proposed protective function of apoA-I, proteins modified by nitrating oxidants were evaluated in the aortic tissue and plasma of mice lacking the low-density lipoprotein receptor and apobec (LA) and LA mice with genetic deletion of apoA-I (LA-apoA-I(-/-)). The levels of nitrated proteins in aortic tissue quantified by liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) were 6-fold higher in the LA-apoA-I(-/-) as compared with the LA mice. The quantitative analyses were corroborated by immunohistochemical and high-resolution immunoelectron microscopic evaluation of the lesions, which revealed abundant staining for nitrated proteins in the aortic root lesions of LA-apoA-I(-/-) as compared with the LA mice. Proteomic approaches based on affinity enrichment and site-specific adduct mapping identified unique specific protein targets for nitration in the plasma of LA-apoA-I(-/-) that were not present in the plasma of LA mice. In particular the nitration of fibrinogen was shown to accelerate fibrin clot formation. Another consequence of the augmented levels of nitrated proteins was the induction of humoral responses documented by the increased circulating immunoglobulins that recognize nitrotyrosine in LA-apoA-I(-/-) as compared with the LA mice. These data collectively support a protective function of apoA-I diminishing the burden of nitrative oxidants in these mice models of atherosclerosis.

Role of membrane cholesterol in platelet calcium signalling in response to VWF and collagen under stasis and flow.

Several studies have highlighted a specific role for membrane cholesterol domains in platelet signalling. Upon adhesion to von Willebrand factor (VWF) or collagen, cholesterol-rich domains (CRDs) accumulate in filopodial extensions and selectively harbour counterpart receptors (GPIb and GPVI) and associated signalling molecules. In the present study we have addressed the role of membrane cholesterol in Ca(2+) signalling and secretion during the interaction of platelets with VWF and collagen. VWF/ristocetin-induced platelet aggregation was delayed after treatment with methyl beta-cyclodextrin (mbCD), but the maximal aggregation response was not affected. Platelet spreading but not adhesion to immobilised VWF under flow was attenuated by cholesterol removal, and accompanied by moderate lowering in the spiking Ca(2+) response. On the other hand, platelet interaction with collagen was quite sensitive to cholesterol depletion. Platelet aggregation decreased after treatment with mbCD, and Ca(2+) responses were decreased, both under static and flow conditions. Cholesterol depletion affected the secondary feedback activation via release of thromboxane A(2) and ADP. The collagen-induced secretion of alpha granules and surface translocation of P-selectin and CD63 was also critically affected by cholesterol depletion. Confocal microscopy showed localization of p-Tyr at sites of contact with substrate and other platelets, where also CRDs accumulate. Our data thus reveal a more critical role for membrane cholesterol in collagen-induced than in VWF-induced Ca(2+) signalling, and furthermore support the concept that secondary activation responses are dependent on intact CRDs.

Electron Tomography and Correlative Approaches in Platelet Studies.

Blood platelets play a central role in the arrest of bleeding and the development of thrombosis. Unraveling the complex processes of platelet biogenesis from megakaryocytes, platelet adhesion, aggregation, and secretory responses are important topics in the field of hemostasis and thrombosis. Analysis of the ultrastructural changes that occur during these processes is essential for understanding the rapid membrane dynamics and has contributed substantially to our present knowledge of platelet formation and functioning. Recent developments in real-time imaging, correlative light and electron microscopy imaging (CLEM), and 3D (cryo) electron microscopy and tomography offer exciting opportunities to improve studies of the platelet adhesive responses and secretion at the ultrastructural level in a close to native environment. In this chapter we discuss and illustrate cryo preparation techniques (high-pressure freezing, vitrification), correlative LM and EM workflows, and 3D cryo-electron tomography that we apply in our current research projects.

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow.

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions. A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

Subcellular localization of tyrosine-nitrated proteins is dictated by reactive oxygen species generating enzymes and by proximity to nitric oxide synthase.

Using high-resolution immuno-electron microscopy the steady-state subcellular distribution of tyrosine-nitrated proteins in different cells and tissues was evaluated. In quiescent eosinophils and neutrophils in the bone marrow intracellular nitrated proteins were mainly restricted to the peroxidase-containing secretory granules. The inducible nitric oxide synthase (iNOS) was expressed in the same granules. Proteins nitrated on tyrosine residues were also abundant in the cytosol of circulating erythrocytes. In the vasculature, nitrated proteins were mainly located in mitochondria and endoplasmic reticulum of the endothelial cells, fibroblasts, and smooth muscle cells. Endogenous nitrated proteins were also found in chondrocytes in cartilage, where it was typically associated with the cytoplasmic interface of the endoplasmic reticulum membrane. Nitrated proteins were also prominent in the peroxisomes of liver hepatocytes and of secretory cells in the lacrimal gland. Challenge of mouse dendritic cells with lipopolysaccharide induced iNOS protein expression in cytosol and peroxisomes and was associated with an increased 3-nitrotyrosine formation in cytosol, mitochondria, and peroxisomes. These data indicate that nitric oxide-dependent protein tyrosine nitration is a physiologically relevant process localized within specific subcellular compartments in close proximity to iNOS and to enzymes capable of peroxidative chemistry and reactive oxygen species production.

Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI.

The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C- macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.

Colocalization of eNOS and the catalytic subunit of PKA in endothelial cell junctions: a clue for regulated NO production.

Localization and coordinate phosphorylation/dephosphorylation of endothelial nitric oxide synthase (eNOS) are critical determinants for the basal and stimulated production of nitric oxide. Several phosphorylation sites in eNOS have been identified as targets of the cAMP-dependent protein kinase A (PKA). Basal eNOS activity is also regulated by interaction with caveolin-1, the major coat protein of caveolae. In the present study we have examined in rat aorta endothelium the subcellular steady-state distribution of eNOS, the catalytic subunit of PKA (PKA-c), and caveolin-1. Basal eNOS expression was found in two distinct locations, the endothelial cell surface and the Golgi complex. Cell surface eNOS was equally distributed over caveolar and non-caveolar membranes but was 2.5-fold enriched on luminal lamellipodia located at endothelial cell contacts. PKA-c colocalized with eNOS in the lamellipodia, whereas caveolin-1 was absent from these membrane domains. PKA-c was also found associated with cell surface caveolae and with tubulovesicular membranes of Golgi complex and endosomes. The topological proximity of eNOS with the catalytic subunit of PKA in restricted intracellular locations may provide mechanisms for differential PKA-mediated eNOS regulation.

Immunoelectron microscopic localization of cholesterol using biotinylated and non-cytolytic perfringolysin O.

We used a proteolytically modified and biotinylated derivative of the cholesterol-binding Theta-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-beta-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the Theta-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections.

Lysosome-related organelles: unusual compartments become mainstream.

Lysosome-related organelles (LROs) comprise a group of cell type-specific subcellular compartments with unique composition, morphology and structure that share some features with endosomes and lysosomes and that function in varied processes such as pigmentation, hemostasis, lung plasticity and immunity. In recent years, studies of genetic diseases in which LRO functions are compromised have provided new insights into the mechanisms of LRO biogenesis and the regulated secretion of LRO contents. These insights have revealed previously unappreciated specialized endosomal sorting processes in all cell types, and are expanding our views of the plasticity of the endosomal and secretory systems in adapting to cell type-specific needs.