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The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view. In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification. In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.

We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50-150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications., Competing Interests: The endocytic fBSA-Au5 and fBSA-Au10 fiducials reported here are available in the product listing of Cell Microscopy Core, UMC Utrecht., (© 2022 The Author(s).)

Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41 S285P and VPS41 R662 * ; VPS41 c.1423-2A>G and VPS41 R662 enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41 * ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41 S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41 S285P /VPS41 R662 * abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Recycling endosomes maintain plasma membrane homeostasis and are important for cell polarity, migration, and cytokinesis. Yet, the molecular machineries that drive endocytic recycling remain largely unclear. The CORVET complex is a multi-subunit tether required for fusion between early endosomes. Here we show that the CORVET-specific subunits Vps3 and Vps8 also regulate vesicular transport from early to recycling endosomes. Vps3 and Vps8 localise to Rab4-positive recycling vesicles and co-localise with the CHEVI complex on Rab11-positive recycling endosomes. Depletion of Vps3 or Vps8 does not affect transferrin recycling, but delays the delivery of internalised integrins to recycling endosomes and their subsequent return to the plasma membrane. Consequently, Vps3/8 depletion results in defects in integrin-dependent cell adhesion and spreading, focal adhesion formation, and cell migration. These data reveal a role for Vps3 and Vps8 in a specialised recycling pathway important for integrin trafficking.

Lysosomes are the main degradative compartments of eukaryotic cells. The CORVET and HOPS tethering complexes are well known for their role in membrane fusion in the yeast endocytic pathway. Yeast Vps33p is part of both complexes, and has two mammalian homologues: Vps33A and Vps33B. Vps33B is required for recycling of apical proteins in polarized cells and a causative gene for ARC syndrome. Here, we investigate whether Vps33B is also required in the degradative pathway. By fluorescence and electron microscopy we show that Vps33B depletion in HeLa cells leads to significantly increased numbers of late endosomes that together with lysosomes accumulate in the perinuclear region. Degradation of endocytosed cargo is impaired in these cells. By electron microscopy we show that endocytosed BSA-gold reaches late endosomes, but is decreased in lysosomes. The increase in late endosome numbers and the lack of internalized cargo in lysosomes are indicative for a defect in late endosomal-lysosomal fusion events, which explains the observed decrease in cargo degradation. A corresponding phenotype was found after Vps33A knock down, which in addition also resulted in decreased lysosome numbers. We conclude that Vps33B, in addition to its role in endosomal recycling, is required for late endosomal-lysosomal fusion events., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Olanzapine pamoate injection is an anti-psychotic depot to be administered intramuscularly once every 2-4 weeks. A post-injection syndrome may occur shortly after administration, resulting in an acute intoxication with olanzapine., Case Description: A 42-year-old patient with a schizophrenic disorder lost consciousness 30 min after administration of olanzapine pamoate. He was admitted to a nearby hospital with tachycardia, hypotension, pin-point pupils and respiratory distress leading to reduced oxygen saturation. He was ventilated during one night and recovered within 2 days., Conclusion: A post-injection syndrome may develop after administration of olanzapine pamoate when the entire dose olanzapine is released at once from the muscle. Therefore, the patient should be observed for at least 3 hours after every injection. The risk of a post-injection syndrome and the necessary observation period should to be taken into account when deciding to start treatment with olanzapine pamoate depot.

The homotypic fusion and protein sorting (HOPS) complex is a multisubunit tethering complex that in yeast regulates membrane fusion events with the vacuole, the yeast lysosome. Mammalian homologs of all HOPS components have been found, but little is known about their function. Here, we studied the role of hVps41 and hVps39, two components of the putative human HOPS complex, in the endo-lysosomal pathway of human cells. By expressing hemagglutinin (HA)-tagged constructs, we show by immunoelectron microscopy (immunoEM) that both hVps41 and hVps39 associate with the limiting membrane of late endosomes as well as lysosomes. Small interference RNA (siRNA)-mediated knockdown of hVps41 or hVps39 resulted in an accumulation of late endosomes, a depletion in the number of lysosomes and a block in the degradation of endocytosed cargo. Lysosomal pH and cathepsin B activity remained unaltered in these conditions. By immunoEM we found that hVps41 or hVps39 knockdown impairs homotypic fusion between late endosomes as well as heterotypic fusion between late endosomes and lysosomes. Thus, our data show that both hVps41 and hVps39 are required for late endosomal-lysosomal fusion events and the delivery of endocytic cargo to lysosomes in human cells., (© 2012 John Wiley & Sons A/S.)

Targeted delivery of lysosome-associated membrane proteins is important for lysosome stability and function. Here we identify a pathway for transport of lysosome-associated membrane proteins directly from the trans-Golgi network to late endosomes, which exists in parallel to mannose 6-phosphate receptor and clathrin-dependent transport of lysosomal enzymes to early endosomes. By immunoelectron microscopy we localized endogenous LAMP-1 and -2 as well as LAMP-1-mGFP to non-coated, biosynthetic carriers at the trans-Golgi network and near late endosomes. These LAMP carriers were negative for mannose 6-phosphate receptor, adaptor-protein complex-1, secretory albumin and endocytic markers, but contained the homotypic fusion and protein sorting complex component hVps41 and the soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein VAMP7. Knockdown of hVps41 or VAMP7 resulted in the accumulation of lysosome-associated membrane protein carriers, whereas knockdown of hVps39 or hVps18 did not, indicating that the effect of hVps41 is independent of CORVET/HOPS. Mannose 6-phosphate receptor carriers remained unaffected upon hVps41 or VAMP7 knockdown, implicating that hVps41 and VAMP7 are specifically involved in the fusion of trans-Golgi network-derived lysosome-associated membrane protein carriers with late endosomes.

In metazoans, lysosomes are characterized by a unique tubular morphology, acidic pH, and specific membrane protein (LAMP) and lipid (cholesterol) composition as well as a soluble protein (hydrolases) composition. Here we show that perturbation to the eye-color gene, light, results in impaired lysosomal acidification, sterol accumulation, altered endosomal morphology as well as compromised lysosomal degradation. We find that Drosophila homologue of Vps41, Light, regulates the fusion of a specific subset of biosynthetic carriers containing characteristic endolysosomal membrane proteins, LAMP1, V0-ATPase and the cholesterol transport protein, NPC1, with the endolysosomal system, and is then required for the morphological progression of the multivesicular endosome. Inhibition of Light results in accumulation of biosynthetic transport intermediates that contain these membrane cargoes, whereas under similar conditions, endosomal delivery of soluble hydrolases, previously shown to be mediated by Dor, the Drosophila homologue of Vps18, is not affected. Unlike Dor, Light is recruited to endosomes in a PI3P-sensitive fashion wherein it facilitates fusion of these biosynthetic cargoes with the endosomes. Depletion of the mammalian counterpart of Light, hVps41, in a human cell line also inhibits delivery of hLAMP to endosomes, suggesting an evolutionarily conserved pathway in metazoa., (© 2011 John Wiley & Sons A/S.)

In this report, the use of DNA vaccination to induce class II restricted antigen specific proliferative responses was studied. To this end, a construct encoding the invariant chain (Ii) was engineered in which the Class II associated invariant chain peptide (CLIP) sequence was replaced by an immunogenic epitope derived form Heat Shock Protein 60, HSP60 178-186. Transfection studies in vitro showed that this construct can be used to efficiently load MHC class II molecules and present epitopes to MHC class II restricted antigen specific T cells. In addition, we showed that intradermal immunisation of Lewis rats with these constructs induced antigen specific T cells in vivo. Therefore, our Ii-gene constructs can be used to immunise for defined CD4+ T cell epitope sequences.

In recent years, the measurement of soluble CD44 levels in the circulation of patients with malignant diseases has been introduced as a new and simple diagnostic tool for the detection of human cancer. The high CD44v6 expression in head and neck squamous cell carcinoma (HNSCC) would enable the use of soluble CD44v6 proteins present in the circulation of HNSCC patients as a marker of disease. In the present study, we determined CD44v6 plasma levels using a domain-specific ELISA in healthy volunteers, non-cancer patients, and HNSCC patients before and after surgical removal of the tumor. A difference between the CD44v6 plasma levels of HNSCC patients and controls could not be observed. Moreover, surgical removal of the tumor did not result in a reduction of the CD44v6 plasma level in the HNSCC patients. In addition, the spectrum of soluble v6-containing CD44 proteins present in the plasma of HNSCC patients and controls was determined by immunoprecipitation experiments, but again, tumor-related isoforms could not be distinguished in patient samples. Additional experiments to unravel the biological source of these circulating proteins indicated surprisingly that the v6-containing proteins present in the circulation of healthy individuals are only released in part, if at all, by activated lymphocytes or other nucleated blood cells. Most circulating CD44v6 proteins seem to be derived from the normal epithelial cell compartments, including breast cells, colon cells, and squamous cells. Taken together, these data do not support the use of soluble CD44v6 as a tumor marker in HNSCC or any other tumor type that has developed from tissues producing soluble isoforms.

In previous studies, we described the selective reactivity of monoclonal antibody E48 with normal squamous and transitional epithelia and their malignant counterparts and the capacity of monoclonal antibody E48 for selective tumor targeting in head and neck cancer patients. Cloning of the E48 encoding cDNA and elucidation of the gene structure enabled the selection of an intron-spanning primer set for the detection of circulating tumor cells in blood and bone marrow of head and neck cancer patients. Extensive optimizations led to a reproducible reverse transcriptase-PCR assay with an internal standard for RNA quality control and an external standard for sensitivity control. In reconstruction experiments, we were able to reach a reproducible sensitivity of one single tumor cell per 7 ml of blood (2 x 10(7) nucleated cells). When applying this method to patient material, we were able to detect positive signal in 35% of the bone marrow samples (0 of 2 stage II, 0 of 4 stage III, 4 of 11 stage IV, and 4 of 6 recurrences) and 10% of the blood samples (2 of 21) of patients with squamous cell carcinoma of the head and neck. The specificity of the method was demonstrated on 29 blood and bone marrow samples of noncancer controls, which were all negative. Our study shows the feasibility of E48 reverse transcriptase-PCR for the detection of squamous cells in nonsquamous tissues.

The improvement of detection and eradication of minimal residual disease, to reduce local and distant relapse after primary therapy, is one of the major challenges in the management of squamous cell carcinoma of the head and neck (HNSCC). This paper describes perspectives arising from the use of monoclonal antibodies (MAbs) E48 and U36 directed against HNSCC-associated antigens, and the molecular characterization of these antigens. Novel strategies for the detection of minimal residual disease are outlined and comprise the use of immunocytochemistry in combination with automated image analysis, and the use of an E48-specific reverse transcriptase-polymerase chain reaction (RT-PCR) method. These methods have potential for the detection of single HNSCC cells in lymph nodes, bone marrow and peripheral blood, and may contribute to the better staging of head and neck cancer in the near future. Besides this, preclinical and clinical data on HNSCC targeting with radiolabelled MAbs E48 and U36 are summarized, illustrating the perspectives of systemic adjuvant radioimmunotherapy with these MAbs.

Brain-stem auditory evoked responses (BAERs) were recorded both to rarefaction and condensation click stimuli in 92 normal hearers and 78 patients with varying degrees of cochlear hearing loss (N = 340 ears). Frequency distributions of rarefaction minus condensation (R - C) latency and amplitude differences revealed clinically significant polarity effects in a substantial percentage of the patients studied. Bivariate plots of R - C latency and amplitude differences versus average high frequency hearing loss (PTA 3) demonstrated that the magnitude of the R - C latency and amplitude differences also seemed to be influenced by degree of high frequency hearing loss. Results are discussed relative to the phase-locking properties of the afferent auditory nerve fibers and the possible electrodiagnostic consequences of recording the BAER either to alternating or condensation clicks.