In this thesis I explored the ins and outs of the multivesicular body, the pathways that intersect at this central endocytic compartment, the dynamics governing its lumen and how such processes... Show moreIn this thesis I explored the ins and outs of the multivesicular body, the pathways that intersect at this central endocytic compartment, the dynamics governing its lumen and how such processes impact various fields of cell biology and disease. Show less
Dit proefschrift bevat onderzoek over verschillende processen binnen het intracellulaire domein. Als eerst wordt er onderzocht hoe een cel zijn oplosbare eiwitten in de correcte compartimenten... Show moreDit proefschrift bevat onderzoek over verschillende processen binnen het intracellulaire domein. Als eerst wordt er onderzocht hoe een cel zijn oplosbare eiwitten in de correcte compartimenten krijgt na mitosis. Het tweede omschrijft een mechanisme dat verantwoordelijk is voor het structureren van het endoplasmatisch reticulum. Als derde werd onderzocht wat voorn rol het eiwit MOSPD2 vervult binnen contacten tussen het ER, Golgi apparatus en lysosomen. Show less
The lysosomal β-glucosidase named glucocerebrosidase (GCase) is a retaining β-glucosidase that hydrolyzes the glycosphingolipid glucosylceramide (GlcCer) to ceramide and glucose at acid pH.... Show moreThe lysosomal β-glucosidase named glucocerebrosidase (GCase) is a retaining β-glucosidase that hydrolyzes the glycosphingolipid glucosylceramide (GlcCer) to ceramide and glucose at acid pH. Inherited deficiency of GCase causes Gaucher disease (GD), a relatively common lysosomal storage disorder. GCase fulfills another crucial function beyond lysosomes. The enzyme generates ceramides from GlcCer molecules in the outer part of the skin, the stratum corneum. This is essential for skin barrier properties compatible with terrestrial life. GCase is catalytically versatile and can hydrolyze as well as catalyze transglycosylation.In this thesis a novel sensitive in situ method for the detection of active GCase in skin sections is described. Followed by a study of skin sections of patiens with atopic dermatitis revealing that the localization and activity of GCase and acid sphingomyelinase (ASM) was abnormal in skin of AD patients, particularly at lesional skin sites.It is demonstrated that GCase not only cleaves 4-methylumbelliferyl-β-D-glucose, but also 4-methylumbelliferyl-β-D-xylose. It is reported for the first time that GCase is able to transxylosylate cholesterol to render xylosyl-β-cholesterol (XylChol). The formed XylChol can act as a subsequent acceptor for further transxylosylation, rendering di-xylosyl-cholesterol. And finally the discovery of of GlcChol as novel component of human epidermis is reported. Show less
This thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an... Show moreThis thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an atypical and a conventional chemokine receptor during chemotaxis in macrophages, the role of chemotactic signaling in cell polarization and explores an in vivo screening workflow for human anti-inflammatory drugs using zebrafish. Show less
Diabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of... Show moreDiabetes mellitus type 2 (DM) is a major risk factor for developing active tuberculosis (TB) disease, yet the causal mechanisms driving this association remain largely elusive. As the incidence of DM is rising, especially in TB endemic countries, it is important to identify the relevant immunological and metabolic processes that underlie TB-DM comorbidity, because such insights will facilitate optimal treatment, diagnosis and prevention. In this thesis, we have started to unravel key factors underlying the association between TBand DM using two approaches. Firstly, we identified and analyzed human macrophage subsets and studied the interactions between these human cells and a major pathogen, Mycobacterium tuberculosis (Mtb), and the specific metabolic changes involved using well-controlled in vitro systems. Next, we employed metabolomics to determine the impact of concurrent TB-DM on circulating metabolites in patient cohorts ex vivo. In this thesis we present evidence derived from in vitro experiments and from ex vivo observational data which collectively suggest a pathogenic role of atherogenic lipid species during TB development. Show less
Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer__s disease. This thesis describes the regulation of late endosomal dynamics by... Show moreLate endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer__s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the direction of late endosomal transport by dynein-motors, and that this process is disrupted in Niemann-Pick disease. Furthermore cholesterol also regulates tethering via the multi-subunit HOPS complex. Homologues of this complex are mutated in ARC syndrome patients and the cellular consequences are described in this thesis. We find that the cholesterol sensing proteins ORP1L and MLN64 are major regulators of late endosomal transport, and that this transport is further regulated by the endoplasmatic reticulum. Show less
The most common neurodegenerative genetic childhood disease, juvenile neuronal ceroid lipofuscinosis (JNCL), is a lysosomal storage disorder that is caused by mutations in the CLN3 gene. In... Show moreThe most common neurodegenerative genetic childhood disease, juvenile neuronal ceroid lipofuscinosis (JNCL), is a lysosomal storage disorder that is caused by mutations in the CLN3 gene. In patients' lysosomes material accumulates primarily composed of Subunit c. How mutations in CLN3 lead to this disease is unknown. The nematode Caenorhabditis elegans is useful for researching genetic neurodegenerative diseases. C. elegans has three CLN3 homologous proteins, cln-3.1, cln-3.2, and cln-3.3. Nematode cln-3 mutants were generated, crossed to obtain double and triple mutants, and their phenotypes were studied. Cln-3 triple mutants have slightly decreased life span and little decreased brood size. This triple mutant is viable, indicating that cln-3 genes are not essential for nematode life under laboratory conditions. These phenotypes are not useable in genetic screens and no neurological or other robust phenotypes were present. Expression analysis was performed to focus the phenotypic analysis. The short life span of C. elegans may explain the lack of accumulated materials. Therefore, Subunit c was inducibly overexpressed in cln-3 triple mutants. This is deleterious to nematodes, appearing to affect mitochondrial ultrastructure, although similar in wildtype and cln-3 mutants. Additional experiments with this nematode model may increase our understanding of the function of the cln-3 genes. Show less
Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of... Show moreMany intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is largely unclear how these motor proteins are targeted to specific compartments. Rab GTPases recruit and/or activate several proteins involved in membrane fusion and vesicular transport. They associate with specific compartments and therefore are ideal candidates for controlling motor protein recruitment. This work shows that dynein-dynactin motor recruitment to lysosomal compartments requires activation of the GTPase Rab7 that subsequently associates with its effector protein, RILP (for Rab7-Interacting Lysosomal Protein). RILP maintains Rab7 in the vesicle-bound, activated state and transmits a signal for specific recruitment of the dynein-dynactin motor. As a consequence, lysosomes are transported towards the minus-end of microtubules. This signalling cascade thus regulates lysosomal transport. In addition, we showed that this pathway also regulates transport of several other lysosomal compartments, including Salmonella-containing vacuoles and melanosomes. Show less
