The bacterial cell wall is a nearly universal structure that offers protection and gives the cell its shape. However, environmental stressors, such as cell wall-targeting antibiotics and... Show moreThe bacterial cell wall is a nearly universal structure that offers protection and gives the cell its shape. However, environmental stressors, such as cell wall-targeting antibiotics and hyperosmotic conditions, can induce bacteria to shift to a wall-deficient state. It is unknown whether the lack of this cell wall ‘barrier’ can enable DNA exchange via horizontal gene transfer (HGT), a process that facilitates the spread of antibiotic resistance amongst bacteria. The research in this thesis confirms that wall-deficiency enables HGT in the actinobacterium Kitasatospora viridifaciens. Specifically, we show that wall-deficient L-forms take up extracellular material including DNA, sugar polymers and lipid nanoparticles via an endocytosis-like mechanism, which in case of DNA uptake leads to genetic transformation. Moreover, wall-less cells exchange genomic DNA in a DNAse-resistant manner, most likely via cell-cell fusion and/or production of extracellular vesicles. We also isolated vesicle-producing actinobacteria from a wastewater treatment plant, an environment that is considered a hotspot for the spread of antibiotic resistance genes. Lastly, growth of K. viridifaciens under hyperosmotic stress conditions leads to genetic instability, which together with HGT can contribute to enhanced genome plasticity. In summary, this thesis provides important insights into the consequences of bacterial cell wall-deficiency for HGT and evolution. Show less
Biological cells, the basic building blocks of all life forms, are surrounded by a lipid membrane. More than half of the membrane is occupied by membrane proteins, which can regulate the cell... Show moreBiological cells, the basic building blocks of all life forms, are surrounded by a lipid membrane. More than half of the membrane is occupied by membrane proteins, which can regulate the cell functionality through specific arrangements. To regulate the arrangements several proteins have to work together. In addition to direct forces, there exists an indirect force between the proteins, which stems from their deformation of the membrane and contributes to their self-organization. Since the actual membrane is very crowded and proteins are too tiny and complex to measure this interaction, in this thesis we used a model system consisting of lipid membranes and solid particles to study the deformation-mediated interaction. We experimentally confirmed for the first time that, unlike many known forces, this deformation-mediated interaction is not additive, i.e. the strength and range of three (or more) deformations cannot be obtained by simple addition of the interactions between pairs of deformations. We found that the interaction weakens with increasing number of membrane-deforming particles and that the particle become less ordered. We investigated deformations in both directions of the membrane and found that the interaction can be both repulsive and attractive, and furthermore depends on the shape of the deformation. This thesis helps to better understand the organization of proteins that deform cellular membranes. Show less
Endocytosis is a crucial mechanism for the cell to maintain homeostasis. By vesicular transport the cell is able to take up cargo from the cell surface, send molecules to the plasma membrane for... Show moreEndocytosis is a crucial mechanism for the cell to maintain homeostasis. By vesicular transport the cell is able to take up cargo from the cell surface, send molecules to the plasma membrane for excretion or to regulate positioning of internalized cargo in the cell. This thesis describes the regulation of transport complexes associated to these vesicles that determines their movement in the cell. Through recruitment of RAB7GAP the GTPase Rab7 can be removed from the vesicular membrane, allowing for transport mediated by another GTPase called Arl8b.The second part of this thesis focusses on the bacterium Salmonella which manipulates the endosomal system in order to create a niche that supports its own survival. By excretion of effector molecules Salmonella imodulates the membrane content of vesicles and thereby prevents its own lysosomal degradation. This allows Salmonella to create a Salmonella containing vesicle compartment that benefits its own survival in the host cell. This thesis describes how release of these effector molecules can support transformation of host cells. These results explain the strrong correlation between gallbladder cancer and chronic S. Typhi infections observed in patients from countries like India, where S. Typhi infections are still common. Show less
Understanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation... Show moreUnderstanding of the regulation mechanisms of CXCR4 signaling is essential for revealing its role in physiological and pathological processes. Though biochemical pathways following CXCR4 activation by its ligand CXCL12 are well established, knowledge about the receptor dynamics on the plasma membrane remains limited. Here we used Ewing sarcoma-derived cells to unravel the processes that are involved in regulating CXCR4 dynamics on the plasma membrane during receptor signaling. Single-molecule epi-fluorescence microscopy showed that CXCR4 was present in monomeric state on the plasma membrane independent of receptor stimulation. However, upon activation freely diffusing receptors were immobilized in a ligand concentration-dependent manner. CXCR4 immobilization was strongly correlated with the ability for G-protein signaling and was a precursor of subsequent endocytotic events. Our data suggest that, a balanced regulation of G-protein dependent and independent pathways is required for controlling CXCR4 receptor mobility, and potentially subsequent controlled signal transduction. (C) 2015 Published by Elsevier B.V. Show less
Graauw, M. de; Cao, L.; Winkel, L.; Miltenburg, M.H.A.M. van; Dévédec, S.E. le; Klop, M.; ... ; Water, B. van de 2014
Endocytosis can be conceptually broken down into two stages: The formation of the nascent vesicle containing the ingested proteins and lipids, and its subsequent maturation into a degradative... Show moreEndocytosis can be conceptually broken down into two stages: The formation of the nascent vesicle containing the ingested proteins and lipids, and its subsequent maturation into a degradative compartment. During this maturation process proteins can be recycled back to the plasma membrane to prevent degradation. The vesicular membrane and the luminal contents undergo considerable remodeling to transform this compartment into a degradative one. This stops signal transduction by degrading the receptors within the vesicle and also generates antigens for presentation on MHC class II molecules in order to elicit an adaptive immune response. The engulfment of large particles and microorganisms (e.g. pathogens) is referred to as phagocytosis. The vesicle created by phagocytosis is called the phagosome. Phagosome maturation is considered to involve a complex sequence of fusion and fission events with the sub compartments of the endocytic pathway. In many ways, phagosome maturation recapitulates the progression of cargo along the endocytic pathway. In this thesis we have described molecular mechanisms that regulate the endocytic route and how pathogens explore this endocytic route to ensure survival. Show less
Antigen presentation by MHC class II is critical for immune responses against pathogens and tumors. Antigen loading occurs primarily in lysosomal-related organelles (LROs) known as MIICs.... Show moreAntigen presentation by MHC class II is critical for immune responses against pathogens and tumors. Antigen loading occurs primarily in lysosomal-related organelles (LROs) known as MIICs. Ultimately, the MHC II-peptide complexes are transported for cell surface display. Here, we study intracellular transport of MIICs, a poorly understood process in MHC II antigen presentation. We propose that Rab7 lies at the heart of transport by assembling a specific receptor (Rab7-RILP-ORP1L) for the minus end-directed dynein-dynactin motor on the cytosolic face of MIICs/LROs. Full activation of transport requires a second receptor, _III spectrin. Whereas this model explains how Rab7 controls minus end-directed transport, it does not suffice to explain the characteristic pattern of bidirectional motility exhibited by MIICs/LROs. Here, we propose that cholesterol dictates ORP1L conformation which acts as a switch controlling access of dynein-dynactin to Rab7-RILP, thereby regulating LRO positioning, as observed in NPC disease. Rab7-RILP-ORP1L may also integrate transport and tethering of MIICs/LROs, consecutive processes within the endocytic pathway. Show less
Filamentous fungi are multicellular eukaryotic organisms, which represent a separate taxonomic group organisms within the fungal kingdom, apart from the yeasts. These fungi always need a substrate... Show moreFilamentous fungi are multicellular eukaryotic organisms, which represent a separate taxonomic group organisms within the fungal kingdom, apart from the yeasts. These fungi always need a substrate to grow on, this can be living or dead material. Fungi possess the capacity to secrete high levels of enzymes. Because of this specific property, fungi are already used for centuries as miniature factories for the production of extracellular proteins. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of, heterologous proteins. This low protein yield is likely to be caused by impaired secretion of the heterologous protein. Evidence support the idea that a bottleneck for protein production is post-translational, possibly within the protein secretion pathway. To be able to improve heterologous protein secretion in fungi the (molecular) mechanism(s) of the protein secretion pathway was studied and resulted in this thesis. In Chapter 1 the knowledge about the secretory pathway in the yeast S. cerevisiae has been used as starting point to discuss and review different aspects of protein secretion in filamentous fungi. Special focus is on the comparison of the presence and function of secretion related small GTPases in yeasts, mammalian cells and filamentous fungi. Chapter 2 describes the visualisation of different cell organelles from A. niger using GFP-reporter proteins. To target GFP to a specific organelle, the GFP was fused to an organelle specific protein, or part of such a protein. In this way it was possible to visualize, nuclei, the endoplasmic reticulum (ER) and vacuoles. In addition, by fusing GFP to a protein that is efficiently secreted (glucoamylase), also the protein secretion process could be visualised. Chapter 3 describes the identification of several small GTPases in A. niger. The function of one of them, srgA, has been studied in more detail. In Chapter 4 the in depth functional characterisation of a second secretion related GTPase from A. niger named srgC is described. Here it is shown that this secretion related GTPase is specifically important for vacuolar biosynthesis which is visualized by the GFP-reporters described in chapter 2. In Chapter 5 a study on heterologous protein production in A. niger is described. Here a novel screening method is used based on a fusion protein between the well secreted A. niger glucoamylase protein fused with a laccase from Pleurotus ostreatus. With this method laccase hyper-secretion mutants were isolated. 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
