This thesis describes the cryo-electron microscopic (cryo-EM) analyses of intracellular viral replication structures. The main methodology used for the analyses was cellular electron cryo... Show moreThis thesis describes the cryo-electron microscopic (cryo-EM) analyses of intracellular viral replication structures. The main methodology used for the analyses was cellular electron cryo-tomography (cryo-ET) to study the replication organelles induced by cornoa- and arteriviruses at macromolecular resolution. Cellular cryo-ET is a young technique incorporating focused ion beam (FIB) milling of 100-300 nm thin cryo-lamellae containing intracellular material readily imageble by high-resolution cryo-EM. However, this workflow is low in throughput and error-prone. Part of the thesis describes an improved sample-preparation strategy that successfully increased the throughput of this workflow. Furthermore, this method was applied on cells infected by coronaviruses and unveiled so far unseen protein complexes that seem to shuttle viral RNA across the double-membranes of the viral replication organelles. It was found that these molecular pores are (partially) formed by viral proteins containing transmembrane-domains. The molecular pores appear to be a central component in the replication cycle of corona- and arteriviruses, two virus families united in the virus order Nidovirales. Finally, the structure and function of the viral replication organelles are reviewed and how past and future technical developments in EM did and likely will contribute to our growing understanding of these. Show less
Replication organelles (ROs) are novel membrane rearrangements formed in the host cell cytosol during infections of positive-sense RNA (+RNA) viruses, which are utilised as platforms for viral... Show moreReplication organelles (ROs) are novel membrane rearrangements formed in the host cell cytosol during infections of positive-sense RNA (+RNA) viruses, which are utilised as platforms for viral replication. This thesis addresses several open questions regarding the ROs of picornaviruses, including the host factor requirements for enterovirus RO formation, their potential functions, and the on-going uncertainty surrounding their origin. It extends our detailed understanding of enterovirus ROs to a second picornavirus genus, the cardioviruses, with broader ramifications for the development of antiviral strategies. The resulting data highlight an unexpected degree of flexibility in the use of membranes for vRNA synthesis, asking for a re-examination of how we define the replication organelles of +RNA viruses. Show less
The research in this thesis is focused on tomographic reconstruction based on two imaging modalities in electron microscopy. The first modality is high angle annular dark field scanning... Show moreThe research in this thesis is focused on tomographic reconstruction based on two imaging modalities in electron microscopy. The first modality is high angle annular dark field scanning transmission microscopy (HAADF-STEM), and the second modality is energy-dispersive X-ray spectroscopy (EDS). In this Ph.D. thesis, we propose several approaches to pave the way for HAADF- STEM + EDS tomography: (1) the HAADF-EDS bimodal tomographic reconstruction technique, which is based on jointly modeling the consistency of the two imaging modalities; (2) TNV-regularized joined reconstruction which allows to incorporate the prior knowledge that common edges exist in the reconstructions from HAADF and EDS data respectively; (3) a set of algorithmic recipes to tailor various reconstruction algorithms for given experimental conditions and sample properties; (4) an algorithm for automatically correcting the nonlinear damping effects in HAADF-STEM tomographic data. Show less
Electron microscopy has become an extremely important techniquein a wide variety of elds. The resolving power is vastly superiorto light microscopes and electron microscopy has proven tobe valuable... Show moreElectron microscopy has become an extremely important techniquein a wide variety of elds. The resolving power is vastly superiorto light microscopes and electron microscopy has proven tobe valuable in elds ranging from archaeology and geology to biology andcondensed-matter physics.A major disadvantage is that the electron energy used in conventional ElectronMicroscopy (EM) ranges from 10’s to 100’s of keV. Such energetic electronscan signicantly damage the specimen. This is especially relevant in thestudy of biological samples and organic materials in general. Major eorts arebeing made to avoid this radiation damage from interfering with the studyof such materials. There are several approaches to minimize damage in EM.These include developing better detectors such that lower electron doses aresucient to form an image, and lowering the electron energies to several keV.In this dissertation I present the development of, and measurements with, atransmission electron microscope that uses electron energies ve orders ofmagnitude lower than in conventional Transmission Electron Microscopes(TEMs). The energies we use are in the order of a few eV. Hence, we call ourtechnique ’eV-TEM’. Show less
In this thesis the combinatorial use of bioorthogonal labelling and Electron Microscopy (EM)-based imaging techniques is explored to enable observations of specific molecular targets in their... Show moreIn this thesis the combinatorial use of bioorthogonal labelling and Electron Microscopy (EM)-based imaging techniques is explored to enable observations of specific molecular targets in their ultrastructural context within the cell. Show less
Lenchenkov, N.S.; Glasbergen, G.; Kruijsdijk, C. van; Vulovic, M.; Bos, E.; Koning, R.I.; Cassiola, F. 2017
ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM... Show moreORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure. Show less
Koning, R.I.; Faas, F.G.; Boonekamp, M.; Visser, B. de; Janse, J.; Wiegant, J.C.; ... ; Koster, A.J. 2014
In this thesis, the mode of action of stratum corneum moisturizers is studied using a variety of techniques: cryo-scanning electron microscopy, freeze fracture transmission electron microscopy,... Show moreIn this thesis, the mode of action of stratum corneum moisturizers is studied using a variety of techniques: cryo-scanning electron microscopy, freeze fracture transmission electron microscopy, small angle X-ray diffraction and Fourier transform infrared spectroscopy. Show less
During developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome... Show moreDuring developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome condensation and segregation. Several unique protein families are involved in the control of this process, including that of the SsgA-like proteins (SALPs). While SsgA and SsgB are essential for sporulation-specific cell division in S. coelicolor, SsgC-G are responsible for correct DNA segregation/condensation, spore wall synthesis, autolytic spore separation, or exact septum localisation. The SALPs are a novel protein family that acts through timing and localisation of the activity of penicillin-binding proteins and autolysins, thus controlling important steps during the initiation and the completion of sporulation. The formation of septa is initiated by the formation of a ring of the tubulin-like protein FtsZ (the Z-ring), functioning as a scaffold for the construction of septa. Subsequently, other cell division proteins are recruited to the Z-ring, forming the divisome. In S. coelicolor, the cell division proteins FtsE and FtsX participate during autolytic spore separation, and most likely function by re-importing peptidoglycan subunits for recycling. The cytoskeletal protein MreB is involved in cell shape determination and chromosome segregation in many rod-shaped bacteria. In S. coelicolor, the actin-like proteins MreB and Mbl are not essential for vegetative growth but exert their function in the formation of environmentally stable spores, thereby primarily influencing the assembly of the spore wall. Show less