Cellular maturation of mitochondrial molybdoenzymes

The molybdenum cofactor (Moco) is an essential component present in nearly all domains of life. In mammals, Moco is part of four currently known enzymes and constitutes a crucial redox-active center involved in a number of fundamental cellular reactions. Moco-dependent enzymes are present in the cytosol but also in or at mitochondria, where Moco is integrated into sulfite oxidase (SO) and the mitochondrial amidoxime-reducing component (mARC), respectively. The family of mitochondrial Moco-enzymes is of particular interest considering the cytosolic synthesis of enzymes and cofactor, which requires a coordinated mitochondrial transport and assembly process. In the current study, the mitochondrial maturations of SO and mARC1 were thus analyzed to obtain a mechanistic understanding of the processes starting with the cytosolic syntheses of apo-proteins all the way to the formation of the mature mitochondrial enzymes. The first part of this work uncovered the cellular assembly of SO, a soluble protein of the mitochondrial intermembrane space, and revealed a Moco-dependent mitochondrial targeting mechanism. In spite of its functional bipartite N-terminal targeting signal, about 70% of SO mislocalized to the cytosol if Moco was not present. Following the identification of SO processing by the inner membrane peptidase (IMP) complex, prevention of this cleavage and thus anchoring of SO in the inner mitochondrial membrane resulted in an efficient mitochondrial targeting even in absence of Moco. SO was thereby identified to undergo a reverse translocation to the cytosol in absence of Moco, which is required to trap SO in the intermembrane space and to constitute in addition a vectorial driving force for completion of SO translocation across the TOM complex. The integration of Moco is not only essential for correct sub-mitochondrial localization, but also a prerequisite for in vivo heme integration and homodimerization of SO. In conclusion, the identified molecular hierarchy of SO maturation represents a novel link between the canonical pre-sequence pathway and folding-trap mechanisms of mitochondrial import. The other mitochondrial Moco-enzyme mARC1 was recently discovered and its sub-mitochondrial localization had remained unclear. In the second part of this study, mARC1 was shown to be localized to the outer mitochondrial membrane. As a result of the translocation process, the C-terminal catalytic core of the protein remains exposed to the cytosol and confers an N(in)-C(out) membrane orientation of mARC1. This localization is mediated by the N-terminal domain of the enzyme, being composed of a classical but weak N-terminal targeting signal and a downstream transmembrane domain. Thereby, the transmembrane domain of mARC1 is sufficient for mitochondrial targeting, while the N-terminal targeting signal seems to function as a supportive receptor for the outer mitochondrial membrane. According to its localization and targeting mechanism, mARC1 is classified as a novel signal-anchored protein. Considering the membrane integration of mARC1, an SO-similar demand of Moco for mitochondrial retention of mARC1 is not required and its N-terminal targeting motifs are sufficient for adequate mitochondrial localization. During mitochondrial import, mARC1 is not processed and membrane integration proceeds membrane potential independently but requires external ATP, which finally results in the assembly of mARC1 into high-oligomeric protein complexes

Algebraic, Block and Multiplicative Preconditioners based on Fast Tridiagonal Solves on GPUs

This thesis contributes to the field of sparse linear algebra, graph applications, and preconditioners for Krylov iterative solvers of sparse linear equation systems, by providing a (block) tridiagonal solver library, a generalized sparse matrix-vector implementation, a linear forest extraction, and a multiplicative preconditioner based on tridiagonal solves. The tridiagonal library, which supports (scaled) partial pivoting, outperforms cuSPARSE's tridiagonal solver by factor five while completely utilizing the available GPU memory bandwidth. For the performance optimized solving of multiple right-hand sides, the explicit factorization of the tridiagonal matrix can be computed. The extraction of a weighted linear forest (union of disjoint paths) from a general graph is used to build algebraic (block) tridiagonal preconditioners and deploys the generalized sparse-matrix vector implementation of this thesis for preconditioner construction. During linear forest extraction, a new parallel bidirectional scan pattern, which can operate on double-linked list structures, identifies the path ID and the position of a vertex. The algebraic preconditioner construction is also used to build more advanced preconditioners, which contain multiple tridiagonal factors, based on generalized ILU factorizations. Additionally, other preconditioners based on tridiagonal factors are presented and evaluated in comparison to ILU and ILU incomplete sparse approximate inverse preconditioners (ILU-ISAI) for the solution of large sparse linear equation systems from the Sparse Matrix Collection. For all presented problems of this thesis, an efficient parallel algorithm and its CUDA implementation for single GPU systems is provided

Pollinator monitoring in agroecosystems – general methods for evaluations in field studies

Extensive knowledge of the occurrence, condition and population changes of wild bee communities in agroecosystems is important. The knowledge is needed to understand the complexity of potential exposure routes to plant protection products in specific crops and agricultural scenarios or to evaluate possible impacts of treatments at a landscape scale taking into account other influencing parameters like the cultivation system or management practices.Extensive knowledge of the occurrence, condition and population changes of wild bee communities in agroecosystems is important. The knowledge is needed to understand the complexity of potential exposure routes to plant protection products in specific crops and agricultural scenarios or to evaluate possible impacts of treatments at a landscape scale taking into account other influencing parameters like the cultivation system or management practices

A Calibration Scheme for Non-Line-of-Sight Imaging Setups

The recent years have given rise to a large number of techniques for "looking around corners", i.e., for reconstructing occluded objects from time-resolved measurements of indirect light reflections off a wall. While the direct view of cameras is routinely calibrated in computer vision applications, the calibration of non-line-of-sight setups has so far relied on manual measurement of the most important dimensions (device positions, wall position and orientation, etc.). In this paper, we propose a semi-automatic method for calibrating such systems that relies on mirrors as known targets. A roughly determined initialization is refined in order to optimize a spatio-temporal consistency. Our system is general enough to be applicable to a variety of sensing scenarios ranging from single sources/detectors via scanning arrangements to large-scale arrays. It is robust towards bad initialization and the achieved accuracy is proportional to the depth resolution of the camera system. We demonstrate this capability with a real-world setup and despite a large number of dead pixels and very low temporal resolution achieve a result that outperforms a manual calibration

Light-induced cell damage in live-cell super-resolution microscopy

Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities