Raimund J. Ober

Localization Accuracy in Single-Molecule Microscopy

One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by, lambda(em)/2pi n(a) square root of gammaAt, where lambda(em), n(a), gamma, A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.

Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn.

The MHC class I-related receptor, FcRn, plays a central role in regulating the serum levels of IgG. FcRn is expressed in endothelial cells, suggesting that these cells may be involved in maintaining IgG levels. We have used live cell imaging of FcRn-green fluorescent protein transfected human endothelial cells to analyze the intracellular events that control IgG homeostasis. We show that segregation of FcRn-IgG complexes from unbound IgG occurs in the sorting endosome. FcRn or FcRn-IgG complexes are gradually depleted from sorting endosomes to ultimately generate multivesicular bodies whose contents are destined for lysosomal degradation. In addition, the pathways taken by FcRn and the transferrin receptor overlap, despite distinct mechanisms of ligand uptake. The studies provide a dynamic view of the trafficking of FcRn and its ligand and have relevance to understanding how FcRn functions to maintain IgG homeostasis.

High Accuracy 3D Quantum Dot Tracking with Multifocal Plane Microscopy for the Study of Fast Intracellular Dynamics in Live Cells

Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.

Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG homeostasis in mice

The MHC class I-related receptor FcRn regulates the levels and persistence of IgG in vivo. This receptor salvages IgG from lysosomal degradation within cells, and the binding properties of an IgG for FcRn correlate with in vivo half-life. FcRn is expressed at multiple different sites throughout adult life. However, the cell types and sites at which FcRn maintains IgG homeostasis are not well defined. Toward understanding the sites of FcRn function, we have generated a mouse strain in which this Fc receptor can be conditionally deleted. In combination with mice that express Cre recombinase under the control of the Tie2 promoter (Tie2-Cre), the effect of site-specific deletion of floxed FcRn in endothelial and hematopoietic cells on IgG persistence was analyzed. The pharmacokinetics and steady-state levels of IgG in Tie2-Cre mice that are homozygous for the floxed FcRn allele reveal a complete loss of FcRn function in regulating the half-lives of wild-type IgG. The primary sites for the maintenance of endogenous IgGs in mice are therefore endothelial and hematopoietic cells.

Balanced parametrization of classes of linear systems

Canonical forms and parametrizations are presented for several sets of minimal systems of given dimension: asymptotically stable systems, allpass systems, bounded real systems, positive real systems, minimum-phase systems, and the class of all minimal systems. The approach is based on balancing techniques for these classes of systems. Applications of these results to Hankel operators and model reduction are discussed.

Chapter 4 Multitasking by Exploitation of Intracellular Transport Functions: The Many Faces of FcRn

The MHC Class I-related receptor, FcRn, transports antibodies of the immunoglobulin G (IgG) class within and across a diverse array of different cell types. Through this transport, FcRn serves multiple roles throughout adult life that extend well beyond its earlier defined function of transcytosing IgGs from mother to offspring. These roles include the maintenance of IgG levels and the delivery of antigen in the form of immune complexes to degradative compartments within cells. Recent studies have led to significant advances in knowledge of the intracellular trafficking of FcRn and (engineered) IgGs at both the molecular and cellular levels. The engineering of FcRn-IgG (or Fc) interactions to generate antibodies of increased longevity represents an area of active interest, particularly in the light of the expanding use of antibodies in therapy. The strict pH dependence of FcRn-IgG interactions, with binding at pH 6 that becomes essentially undetectable as near neutral pH is approached, is essential for efficient transport. The requirement for retention of low affinity at near neutral pH increases the complexity of engineering antibodies for increased half-life. Conversely, engineered IgGs that have gained significant binding for FcRn at this pH can be potent inhibitors of FcRn that lower endogenous IgG levels and have multiple potential uses as therapeutics. In addition, molecular studies of FcRn-IgG interactions indicate that mice have limitations as preclinical models for FcRn function, primarily due to cross-species differences in FcRn-binding specificity.

Elucidation of intracellular recycling pathways leading to exocytosis of the Fc receptor, FcRn, by using multifocal plane microscopy.

The intracellular events on the recycling pathway that lead from sorting endosomes to exocytosis at the plasma membrane are central to cellular function. However, despite intensive study, these processes are poorly characterized in spatial and dynamic terms. The primary reason for this is that, to date, it has not been possible to visualize rapidly moving intracellular compartments in three dimensions in cells. Here, we use a recently developed imaging setup in which multiple planes can be simultaneously imaged within the cell in conjunction with visualization of the plasma membrane plane by using total internal reflection fluorescence microscopy. This has allowed us to track and characterize intracellular events on the recycling pathway that lead to exocytosis of the MHC Class I-related receptor, FcRn. We observe both direct delivery of tubular and vesicular transport containers (TCs) from sorting endosomes to exocytic sites at the plasma membrane, and indirect pathways in which TCs that are not in proximity to sorting endosomes undergo exocytosis. TCs can also interact with different sorting endosomes before exocytosis. Our data provide insight into the intracellular events that precede exocytic fusion.

Kinetics and thermodynamics of T cell receptor– autoantigen interactions in murine experimental autoimmune encephalomyelitis

In the current study, cellular and molecular approaches have been used to analyze the biophysical nature of T cell receptor (TCR)–peptide MHC (pMHC) interactions for two autoreactive TCRs. These two TCRs recognize the N-terminal epitope of myelin basic protein (MBP1–11) bound to the MHC class II protein, I-Au, and are associated with murine experimental autoimmune encephalomyelitis. Mice transgenic for the TCRs have been generated and characterized in other laboratories. These analyses indicate that the mice either develop encephalomyelitis spontaneously (172.10 TCR) or only if immunized with autoantigen in adjuvant (1934.4 TCR). Here, we show that the 172.10 TCR binds MBP1–11:I-Au with a 4–5-fold higher affinity than the 1934.4 TCR. Consistent with the higher affinity, 172.10 T hybridoma cells are significantly more responsive to autoantigen than 1934.4 cells. The interaction of the 172.10 TCR with cognate ligand is more entropically unfavorable than that of the 1934.4 TCR, indicating that the 172.10 TCR undergoes greater conformational rearrangements upon ligand binding. The studies therefore suggest a correlation between the strength and plasticity of a TCR–pMHC interaction and the frequency of spontaneous disease in the corresponding TCR transgenic mice. The comparative analysis of these two TCRs has implications for understanding autoreactive T cell recognition and activation.

Quantitative study of single molecule location estimation techniques

Estimating the location of single molecules from microscopy images is a key step in many quantitative single molecule data analysis techniques. Different algorithms have been advocated for the fitting of single molecule data, particularly the nonlinear least squares and maximum likelihood estimators. Comparisons were carried out to assess the performance of these two algorithms in different scenarios. Our results show that both estimators, on average, are able to recover the true location of the single molecule in all scenarios we examined. However, in the absence of modeling inaccuracies and low noise levels, the maximum likelihood estimator is more accurate than the nonlinear least squares estimator, as measured by the standard deviations of its estimates, and attains the best possible accuracy achievable for the sets of imaging and experimental conditions that were tested. Although neither algorithm is consistently superior to the other in the presence of modeling inaccuracies or misspecifications, the maximum likelihood algorithm emerges as a robust estimator producing results with consistent accuracy across various model mismatches and misspecifications. At high noise levels, relative to the signal from the point source, neither algorithm has a clear accuracy advantage over the other. Comparisons were also carried out for two localization accuracy measures derived previously. Software packages with user-friendly graphical interfaces developed for single molecule location estimation (EstimationTool) and limit of the localization accuracy calculations (FandPLimitTool) are also discussed.

Divergent activities of an engineered antibody in murine and human systems have implications for therapeutic antibodies

The MHC class I-related receptor, neonatal Fc receptor (FcRn), plays a central role in regulating the transport and in vivo persistence of immunoglobulin G (IgG). IgG–FcRn interactions can be targeted for engineering to modulate the in vivo longevity and transport of an antibody, and this has implications for the successful application of therapeutic IgGs. Although mice are widely used to preclinically test antibodies, human and mouse FcRn have significant differences in binding specificity. Here we show that an engineered human IgG1 has disparate properties in murine and human systems. The mutant shows improved transport relative to wild-type human IgG1 in assays of human FcRn function but has short in vivo persistence and competitively inhibits FcRn activity in mice. These studies indicate potential limitations of using mice as preclinical models for the analysis of engineered antibodies. Alternative assays are proposed that serve as indicators of the properties of IgGs in humans.