Studying Large Multi-Protein Complexes Using Single Molecule Localization Microscopy

Biology would not be where it is today without fluorescence microscopy. It is arguably one of the most commonly used tools in the biologists toolbox and it has helped scientists study the localization of cellular proteins and other small things for decades, but it is not without its limitations. Due to the diffraction limit, conventional fluorescence microscopy is limited to micrometer-range structures. Science has long relied upon electron microscopy and X-ray crystallography to study phenomena that occur below this limit. However, many of lifes processes occur between these two spatial domains. Super-resolution microscopy, the next stage of evolution of fluorescence microscopy, has the potential to bridge this gap between micro and nano. It combines superior resolutions of down to a few nanometers with the ability to view objects in their natural environments. It is the ideal tool for studying the large, multi-protein complexes that carry out most of lifes functions, but are too complex and fragile to put on an electron microscope or into a synchrotron. A form of super-resolution microscopy called SMLM Microscopy shows especially high promise in this regard. With its ability to detect individual molecules, it combines the high resolution needed for structural studies with the quantitative readout required for obtaining data on the stoichiometry of multi-protein complexes. This thesis describes new tools which expand the toolbox of SMLM with the specific aim of studying multi-protein complexes. First, the development of a novel fluorescent tagging system that is a mix of genetic tagging and immuno-staining. The system, termed BC2, consists of a short, genetically encodable peptide that is targeted by a nanobody (BC2 nanobody). The system brings several advantages. The small tag is not disruptive to the protein it is attached to and the small nanobody can get into tight spaces, making it an excellent tag for dense multi-protein structures. Next, several new variants of some commonly used green-to-red fluorescent proteins. The novel variants, which can be converted with a combination of blue and infrared light are especially useful for live-cell imaging. The developed fluorescent proteins can also be combined with photo-activatable fluorescent proteins to enable imaging of several targets with the same color protein. Finally, an application of the latter technique to study the multi-protein kinetochore complex and gain first glimpses into its spatial organization and the stoichiometry of its subunits

Exploiting nanobodies and Affimers for superresolution imaging in light microscopy

Antibodies have long been the main approach used for localizing proteins of interest by light microscopy. In the past 5 yr or so, and with the advent of superresolution microscopy, the diversity of tools for imaging has rapidly expanded. One main area of expansion has been in the area of nanobodies, small single-chain antibodies from camelids or sharks. The other has been the use of artificial scaffold proteins, including Affimers. The small size of nanobodies and Affimers compared with the traditional antibody provides several advantages for superresolution imaging

Combining Primed Photoconversion and UV-Photoactivation for Aberration-Free, Live-Cell Compliant Multi-Color Single-Molecule Localization Microscopy Imaging

Super-resolution fluorescence microscopy plays a major role in revealing the organization and dynamics of living cells. Nevertheless, single-molecule localization microscopy imaging of multiple targets is still limited by the availability of suitable fluorophore combinations. Here, we introduce a novel imaging strategy which combines primed photoconversion (PC) and UV-photoactivation for imaging different molecular species tagged by suitable fluorescent protein combinations. In this approach, the fluorescent proteins can be specifically photoactivated/-converted by different light wavelengths using PC and UV-activation modes but emit fluorescence in the same spectral emission channel. We demonstrate that this aberration-free, live-cell compatible imaging method can be applied to various targets in bacteria, yeast and mammalian cells and can be advantageously combined with correlative imaging schemes

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color SMLM imaging.

Funder: Boehringer Ingelheim FondsFunder: Bonn UniversityFunder: Max Planck SocietyFunder: Carnegie Mellon UniversityThe key to ensuring proper chromosome segregation during mitosis is the kinetochore (KT), a tightly regulated multiprotein complex that links the centromeric chromatin to the spindle microtubules and as such leads the segregation process. Understanding its architecture, function, and regulation is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in structural detail so far. In this study, we construct a nanometer-precise in situ map of the human-like regional KT of Schizosaccharomyces pombe using multi-color single-molecule localization microscopy. We measure each protein of interest (POI) in conjunction with two references, cnp1CENP-A at the centromere and sad1 at the spindle pole. This allows us to determine cell cycle and mitotic plane, and to visualize individual centromere regions separately. We determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI and, consequently, build an in situ KT model with unprecedented precision, providing new insights into the architecture

One-step versus two-step culture of mouse preimplantation embryos

David K. Gardner and Michelle Lan

Embryo culture medium: which is the best?

Copyright © 2006 Published by Elsevier Ltd.With the growing move in in-vitro fertilization (IVF) clinics to transfer fewer embryos to women, there is an increasing reliance on the IVF laboratory to maximize embryo viability. Subsequently, there is justified scrutiny on the culture system and the media used to sustain the human embryo in vitro. The transfer of fewer embryos to patients also creates an increased dependence on the ability to cryopreserve embryos successfully. Therefore, in addition to the ability of a culture system to produce a single top-quality embryo for transfer, it is also necessary to enhance the cryotolerance of sibling embryos so that they can survive freezing or vitrification. Therefore, when examining which culture media is the best, it is prudent to not only examine the ability of a culture system to produce a pregnancy with the one or two highest-grade embryos, but also to determine how many embryos from the entire cohort (both fresh and frozen embryos) are capable of producing a live birth. Additionally, research on animal models has demonstrated that stress, and the resultant adaptation to conditions during pre-implantation stages, can affect pregnancy loss and fetal growth. It is therefore important to understand the role of each medium component and to identify possible sources of cellular stress to the embryo that will ultimately affect the function and viability of the conceptus.Michelle Lane, and David K. Gardnerhttp://www.elsevier.com/wps/find/journaldescription.cws_home/623004/description#descriptio