Felix R.M. Beinlich

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Timo Appelhans, Felix R.M. Beinlich, Christian P. Richter, Rainer Kurre, Karin B. Busch

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.


Determining crystal structures through crowdsourcing and coursework

Scott Horowitz, Brian Koepnick, Raoul Martin, Agnes Tymieniecki, Amanda A. Winburn, Seth Cooper, Jeff Flatten, David S. Rogawski, Nicole M. Koropatkin, Tsinatkeab T. Hailu, Neha Jain, Philipp Koldewey, Logan S. Ahlstrom, Matthew R. Chapman, Andrew P. Sikkema, Meredith A. Skiba, Finn P. Maloney, Felix R. M. Beinlich, Foldit Players, University of Michigan students, Zoran Popović, David Baker, Firas Khatib & James C. A. Bardwell

We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality.


Shuttling of PINK1 between Mitochondrial Microcompartments Resolved by Triple-Color Superresolution Microscopy

Felix R. M. Beinlich, Christoph Drees, Jacob Piehler, and Karin B. Busch

The cytosolic phosphatase and tensin homologue Pten-kinase PINK1 involved in mitochondrial quality control undergoes a proteolytic process inside mitochondria. It has been suggested that the protein is not fully imported into mitochondria during this maturation. Here, we have established live cell triple-color super-resolution microscopy by combining FPALM and tracking and localization microscopy (TALM) in order to unravel the spatiotemporal organization of the C-terminal kinase domain of PINK1 during this process. We find that the kinase domain is imported into active mitochondria and colocalizes with respiratory complex I at the inner mitochondrial membrane. When the processing step inside mitochondria is inhibited or mitochondria are de-energized, full length PINK1 distributes between the outer and the inner mitochondrial membranes, indicating a holdup of import. These findings give the molecular base for a dual role of PINK1—inside energized mitochondria and outside of de-energized mitochondria.


Personality variation in a clonal insect: The pea aphid, Acyrthosiphon pisum

Schuett W, Dall SR, Baeumer J, Kloesener MH, Nakagawa S, Beinlich F, Eggers T.

Individuals are often consistent in their behavior but vary from each other in the level of behavior shown. Despite burgeoning interest in such animal personality variation, studies on invertebrates are scarce, and studies on clonal invertebrates nonexistent. This is surprising given the obvious advantages of using invertebrates/clones to tackle the crucial question why such consistent behavioral differences exist. Here we show that individuals of clonal pea aphids exhibit consistent behavioral differences in their escape responses to a predator attack (dropping vs. nondropping off a plant). However, behavior was not repeatable at the clonal level. Genetically identical clones expressed various phenotypes but different clones produced different proportions of each phenotype (dropper, nondropper, and inconsistent). Manipulations of early environmental conditions had little qualitative impact on such patterns. We discuss the importance of our findings for future studies of the evolutionary and ecological consequences of personality variation.