Max Planck Institute of Biochemistry

8 ARTICLES PUBLISHED IN JoVE

Bioengineering

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

Felix Spira¹, Julia Dominguez-Escobar¹, Nikola Müller^1,2, Roland Wedlich-Söldner¹

¹AG Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, ²Helmholtz Zentrum München

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

Biology

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Jacek R. Wiśniewski¹

¹Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Chemistry

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

Yannig Gicquel^*1,2, Robin Schubert^*3,4,5, Svetlana Kapis³, Gleb Bourenkov⁶, Thomas Schneider⁶, Markus Perbandt^3,4, Christian Betzel^3,4,5, Henry N. Chapman^1,2,4, Michael Heymann^1,7

¹Center for Free Electron Laser Science, DESY, ²Department of Physics, University of Hamburg, ³Institute for Biochemistry and Molecular Biology, Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, ⁴The Hamburg Center for Ultrafast Imaging, University of Hamburg, ⁵Integrated Biology Infrastructure Life-Science Facility at the European XFEL (XBI), ⁶European Molecular Biology Laboratory, EMBL c/o DESY, ⁷Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

Developmental Biology

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Sandra B. Lemke¹, Frank Schnorrer^1,2

¹Muscle Dynamics Group, Max Planck Institute of Biochemistry, ²Aix Marseille University, CNRS, IBDM

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Biochemistry

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Beatrice Ramm^*1, Philipp Glock^*1, Petra Schwille¹

¹Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Biology

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography

Anna Bieber^*1, Cristina Capitanio^*1, Florian Wilfling^1,2, Jürgen Plitzko¹, Philipp S. Erdmann^1,3

¹Max Planck Institute of Biochemistry, ²Max Planck Institute for Biophysics, ³Fondazione Human Technopole

Here, we present a pipeline for 3D-correlative focused ion beam milling on guiding the preparation of cellular samples for cryo-electron tomography. The 3D position of fluorescently tagged proteins of interest is first determined by cryo-fluorescence microscopy, and then targeted for milling. The protocol is suitable for mammalian, yeast, and bacterial cells.

Bioengineering

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Yashar Bashirzadeh^*1, Nadab Wubshet^*1, Thomas Litschel², Petra Schwille³, Allen P. Liu^1,4,5,6

¹Department of Mechanical Engineering, University of Michigan, Ann Arbor, ²John A. Paulson School of Engineering and Applied Sciences, Harvard University, ³Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, ⁴Department of Biomedical Engineering, University of Michigan, Ann Arbor, ⁵Department of Biophysics, University of Michigan, Ann Arbor, ⁶Cellular and Molecular Biology Program, University of Michigan, Ann Arbor

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

Biochemistry

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Frederik Steiert^1,2, Tamara Heermann¹, Nikolas Hundt³, Petra Schwille¹

¹Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, ²Department of Physics, Technical University Munich, ³Department of Cellular Physiology, Biomedical Center (BMC), Ludwig-Maximilians-Universität München

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.