Max Planck Institute of Biochemistry

3 ARTICLES PUBLISHED IN JoVE

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.