Biology subjects

Zimmermann, T.

Publications and source records attributed to Zimmermann, T..

3 recordsLinked to original sources

bioRxiv2023

Automated 3D multi-color single-molecule localization microscopy

Since its inception, single molecule localization microscopy (SMLM) has enabled imaging scientists to visualize biological structures with unprecedented resolution. Particularly powerful implementations capable of 3D, multi-color and high-throughput imaging have yielded key biological insights although widespread access to such technologies has been limited. The purpose of this protocol is to provide a guide for interested researchers to establish high-end SMLM in their laboratories. We detail the initial configuration and subsequent assembly of the SMLM, including instructions for alignment of all optical pathways, software/hardware integration and operation of the instrument. We describe validation steps including the preparation and imaging of test- and biological samples with structures of well-defined geometry and assist the user in troubleshooting and benchmarking performance. Additionally, we provide a walkthrough of the reconstruction of a super-resolved dataset from acquired raw images using the Super-resolution Microscopy Analysis Platform (SMAP). Depending on the instrument configuration, the cost of components is in the range $80,000 - 160,000, a fraction of the cost of a commercial instrument. A builder with some experience of optical systems is expected to require 3 - 6 months from the start of system construction to attain high-quality 3D and multi-color biological images.

biophysics↗

bioRxiv2022

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates elongation and stability of ciliary axonemes and other microtubule-based structures.

cell biology↗

bioRxiv2021

Impact of charge patches on tumor disposition and biodistribution of therapeutic antibodies

This study explores the impact of antibody surface charge on tissue distribution into various tissues including tumor. Tumor-bearing mice were dosed intravenously with a mixture comprising three antibodies engineered to carry negative charge patches, a balanced charge distribution, or positive patches, respectively. Tissue levels were analyzed with a specific LC-MS/MS method. In addition, the antibody mix was administered to non-tumor bearing mice. Muscle and skin interstitial fluid were obtained by centrifugation and analyzed by LC-MS/MS. An in-vitro endothelium model was explored for its feasibility to mimic the observed distribution differences. A balanced charge distribution was optimal in terms of total tumor exposure, while in other tissues negatively charged and balanced charged antibodies gave similar results. In contrast, positive charge patches generally result in increased serum clearance but markedly enhance tumor and organ uptake, leading to higher tissue-to-serum ratios. The uptake and availability in the interstitial space were confirmed by specific assessment of antibody levels in the interstitial fluid of muscle and skin, with similar charge impact as in total tissue. The in vitro model was able to differentiate the transport propensity of this series of antibody variants. In summary, our results show the differential effects of charge patches on an antibody surface on biodistribution and tumor uptake. These insights may help in the design of molecules with biodistribution properties tailored to their purpose and an optimized safety profile.

cancer biology↗