live-cell-imaging-endocytic-transport-using-functionalized-nanobodies

Live-cell Imaging of Endocytic Transport using Functionalized Nanobodies in Cultured Cells

Endocytosis of receptors and other transmembrane proteins from the cell surface to endosomes and beyond is critical for homeostasis, physiology, and ...

University of Basel

The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated  malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated , parasites that also proliferate inside erythrocytes but, in contrast to , do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the  proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in .

Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel).

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.

Super-resolution microscopy demystified.

Confocal microscopy is used today on a daily basis in life science labs. This "routine" technique contributes to the progress of scientific projects across many fields by revealing structural details and molecular localization, but researchers need to be aware that detection efficiency and emission light path performance is of major influence in the confocal image quality. By design, a large portion of the signal is discarded in confocal imaging, leading to a decreased signal-to-noise ratio (SNR) which in turn limits resolution. A well-aligned system and high performance detectors are needed in order to generate an image of best quality. However, a convenient method to address system status and performance on the emission side is still lacking. Here, we present a complete method to assess microscope and emission light path performance in terms of SNR, with a comprehensive protocol alongside NoiSee, an easy-to-use macro for Fiji (available via the corresponding update site). We used this method to compare several confocal systems in our facility on biological samples under typical imaging conditions. Our method reveals differences in microscope performance and highlights the various detector types used (multialkali photomultiplier tube (PMT), gallium arsenide phosphide (GaAsP) PMT, and Hybrid detector). Altogether, our method will provide useful information to research groups and facilities to diagnose their confocal microscopes.

Using the NoiSee workflow to measure signal-to-noise ratios of confocal microscopes.

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.

Article	Year
Monoclonal Antibodies That Recognize the Alkylation Signature of Antimalarial Ozonides OZ277 (Arterolane) and OZ439 (Artefenomel). ACS infectious diseases\| PubMed ID: 26819968	2016
Super-resolution microscopy demystified. Nature cell biology\| PubMed ID: 30602772	2019
Using the NoiSee workflow to measure signal-to-noise ratios of confocal microscopes. Scientific reports\| PubMed ID: 30718583	2019
Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells. Cell reports\| PubMed ID: 32905777	2020

Oliver Biehlmaier

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