A bioorthogonal chemistry approach to the study of biomolecules in their ultrastructural cellular context

In this thesis the combinatorial use of bioorthogonal labelling and Electron Microscopy (EM)-based imaging techniques is explored to enable observations of specific molecular targets in their ultrastructural context within the cell.Bio-organic Synthesi

The potential of bioorthogonal chemistry for correlative light and electron microscopy: a call to arms

Bio-organic Synthesi

Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

Bio-organic Synthesi

Super-resolution correlative light-electron microscopy using a click-chemistry approach for studying intracellular trafficking

Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.Microscopic imaging and technolog

A bioorthogonal chemistry approach to the study of biomolecules in their ultrastructural cellular context

In this thesis the combinatorial use of bioorthogonal labelling and Electron Microscopy (EM)-based imaging techniques is explored to enable observations of specific molecular targets in their ultrastructural context within the cell.</p

Imaging Bioorthogonal Groups in Their Ultrastructural Context with Electron Microscopy

Chemical Immunolog

Imaging Bioorthogonal Groups in Their Ultrastructural Context with Electron Microscopy.

Spitting image: Herein a recent paper on the imaging of bioorthogonal groups using three-dimensional electron microscopy is discussed. The work has demonstrated electron microscopy imaging as a technique suitable for gaining structural information on bioorthogonal groups in their cellular context

Correlative light and electron microscopy reveals discrepancy between gold and fluorescence labelling

Microscopic imaging and technolog

Detection of bioorthogonal groups by correlative light and electron microscopy allows imaging of degraded bacteria in phagocytes

Bio-organic Synthesi

Chromokinesin KIF4A teams up with stathmin 1 to regulate abscission in a SUMO-dependent manner

Cell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter cells are severed. Here, we present a novel abscission mechanism. We identified chromokinesin KIF4A, which is adjacent to the midbody during cytokinesis, as being required for efficient abscission. KIF4A is regulated by post-translational modifications. We evaluated modification of KIF4A by the ubiquitin-like protein SUMO. We mapped lysine 460 in KIF4A as the SUMO acceptor site and employed CRISPR-Cas9-mediated genome editing to block SUMO conjugation of endogenous KIF4A. Failure to SUMOylate this site in KIF4A delayed cytokinesis. SUMOylation of KIF4A enhanced the affinity for the microtubule destabilizer stathmin 1 (STMN1). We here present a new level of abscission regulation through the dynamic interactions between KIF4A and STMN1 as controlled by SUMO modification of KIF4A