Biology subjects

Mackey, M. R.

Publications and source records attributed to Mackey, M. R..

3 recordsLinked to original sources

bioRxiv2024

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

biochemistry↗

bioRxiv2023

Fe-TAMLs as a new class of small molecule peroxidase probes for correlated light and electron microscopy

We introduce Fe-TAML, a small molecule-based peroxidase as a versatile new member of the correlated fluorescence and electron microscopy toolkit. The utility of the probe is demonstrated by high resolution imaging of newly synthesized DNA (through biorthogonal labeling), genetically tagged proteins (using HaloTag), and untagged endogenous proteins (via immunostaining). EM visualization in these applications is facilitated by exploiting Fe-TAMLs catalytic activity for the deposition of localized osmiophilic precipitates based on polymerized 3,3-diaminobenzidine. Optimized conditions for synthesizing and implementing Fe-TAML based probes are also described. Overall, Fe-TAML is a new chemical biology tool that can be used to visualize diverse biomolecular species along nanometer and micron scales within cells.

cell biology↗

bioRxiv2020

Elemental Mapping of Labeled Biological Specimens at Intermediate Energy Loss in an Energy-Filtered TEM acquired using a Direct Detection Device

The multi-color or single-color EM that was developed previously, by the pseudo-colored overlay of the core-loss or high-loss EFTEM elemental map/s of the lanthanide onto the conventional image, the lanthanide chelates conjugated to diaminobenzidine being sequentially deposited as a result of selective oxidization by orthogonal photosensitizers / peroxidases. The synthesis of the new second generation lanthanide DABs, which contains 4 times more lanthanide per DAB, gives significant signal amplification and enabling collection of elemental maps at much lower energy-loss regions more favorable. Under the same experimental conditions, acquiring EFTEM elemental maps for the lanthanides at the lower energy-loss of N4,5 edge instead of the core-loss M4,5 edge, provides ~4x increase in signal-to-noise and ~2x increase in resolution. The higher signal at the N4,5 edge, also allows for more sophisticated technique of EFTEM spectrum Image for the acquisition of elemental maps with very high signal fidelity.

systems biology↗