Project 1 – Isolation and Visualization of complexes
Any given macromolecule may make stable interactions with other macromolecules, forming a tight complex. Additionally other macromolecules can exchange rapidly in dynamic or transient interactions with these stable complexes. This whole network is surrounded by a macromolecular milieu of many other complexes that interact with each other via local interactions. This is the environment of the cell.
How do we visualize these complexes in such a crowded environment? How do we isolate these complexes? Our pipeline is as follows:
Developing Reagents for Ultra-Efficient Capture of Endogenous Complexes
Epitope tags have proven to have tremendous utility for visualizing and affinity purifying complexes. However, there remain significant challenges that limit the generality of this approach, including tag-induced loss of function, limited utility in humans, and a significant lack of affordable baits with sufficient affinity for studying interactomics.
We here develop baits for:
- tags on targets that can accommodate tags
A nanobody (blue) against ProteinA, Fridy et al (2015).
targets for which tags are not practical: we have a new strategy for antibody production, achieving large-scale, rapid identification and production of large repertoires of bacterially-expressed, high affinity single-domain antibodies, termed nanobodies, against any desired antigen. Our approach is many orders of magnitude more efficient than standard phage display approaches and can pre-select for exceptionally strong affinities. To learn more about our method, click here.
- We also develop tagging and novel affinity purification strategies for chromatin and RNA – both of which are notoriously promiscuous and “sticky” – such that individual RNAs and chromatin regions can be specifically interrogated.
Production of Complexes
To access and then isolate a macromolecule and its micro-environment, we must break open the cell. Breaking open a cell, be it from yeast, mammal or bacteria can result in potential disintegration of the complexes through biochemical or physical forces, or through time-dependent decay. Our challenge is to advance technology, enabling the isolation and preservation, with high fidelity, of defined hierarchical arrangements of complexes/interactions for any chosen macromolecule, from different cell types, tissues, and animals. We have developed techniques allowing us to achieve such goals. To learn more, click here…
Ultrafast Preservation and Preparation Methods for Endogenous Complexes.
A central concept of our Center is rapid preservation and access to any macromolecular complex in its native state by literally freezing the complexes inside the cell, placing them in a state of “suspended animation”, or by adding cell-permeable linkers to instantly chemically preserve the complex upon addition. We have adapted EM technologies for freezing cells and tissues to minimize cellular damage and we explore complementary chemical stabilization methods that compatible with our approaches. To access macromolecular complexes in frozen cells while preserving them, we have developed our cryomilling methods. Our goal is to produce particles with diameters so small, on the order of a few tens of nm, that they are only a little larger than the complexes themselves. To learn more about the cryo-milling technique, take a look at our protocols page!
Rapid, Parallelized, Optimized Affinity Capture of Endogenous Complexes.
Affinity capture of the Trypanosome nuclear pore complex (Obado et al, 2016)
After lysis or thawing, macromolecular complexes begin to disintegrate and non-specific associations begin to form. One solution, is to simply isolate these complexes very rapidly before significant decay occurs; another, is to find isolation conditions that prolong the life of the complexes. We improve the speed of isolations and enable exploration of condition space with new small diameter paramagnetic beads and optimized isolation technology. Potentially, capture times of seconds will become possible. Access our method here!