How do cells build biological machines?
Cells are expert organisers, manufacturers, transporters and recyclers of cellular content.
We are interested in understanding the manufacturing process of macromolecular machines - specifically the biosynthesis of axonemal dynein motors that power the motion of eukaryotic cilia which are critical to human health.
Defects in ciliary dynein assembly also underlie a range of human pathologies making them a clinically important class of motors.
We integrate cell biology, biochemistry and structural methods to
mechanistically understand the assembly of ciliary dynein motors in health and disease.
Virtually all proteins inside a cell work with other proteins as part of large complexes to exert their biological functions. In the case of ciliary dynein motors, a whole host of cellular assembly factors work together to shephard dynein assembly. To understand their molecular mechanisms in detail, we biochemically purify dynein assembly factor complexes directly from cells and study their functions in vitro.
Image: Cellular contents get separated into distinct fractions after cells are spun at very high speeds in an ultracentrifuge. This is an essential first step in isolating pure protein complexes for further study.
Truly understanding the function of protein complexes requires a detailed look not only in vitro but also at how they behave inside a cell i.e. in vivo. We focus on studying the protein factors involved in building dynein motors (Dynein Axonemal Assembly Factors or DNAAFs) using a diverse set of ciliated cell models including human airway cells (image to the right), protists (Tetrahymena) and green algae (Chlamydomonas). Such studies provide mechanistic insights into ciliary dynein assembly in a native cellular context.
Image: Axonemal dynein complexes labeled in green light up the cilia that cover the top of a human lung epithelial cell. In red is a DNAAF protein which resides in the cell cytoplasm and the blue dye marks the DNA housed in the cells nucleus.
Structure informs function in biology. Directly observing a protein complex often provides novel insights into the way it functions.
We use a suite of cutting edge structural biology techniques including cryogenic-electron microscopy (Cryo-EM) and protein structure predictions to generate 3D models of protein complexes that build dynein motors . Models are then interpreted along with functional studies to infer molecular mechanisms.
Image: Single molecules of a major ciliary dynein motor from different angles and orientations are shown. Images of molecules were acquired using transmission electron microscopes. After extensive image processing, a composite of many thousands of such images was used to reconstruct a first 3D structure of this motor which provided many novel functional insights.