Understanding Macromolecular Structure and Function with Cryo-Electron Microscopy

Our current research focuses on discovering the molecular mechanisms that maintain neuronal integrity. Our limited ability to treat diseases such as Alzheimer’s, Parkinson’s, Lou Gehrig’s, and Huntington’s stems from the fact that our molecular understanding of the fundamental events that trigger the onset of these diseases is severely limited. Using multi-scale high-resolution 3D imaging, we are determining the precise neuronal mechanisms that are involved in maintaining neuronal integrity. By determining the structures of the machinery involved in these processes, we are gaining substantial insight into the molecular relationships that give rise to normal neuronal function, which is the first step in understanding progression of neurological disease.

Recent developments in cryo-electron microscopy instrumentation have made it possible to solve structures to near-atomic resolution, and with access to the cutting-edge technologies at TSRI, my group is developing novel strategies for specimen preparation, imaging, and processing that are enabling us to produce atomic structural models of complex biological systems.

Cargo Transport on Microtubules

One of the avenues my lab is exploring centers on the underlying mechanisms by which small molecular motors are responsible for transport of nutrients within neurons. Neuronal development, repair, and function depends on efficient trafficking of vital organelles, proteins, lipids, and signaling molecules along microtubule highways within nuerons. This cargo transport is accomplished by two sets of ATP-driven motor proteins, kinesins and dyneins, which traverse microtubules in opposite directions. Importantly, these motors are involved in clearing away the dangerous protein aggregates that are associated with a large number of neurodegenerative diseases. These same motors are also required for a variety of cellular processes, including cell division.

Using multi-scale cryo-electron microscopy, we are outlining the interplay between molecular motors, microtubules, and the cargo that they transport. This provides a description of the interactions and conformational rearrangments that are required for regulation of cargo binding and motor movement, which in time will enable us to pinpoint events that lead to the manifestation of neurodegeneration. These studies will provide the critical structural data that could one day deliver new avenues to treat these diseases.

The Eukaryotic 26S Proteasome

The proteasome is the major ATP-dependent protease in eukaryotic cells, maintaining cellular homeostatis by recognizing proteins that have been marked for degradation through covalently linked polyubiquitin chains. Proper function of this multicatalytic complex is crucial to cell cycle regulation, and its inhibition is directly linked to cell death. The proteasome’s inherent flexibility and dynamic nature make atomic analysis by traditional crystallography intractible, and as such this system is uniquely suited for study by cryo-electron microscopy.

Our current research focuses on the manner in which unwanted intracellular proteins are recognized and degraded by the 26S proteasome. Although the overall subunit organization of the proteasome has been determined, there are many questions surrounding the manner in which substrates are recognized by or transported to the proteasome, as well as how targeted proteins are unfolded and translocated to the proteolytic chamber. The answers to these questions would significantly improve our understanding of proteasome’s role in cellular homeostatis, potentially revealing novel approaches to detect and suppress the onset of tumorigenesis and neurodegeneration.