How do cells compute?

Cells are faced with external environments that are in constant flux. To survive, they must adaptively respond to changes in the environment, often via regulated production or degradation of the cell’s protein machinery. Determining the appropriate proteomic response requires the cell to perform complex calculations: integrating diverse signals, determining whether a threshold has been passed, or storing information over time, among others. There remain a number of open questions about how the cell performs these calculations, and in particular, much remains to be understood about the structural basis for cellular computation.

Multi-site phosphorylation enables diverse regulatory behaviors

Phosphorylation is one of the central regulatory motifs in biology: huge swaths of the eukaryotic proteome are targeted by phosphorylation, and regulated phosphorylation represents a central mechanism by which the cell receives and transduces information from the external environment. Moreover, it has long been understood that multi-site phosphorylation (functional phosphorylation of a protein at more than one distinct site) has a unique potential to enable complex cellular calculations. Building a mechanistic understanding of how specific phosphoregulatory motifs produce diverse systems-level behaviors, and thus different types of cellular calculations, has been challenging, however: identifying functional phosphorylation sites at scale represents a throughput challenge, and phosphoregulated protein complexes are typically highly dynamic, rendering them unsuitable for study via traditional structural approaches. Substantial advances have been made in recent years in visualizing dynamic protein complexes with heterogeneous cryo- electron microscopy (cryo-EM), and in deploying library-based approaches to perform massively multiple hypothesis testing in parallel. These techniques have substantial potential to address the challenges of studying highly dynamic, multiply phosphorylated systems, and thus to shed light on these fundamental mechanisms of cellular information processing.

Structural approaches to understand systems-level behaviors

The Kinman lab is centrally interested in understanding how multi-site phosphorylation structurally enables diverse systems-level properties within cells. By studying a variety of phosphoregulated systems across evolutionary space, we hope to identify generalized design principles that can be applied to specific phosphoregulated systems implicated in human disease processes. To answer these questions, we deploy a variety of approaches. These include state-of-the-art structural biology tools, such as cryo-EM to visualize highly dynamic protein complexes and cryo- electron tomography (cryo-ET) to assess the structural landscapes of protein complexes in situ, as well as high-throughput and proteomic approaches. We are also interested in understanding the limits of existing approaches, and developing new experimental and computational methods to push these boundaries.

Autophagic initiation as a case study

When exposed to starvation, yeast cells initiate autophagy, a process whereby bulk components of the cytosol are ferried to the vacuole for degradation and cellular recycling. Appropriate regulation of autophagy is critical: while failure to perform autophagy upon starvation leads to a loss of cellular viability, bulk degradation of cytoplasmic contents in nutrient replete conditions is likely to be similarly detrimental. Critically, multiple types of starvation, including starvation for both nitrogen and carbon sources, have the capacity to induce autophagy in yeast. Each of these starvation conditions is sensed by distinct cell signaling pathways that converge at the autophagic machinery, raising questions about how these signals are integrated to produce the autophagy-no autophagy decision, as well as how the functional outputs of autophagy (the sets of proteins that are degraded) compare between different input signals. In the Kinman lab, we are interested in understanding how phosphorylation drives autophagic initiation and cargo selection, with an emphasis on identifying the structural basis for these processes and determining how these phosphoregulatory mechanisms enable effective signal integration by the cell.