Michael McMurray, Ph.D.

Our research focuses on identifying molecular mechanisms underlying the assembly of macromolecular complexes, with a focus on multisubunit complexes formed by septin proteins. All cellular processes require the function of multisubunit complexes, and while much attention has been given to solving the final structures of such assemblies, comparatively little is known about how individual subunits adopt oligomerization-competent conformations and find their partner subunits in the crowded, dynamic cellular milieu. Below is a summary of the past research from our group.

We first used unbiased genetic screening to find evidence that guanine nucleotide binding by septin proteins plays an entirely structural role during filament assembly, which can be bypassed by specific mutations in a key septin-septin oligomerization interface. Considering that one of these mutations has been found to cause male infertility in humans, these findings have compelling clinical relevance.

We then investigated a phenomenon in which, when cells have a choice between a wild-type and a mutant version of a given septin, mutant septins unable to bind nucleotides are discriminated against during higher-order assembly, despite their ability to function normally under the conditions tested. We discovered that prolonged interaction between nascent mutant polypeptides and cytosolic chaperones results in a kinetic delay in the acquisition by the mutant septin of a conformation competent for oligomerization with other septins. Our findings challenged the dominant paradigm that interactions between chaperones and their clients always promote mutant protein function, and provided a new way of thinking about how chaperone interactions might influence the ability of mutant alleles of proteins to perturb the functions of the wild type. This work was published in Molecular Biology of the Cell in 2015 and in Cell Cycle in 2016.

We went on to develop an entirely new technique for monitoring, for the first time in living cells, the step-wise pathway of assembly of protein complexes, and used it to show how nucleotide-induced conformational changes drive the progression of such an ordered pathway for yeast septins. Moreover, we generated and validated a new model for the role of nucleotide hydrolysis by septins during oligomerization, and found that slow GTPase activity by a key septin subunit drives the formation of distinct hetero-oligomers comprised of alternative subunits. Our “GTPase timer” model predicted that changes in the ratio of GTP to GDP in the cytosol would influence the subunit composition of septin complexes. Our experiments supported this model and further provided a functional context for why septin complex composition is tuned to the metabolic state of the cell.

In budding S. cerevisiae cells, septin complexes are restricted to hetero-octamers, yet in other species hetero-hexamers are also found, in which the "central" subunit corresponding to yeast Cdc10 is bypassed during assembly. It was unknown how this alternative assembly pathway worked. Building from a serendipitous discovery that the small molecule guanidine hydrochloride (GdnHCl) restores high-temperature growth to cdc10 mutants, we found that hexamer assembly (and Cdc10 bypass) in other species involves GTPase activity by the Cdc3 homolog. The guanidine group of GdnHCl appears to replace the guanidine group of a key arginine residue that is present in Cdc3 homologs in other species that make Cdc10-less hexamers (including humans), and is missing from S. cerevisiae Cdc3 and the and Kluyveromyces lactis. Thus the hexamer-vs-octamer decision presumably relies on slow GTPase activity by the septin subunit in the Cdc3 position, just like the choice of terminal subinits in yeast octamers relies on slow GTPase activity by the septin subunit in the Cdc12 position.

Finally, our work suggests for the first time that GdnHCl can be used to functionally replace "missing" arginine side chains in living cells. Since GdnHCl is an FDA-approved drug for human use, and since arginine is the most commonly mutated amino acid in missense mutations causing human disease, these findings may pave the way for the use of GdnHCl to treat a variety of human genetic diseases. This work, which was done in collaboration with Aurélie Bertin in Paris and Amy Gladfelter's lab at UNC Chapel Hill, was published in eLife in 2020.

We studied the functions of the gametogenesis-specific septin subunits in yeast and determined that they play important and unique roles in guiding the biogenesis of new membrane and cell wall. As septins also play important, but poorly understood, roles in human gametogenesis, these findings are also relevant to human septin function. This work, partly in collaboration with the labs of Jeremy Thorner and Eva Nogales at UC Berkeley, was published in Molecular Biology of the Cell and the Journal of Cell Biology in 2016.

We also collaborated with the lab of Ravi Manjithaya (Jawaharlal Nehru Centre for Advanced Scientific Research, India) to show that yeast septins play an early role in macro-autophagy in starved cells. Given the similarities between the membrane dynamics involved in autophagy and those involved in prospore membrane biogenesis, these findings extend our understanding of septin function in membrane dynamics. This work was published in the Journal of Cell Science in 2018.

Finally, we studied the molecular basis of action of a small molecule (forchlorfenuron, FCF) thought to act specifically on septin filament assembly in non-plant eukaryotes and therefore used by many researchers as a “septin drug”. We found clear evidence of off-target effects, using four different eukaryotic cell types. This work was published in Eukaryotic Cell in 2014.

Our studies challenge the idea that thermodynamics (i.e., the affinities between proteins randomly colliding in solution) is the main driving force of protein complex assembly, and favor largely unappreciated roles for the kinetics of protein folding and cooperativity between protein-protein interactions. We are now focusing our attention on the mechanisms by which septin proteins fold in living cells, and how the insights we gained from the study of mitotic yeast septins apply to septins in other developmental contexts, and to oligomers composed of non-septin proteins.