Molecular basis for dyneinopathies reveals insight into dynein regulation and dysfunction

Abstract

Cytoplasmic dynein plays critical roles within the developing and mature nervous systems, including effecting nuclear migration, and retrograde transport of various cargos. Unsurprisingly, mutations in dynein are causative of various developmental neuropathies and motor neuron diseases. These ‘dyneinopathies’ define a broad spectrum of diseases with no known correlation between mutation identity and disease state. To circumvent complications associated with dynein studies in human cells, we employed budding yeast as a screening platform to characterize the motility properties of seventeen disease-correlated dynein mutants. Using this system, we determined the molecular basis for several classes of etiologically related diseases. Moreover, by engineering compensatory mutations, we alleviated the mutant phenotypes in two of these cases, one of which we confirmed with recombinant human dynein. In addition to revealing molecular insight into dynein regulation, our data provide additional evidence that the type of disease may in fact be dictated by the degree of dynein dysfunction.

Pac1/LIS1 promotes an uninhibited conformation of dynein that coordinates its localization and activity

“…We previously showed that a K1475Q dynein mutant exhibits phenotypes much like those described for K1475E ( i.e. , increased single molecule run lengths and cortical localization), and leads to compromised dynein function in cells 34 . Thus, the phi particle is an important, highly conserved conformational state that is used by organisms throughout evolution to ensure appropriate dynein activity.…”

“…We previously found that a disease-correlated amino acid substitution within the linker domain – the mechanical element responsible for the powerstroke – leads to an increase in single molecule run length 34 . This residue (K1475) is equivalent to one known to stabilize the autoinhibited conformation of human dynein (R1567) 7 .…”

“…Here, we set out to address the question of whether yeast dynein adopts an autoinhibited phi particle conformation, and if so, what role it plays in regulating in vitro and in vivo dynein activity. Our recent findings suggested the potential for yeast dynein to adopt such a conformation 34 . Specifically, we found that engineering a neurological disease-correlated mutation into yeast dynein leads to increased run lengths of single molecules of dynein, and a localization pattern in cells that is suggestive of an enhanced dynein-dynactin interaction.…”

Modeling disease-correlated TUBA1A mutation in budding yeast reveals a molecular basis for tubulin dysfunction

“…Given the consequences on apparent brain development in the patient with TUBA1A G436R (pachygyria, and severe microcephaly associated with postural delay and poor communication abilities), and the strong link between mutations in dynein activity and motor neuron diseases and developmental brain disorders (Bahi-Buisson et al, 2014;Laquerriere et al, 2017;Marzo et al, 2019;Vissers et al, 2010;Willemsen et al, 2012), our data linking disrupted dynein activity with this mutation are not entirely surprising. For example, dynein activity is critical for various aspects of early neuronal development, in part by promoting interkinetic nuclear migration in neuronal progenitors, and in the subsequent migration of the resulting postmitotic neurons (Del Bene et al, 2008;Hu et al, 2013;Tsai et al, 2010).…”

“…Bars represent weighted means with weighted standard error of proportion. For panels C and, D, P values were calculated using an unpaired Welch's t test; for panels E -G, statistical significance was determined by calculating Z scores as previously described (Marzo et al, 2019). For all plots, diamonds represent mean values obtained from each independent replicate experiment.…”