Kuo, Y.-W. | Search bioRxiv

bioRxiv2020

Cryo-EM structures of outer-arm dynein array bound to microtubule doublet reveal a mechanism for motor coordination

Thousands of outer-arm dyneins (OADs) are arrayed in the axoneme to drive a rhythmic ciliary beat. Coordination among multiple OADs is essential for generating mechanical forces to bend microtubule doublets (MTDs). Using electron microscopy, we determined high-resolution structures of OAD arrays bound to MTD in two different states. OAD preferentially binds to MTD protofilaments with a pattern resembling the native tracks for its distinct microtubule-binding domains. Upon MTD binding, free OADs are induced to adopt a stable parallel conformation, primed for array formation. Extensive tail-to-head (TTH) interactions between OADs are observed, which need to be broken for ATP turnover by the dynein motor. We propose that OADs in an array sequentially hydrolyze ATP to slide MTDs. ATP-hydrolysis in turn relaxes the TTH interfaces to effectuate free nucleotide cycles of downstream OADs. These findings lead to a model explaining how conformational changes in the axoneme produce coordinated action of dyneins.

biophysics↗

bioRxiv2020

Endogenous viral element-derived piRNAs are not required for production of ping-pong-dependent piRNAs from Diaphorina citri densovirus

Partial integrations of DNA and non-retroviral RNA virus genomes, termed endogenous viral elements (EVEs), are abundant in arthropod genomes and often produce PIWI-interacting RNAs (piRNAs) speculated to target cognate viruses through the ping-pong cycle, a post-transcriptional RNA silencing mechanism. Here we describe a Diaphorina citri densovirus (DcDV)-derived EVE in the genome of Diaphorina citri. We found that this EVE gives rise to DcDV-specific primary piRNAs and is unevenly distributed among D. citri populations. Unexpectedly, we found that DcDV is targeted by ping-pong-dependent viral piRNAs (vpiRNAs) in D. citri lacking the DcDV-derived EVE, while four naturally infecting RNA viruses of D. citri are not targeted by vpiRNAs. Furthermore, a recombinant Cricket paralysis virus containing a portion of the DcDV genome corresponding to the DcDV-derived EVE was not targeted by vpiRNAs during infection in D. citri harboring the EVE. These results represent the first report of ping-pong-dependent vpiRNAs outside of mosquitoes.

microbiology↗

bioRxiv2019

Predicted effects of severing enzymes on the length distribution and total mass of microtubules

Microtubules are dynamic cytoskeletal polymers whose growth and shrinkage are highly regulated as eukaryotic cells change shape, move and divide. One family of microtubule regulators includes the ATP-hydrolyzing enzymes spastin, katanin and fidgetin, which sever microtubule polymers into shorter fragments. Paradoxically, severases can increase microtubule number and mass in cells. Recent work with purified spastin and katanin accounts for this phenotype by showing that, in addition to severing, these enzymes modulate microtubule dynamics by accelerating the conversion of microtubules to the growing state and thereby promoting their regrowth. This leads to the observed exponential increase in microtubule mass. Spastin also influences the steady-state distribution of microtubule lengths, changing it from an exponential, as predicted by models of microtubule dynamic instability, to a peaked distribution. This effect of severing and regrowth by spastin on the microtubule length distribution has not been explained theoretically. To solve this problem, we formulated and solved a master equation for the time evolution of microtubule lengths in the presence of severing and microtubule dynamic instability. We then obtained numerical solutions to the steady-state length distribution and showed that the rate of severing and the speed of microtubule growth are the dominant parameters determining the steady-state length distribution. Furthermore, we found that the amplification rate is predicted to increase with severing, which is a new result. Our results establish a theoretical basis for how severing and dynamics together can serve to nucleate new microtubules, constituting a versatile mechanism to regulate microtubule length and mass.\n\nSignificanceThe numbers and lengths of microtubules are tightly regulated in cells. Severing enzymes fragment microtubules into shorter filaments and are important for cell division and tissue development. Previous work has shown that severing can lead to an increase in total microtubule number and mass, but the effect of severing on microtubule length is not understood quantitatively. Combining mathematical modeling and computational simulation, we solve the microtubule length distribution in the presence of severing enzymes and explore how severing activity and microtubule dynamics collectively control microtubule number and length. These results advance our understanding of the physical basis of severing as a regulatory mechanism shaping the cellular cytoskeletal network.

biophysics↗