Kozai, T. | Search bioRxiv

bioRxiv2023

Dynamic molecular mechanism of the nuclear pore complex permeability barrier

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport of specific macromolecules while impeding the exchange of unsolicited material. However, key aspects of this gating mechanism remain controversial. To address this issue, we determined the nanoscopic behavior of the permeability barrier directly within yeast S. cerevisiae NPCs at transport-relevant timescales. We show that the large intrinsically disordered domains of phenylalanine-glycine repeat nucleoporins (FG Nups) exhibit highly dynamic fluctuations to create transient voids in the permeability barrier that continuously shape-shift and reseal, resembling a radial polymer brush. Together with cargo-carrying transport factors the FG domains form a feature called the central plug, which is also highly dynamic. Remarkably, NPC mutants with longer FG domains show interweaving meshwork-like behavior that attenuates nucleocytoplasmic transport in vivo. Importantly, the bona fide nanoscale NPC behaviors and morphologies are not recapitulated by in vitro FG domain hydrogels. NPCs also exclude self-assembling FG domain condensates in vivo, thereby indicating that the permeability barrier is not generated by a self-assembling phase condensate, but rather is largely a polymer brush, organized by the NPC scaffold, whose dynamic gating selectivity is strongly enhanced by the presence of transport factors.

biophysics↗

bioRxiv2022

Biomimetic microstimulation of sensory cortices

BackgroundIntracortical microstimulation (ICMS) is an emerging approach to restore sensation to people with neurological injury or disease. Biomimetic microstimulation, or stimulus trains that mimic neural activity in the brain through encoding of onset and offset transients, could improve the utility of ICMS for BCI applications, but how biomimetic microstimulation affects neural activation is not understood. Stimulus induced depression of neural activity (decreases in evoked intensity over time) is also a potential barrier to clinical implementation of sensory feedback, and biomimetic microstimulation may reduce this effect. ObjectiveWe evaluated how biomimetic trains change the calcium response, spatial distribution, and depression of neurons in the somatosensory and visual cortices. MethodsCalcium responses of neurons were measured in Layer 2/3 of visual and somatosensory cortices of anesthetized GCaMP6s mice in response to ICMS trains with fixed amplitude and frequency (Fixed) and three biomimetic ICMS trains that increased the stimulation intensity during the onset and offset of stimulation by modulating the amplitude (BioAmp), frequency (BioFreq), or amplitude and frequency (BioBoth). ICMS was provided for either 1-s with 4-s breaks (Short) or for 30-s with 15-s breaks (Long). ResultsBioAmp and BioBoth trains evoked distinct onset and offset transients in recruited neural populations, while BioFreq trains evoked population activity similar to Fixed trains. Individual neurons had heterogeneous responses primarily based on how quickly they depressed to ICMS, where neurons farther from the electrode depressed faster and a small subpopulation (1-5%) were modulated by BioFreq trains. Neurons that depressed to Short trains were also more likely to depress to Long trains, but Long trains induced more depression overall due to the increased stimulation length. Increasing the amplitude during the hold phase resulted in an increase in recruitment and intensity which resulted in more depression and reduced offset responses. Biomimetic amplitude modulation reduced stimulation induced depression by 14.6{+/-}0.3% for Short and 36.1{+/-}0.6% for Long trains. Ideal observers were 0.031{+/-}0.009 s faster for onset detection and 1.33{+/-}0.21 s faster for offset detection with biomimetic amplitude encoding. ConclusionsBiomimetic amplitude modulation evokes distinct onset and offset transients, reduces depression of neural calcium activity, and decreases total charge injection for sensory feedback in brain-computer interfaces by lowering recruitment of neurons during long maintained periods of ICMS. In contrast, biomimetic frequency modulation evokes distinct onset and offset transients in a small subpopulation of neurons but also reduces depression in recruited neurons by reducing the rate of activation.

neuroscience↗

bioRxiv2017

Dynamic clustering of dynamin-amphiphysin rings regulates membrane constriction and fission coupled with GTP hydrolysis

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin mediated endocytosis. Dynamin forms washer ring-shaped/helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulates membrane remodeling during fission, but its precise mechanism remains elusive. In this study, we analyze structural changes of dynamin-amphiphysin complexes during membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin rings are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our new approaches using combination of EM and HS-AFM clearly demonstrates dynamics of dynamin-amphiphysin complexes during membrane fission suggesting a novel \"clusterase\" model of dynamin-mediated membrane fission.

biophysics↗