(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/317172 doi: bioRxiv preprint first posted online May. 8, 2018; 3 adult cochlea (Guinan, 1996). However, OHCs are also innervated by Type II afferent fibres 49 that, unlike the Type I fibres contacting IHCs, which encode sound timing, intensity and 62We found that during a narrow, critical period of postnatal development, OHCs fire 63 spontaneous Ca 2+ spikes immediately preceding their functional maturation (~P7-P8 (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/317172 doi: bioRxiv preprint first posted online May. 8, 2018; 4 Results 70The functional development of OHCs was studied primarily in the apical third of the Figure 1C, Movie 1) and room temperature (~20°C: Figure 1D (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.)The copyright holder for this preprint . http://dx.doi.org/10.1101/317172 doi: bioRxiv preprint first posted online May. 8, 2018; 5 stopping a couple of days later ( Figure 1F). This correlated with a decrease in the maximum 95 Ca 2+ -related changes in fluorescence intensity (ΔF/F 0 ) for Ca 2+ measured from active OHCs 96( Figure `1G), which could be due to a progressive reduction of AP bursts with age. 97Calcium transients were abolished in Ca 2+ free solution (Figure 2A, Figure 3C, Movie 5). We quantified the average correlation 119All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. OHCs ( Figure 3D). However, the r s was independent of the amplitude (ΔF/F 0 ) of the Ca 2+ 127 signal ( Figure 3E) measured as a pixel average over the entire spread of the Ca 2+ wave. 128Therefore, the coordination of the electrical activity between nearby OHCs was dependent on 129 the lateral spread, but not the amplitude, of the Ca 2+ waves. 130We then sought to identify how spontaneous Ca 2+ activity from the non-sensory cells of the easier to see that the Ca 2+ waves originating in the GER were able to travel to the LER and 140propagate through Deiters' cells ( Figure 4D; Supplementary Figure 1B). In order to test 141whether Deiters' cells mediate signal transfer from the GER to the OHCs, we analysed Ca 2+ 142 signals after removal of Deiters' cells ( Figure 5C), which was performed using gentle suction 143 via a small pipette (~3-4 µm in diameter). This procedure is widely used to gain access to the 144 All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. OHCs ( Figure 5A,B, Movie 6). We found that Ca 2+ elevation in OHCs associated ...

18The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two 19 protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer 20 deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here 21 we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture 22 two reaction intermediates. In one structure, histone residues near the dyad and in the H2A- 23H2B acidic patch, distal to the active SNF2h protomer, are disordered. The disordered acidic 24 patch is expected to inhibit the second SNF2h promoter, while disorder near the dyad is 25 expected to promote DNA translocation. The other structure doesn't show octamer deformation, 26but surprisingly shows a 2bp translocation. FRET studies indicate that ADP-BeFx predisposes 27 SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for 28 allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric 29 octamer deformation to inhibit the second protomer, while stimulating directional DNA 30 translocation. 31 32 One sentence summary: Cryo-EM structures capture different conformational states of 33 chromatin remodeler-nucleosome complexes. 34 35 65 complex: a state with an unexpectedly translocated nucleosome (Figure 1, Figure 1-supplement 66 1-6), a state with two SNF2h protomers bound to a nucleosome (Figure 2A, Figure 2-67 supplement 1) and a state with one protomer bound to a nucleosome that shows increased 68 disorder within the histone core (Figure 2B). The locations of histone disorder strongly suggest a 69 4 role for octamer deformation in protomer coordination and directional DNA translocation. In 70 addition, we detect new ISWI-histone contacts that make significant contributions to 71 nucleosome sliding and help explain why ISWI may differ in mechanism from Swi2/Snf2 (Figure 72 3) (Liu et al., 2017).73 74 Overview of SNF2h-nucleosome structures 75 Like most ISWI remodelers, SNF2h slides mono-nucleosomes assembled on short stretches of 76 DNA towards the center of the DNA (Clapier and Cairns, 2009; Narlikar et al., 2013; Zhou et al., 77 2016). In previous studies we have found that while a monomer of SNF2h can slide 78 nucleosomes, SNF2h functions most optimally as a dimer (Leonard and Narlikar, 2015; Racki et 79 al., 2009). In these studies, we were able to visualize both singly bound and doubly bound 80 SNF2h using negative stain EM (Racki et al., 2009). Previous studies have further shown that 81 binding of the ATP analog, ADP-BeF x , promotes a restricted conformation of the ATPase active 82 site in a manner that is dependent on the H4 tail (Racki et al., 2014). The restricted 83 conformation is consistent with observations showing an activating role for the H4 tail (Clapier et 84 al., 2001; 2002; Hamiche et al., 2001). Further, binding of ADP-BeF x to SNF2h promotes 85 conformational flexibility of buried histone residues (Sinha et al., 2017). This conformational 86 flexibility is functionally important ...

ATP-dependent chromatin remodelers are essential enzymes that restructure eukaryotic genomes to enable all DNA-based processes. The diversity and complexity of these processes are matched by the complexity of the enzymes that carry them out, making remodelers a challenging class of molecular motors to study by conventional methods. Here we use a single molecule biophysical assay to overcome some of these challenges, enabling a detailed mechanistic dissection of a paradigmatic remodeler reaction, that of sliding a nucleosome towards the longer DNA linker. We focus on how two motors of a dimeric remodeler coordinate to accomplish such directional sliding. We find that ATP hydrolysis by both motors promotes coordination, suggesting a role for ATP in resolving the competition for directional commitment. Furthermore, we show an artificially constitutive dimer is no more or less coordinated, but is more processive, suggesting a cell could modulate a remodeler’s oligomeric state to modulate local chromatin dynamics.