Figure 3. ATP hydrolysis powers mobility of ISWI and prevents ISWI-mediated hardening of condensates.
a, Holotomogram of chromatin condensates. The average nucleosome concentration (225 ± 59 μM) was determined from the refractive index. One of two independent replicates is depicted. b, Left: Partial-condensate FRAP of ISWI-GFP in presence of indicated nucleotides (all 0.77 mM). Only the addition of ATP allowed a fast recovery. Bottom: Quantification of FRAP time courses. Lines are averages, shaded areas SD of 20 condensates for AMPPNP and UTP, 15 for ADP, 25 for ADP-BeFx and 30 for ATP. c, Schematic of condensate fusion experiments with optical tweezers. d, Fusion velocities of condensates measured by optical trapping. ISWI in presence of non-hydrolysable nucleotides but not of ATP, slowed down fusion. Chromatin-only and nucleotide-free ISWI conditions were replicated three times. e, AMPPNP did not slow down fusion when ISWI lacked its HSS domain. f, Addition of ATP to an ATPase dead mutant of ISWI (E257Q) slowed down condensate fusion. g, Addition of ATP to wild type ISWI did not affect condensate fusion. Data in d–g are log10-fold changes in velocities relative to the mean of the chromatin only condition. Unless stated otherwise, all data were independently replicated twice per condition.