Cytoplasmic forces functionally reorganize nuclear condensates in oocytes

Cells remodel their cytoplasm with force-generating cytoskeletal motors1. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic2–4 and germ5–7 cells. These forces are transmitted inside the nucleus4,7, yet their consequences on liquid-like biomolecular condensates8–10 residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include splicing speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of condensates inside the nucleus. We determine that cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Inversely, disrupting the forces decelerates nuclear condensate reorganization on both scales. We link the molecular deceleration found in mRNA-processing splicing speckles to reduced and altered splicing of mRNA, which in oocytes impedes fertility11. We establish that different sources of cytoplasmic forces can reorganize nuclear condensates and that this cytoplasmic aptitude for subnuclear reorganization is evolutionary conserved in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections12.One sentence summaryCytoplasmic random forces in growing oocytes drive multiscale reorganization of nuclear liquid-like biomolecular condensates.

Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors

Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, we describe an alternative, highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors. We applied tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilized a large field-of-view to simultaneously track the velocities, run lengths and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads. Importantly, the 16-μm long DNA tethers between the motors and the beads significantly reduced the vertical component of the applied force pulling the motors away from the microtubule. Our approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.

Structural characterization of the RH1-LZI tandem of JIP3/4 highlights RH1 domains as a cytoskeletal motor-binding motif

JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin1 motors. Both are dimers that are stabilised by two sections of leucine zipper coiled coils. The N-terminal Leucine Zipper I (LZI) belongs to a section that binds dynein-DLIC and kinesin1-KHC, whilst the medial Leucine Zipper II (LZII) binds dynactin-p150glued and kinesin1-KLC. Structural data is available for the LZII, but the LZI section is still uncharacterized. Here we characterize the N-terminal part of JIP3/4 which consists of an RH1 (RILP homology 1) domain followed by the LZI coiled coil using bioinformatical, biophysical and structural approaches. The RH1-LZI tandem of JIP3 associates as a high affinity homodimer exhibiting elongated alpha-helical fold. 3D homology modelling of the RH1-LZI tandem reveals that the kinesin1-KHC binding site mainly overlaps with the RH1 domain. A sequence comparison search indicates that only one other protein family has RH1 domains similar to those of JIP3/4, the RILP (Rab-interacting lysosomal protein) family which consists of adaptor proteins linking Rab GTPases to cytoskeletal motors. RILPL2 is recruited through its RH1 domain by the myosin 5a motor. Here, we showed that the RH1 domain of JIP3 also interacts with myosin 5 A in vitro, highlighting JIP3/4 as possible myosin 5a adaptors. Finally, we propose that JIP3/4 and RILP family members define a unique RH1/RH2-architecture adaptor superfamily linking cytoskeletal motors and Rab GTPases.