A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly.

The linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

TorsinA controls TAN line assembly and the retrograde flow of dorsal perinuclear actin cables during rearward nuclear movement.

The nucleus is positioned toward the rear of most migratory cells. In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, resulting in the orientation of the centrosome in the direction of migration. In this study, we report that the nuclear envelope-localized AAA+ (ATPase associated with various cellular activities) torsinA (TA) and its activator, the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1), are required for rearward nuclear movement during centrosome orientation in migrating fibroblasts. Both TA and LAP1 contributed to the assembly of transmembrane actin-associated nuclear (TAN) lines, which couple the nucleus to dorsal perinuclear actin cables undergoing retrograde flow. In addition, TA localized to TAN lines and was necessary for the proper mobility of EGFP-mini-nesprin-2G, a functional TAN line reporter construct, within the nuclear envelope. Furthermore, TA and LAP1 were indispensable for the retrograde flow of dorsal perinuclear actin cables, supporting the recently proposed function for the nucleus in spatially organizing actin flow and cytoplasmic polarity. Collectively, these results identify TA as a key regulator of actin-dependent rearward nuclear movement during centrosome orientation.

Minus-end-directed Kinesin-14 motors align antiparallel microtubules to control metaphase spindle length.

During cell division, a microtubule-based mitotic spindle mediates the faithful segregation of duplicated chromosomes into daughter cells. Proper length control of the metaphase mitotic spindle is critical to this process and is thought to be achieved through a mechanism in which spindle pole separation forces from plus-end-directed motors are balanced by forces from minus-end-directed motors that pull spindle poles together. However, in contrast to this model, metaphase mitotic spindles with inactive kinesin-14 minus-end-directed motors often have shorter spindle lengths, along with poorly aligned spindle microtubules. A mechanistic explanation for this paradox is unknown. Using computational modeling, in vitro reconstitution, live-cell fluorescence microscopy, and electron microscopy, we now find that the budding yeast kinesin-14 molecular motor Kar3-Cik1 can efficiently align spindle microtubules along the spindle axis. This then allows plus-end-directed kinesin-5 motors to efficiently exert the outward microtubule sliding forces needed for proper spindle bipolarity.