Summary 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 minusend 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.

In fibroblasts and myoblasts polarizing for migration, retrograde actin flow moves the nucleus rearward, orienting the centrosome toward the leading edge. The nucleus engages moving dorsal actin cables through linear arrays of nesprin-2G and SUN2 called TAN lines. In this study, Saunders et al. report that the nuclear envelope–localized AAA+ ATPase torsinA and its activator, LAP1, are required for TAN line assembly and retrograde dorsal actin cable flow.

Fig. 3 (corrected panel). Reconstitution of CFE-synthesized FL SUN1 and SUN2 in ANMs. (A) Schematic of the process of generating ANMs with inserted CFE-synthesized membrane proteins. Fig. 3 (original panel). Reconstitution of CFE-synthesized FL SUN1 and SUN2 in ANMs. (A) Schematic of the process of generating ANMs with inserted CFE-synthesized membrane proteins.

The labels for the N-terminus and C-terminus in Fig. 3A were incorrectly swapped, and Fig. 5 had labels stating that the constructs were from SUN1 when in fact they were from SUN2. The online full-text and PDF versions of the paper have been updated.

Condensed title:Reconstituting functional SUN proteins in artificial nuclear membranes. ABSTRACT (180/180 words)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.Eukaryotic cells are defined by the presence of a genome-containing nucleus, the boundary of which is delineated by the nuclear envelope (NE), a specialized subdomain of the endoplasmic reticulum (ER) (Kite, 1913). The NE consists of concentric inner and outer nuclear membranes (INM and ONM, respectively) separated by a ~30-50 nm perinuclear space (PNS) that is contiguous with the ER lumen (Watson, 1959).While the ONM is an extension of the ER, a unique subset of proteins resides in the INM that interact with the nuclear lamina and chromatin within the nucleoplasm (Burke and Stewart, 2014).Fusion between the INM and ONM creates numerous aqueous channels throughout the NE that are occupied by nuclear pore complexes (NPCs), which are the primary sites of molecular exchange between the cytoplasm and nucleoplasm (Knockenhauer and Schwartz, 2016, Otsuka and Ellenberg, 2018). However, mechanical forces generated by the cytoskeleton within the cytoplasm can also be sensed and transmitted across the NE and into the nucleoplasm by LINC complexes (Alam et al., 2016, Brosig et al., 2010, Guilluy et al., 2014, Lombardi et al., 2011, Tajik et al., 2016. These evolutionarily conserved NE-spanning molecular bridges mediate several fundamental cellular processes including DNA damage repair, meiotic chromosome pairing, mechano-regulation of gene expression, and nuclear positioning (Alam et al., 2016, Chang et al., 2015, Meinke and Schirmer, 2015, Tapley and Starr, 2013. Consistent with their central role in cellular function is a growing list of genetic mutations in LINC complex proteins associated with human diseases such as aging-related hearing l...