Hierarchical integration of mitochondrial and nuclear positioning pathways by the Num1 EF hand

Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.

SUMO-Based Regulation of Nuclear Positioning to Spatially Regulate Homologous Recombination Activities at Replication Stress Sites

DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks.

A nesprin-4/kinesin-1 cargo model for nucleus positioning in cochlear outer hair cells

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, nesprin-4 (encoded by Syne4) was shown to play a crucial role in nucleus positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in-vivo AAV gene delivery, we show that this interaction is critical for nucleus positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Overall, our results suggest that OHCs require unique cellular machinery for proper nucleus positioning at the onset of electromotility. This machinery relies on the interaction between nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nucleus positioning.

The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans independently from its KASH or actin-binding domains

KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER, mitochondria, and lipid droplets were unanchored, moving throughout the cytoplasm. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.

Diverse organizations of actin and nuclei underpin the evolution of indeterminate growth in Chytridiomycota and Dikarya

Indeterminate growth, as in the hyphae of the “Humongous Fungus” of Michigan requires sustained nuclear migration and cell wall remodeling. We compare actin organization and patterns of nuclear positioning among four distantly related, indeterminate species of phylum Chytridiomycota: Cladochytrium replicatum, Physocladia obscura, Nowakowskiella sp., and Polychytrium aggregatum. We combined light microscopy, nuclear staining with DAPI, and actin staining with rhodamine phalloidin to analyze actin distribution and nuclear migration during somatic growth in the four Chytridiomycota species. Actin formed plaques, filaments, cables and perinuclear shells in patterns that varied across the four species. All four species initiated indeterminate growth by extending branching, anucleate rhizomycelium, <1 µm in diameter. Nuclei, some elongated as if migrating, first appear in intercalary segments that widened to diameters >1 µm. After mitosis, an intercalary swelling in C. replicatum became septate and a single, distal nucleus migrated tipwards to a new swelling. In Physocladia obscura, swellings were aseptate and multinucleate, and several nuclei migrated tipwards into a new swelling. Nuclei migrated tipwards from irregularly cylindrical filaments in Nowakowskiella sp., and in Polychytrium aggregatum, from regular, hypha-like filaments. Thus, distantly related lineages of zoosporic fungi deploy ancestral morphogenetic machinery in differing patterns that resulted in convergent, indeterminate growth.

Microtubule-independent movement of the fission yeast nucleus

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.

Escape from mitotic catastrophe by actin-dependent nuclear displacement in fission yeast

SUMMARYProper nuclear positioning is essential for the execution of a wide variety of cellular processes in eukaryotic cells (Gundersen and Worman, 2013; Kopf et al., 2020; Lele et al., 2018). In proliferating mitotic cells, nuclear positioning is crucial for successful cell division. The bipolar spindle, which pulls sister chromatids towards two opposite poles, needs to assemble in the geometrical center of the cell. This ensures symmetrical positioning of the two nuclei that are reformed upon mitotic exit, by which two daughter cells inherit the identical set of the chromosomes upon cytokinesis. In fission yeast, the nucleus is positioned in the cell center during interphase; cytoplasmic microtubules interact with both the nucleus and the cell tips, thereby retaining the nucleus in the medial position of the cell (Daga et al., 2006; Tran et al., 2001). By contrast, how the nucleus is positioned during mitosis remains elusive. Here we show that several cell-cycle mutants that arrest in mitosis all displace the nucleus towards one end of the cell axis. Intriguingly, the actin cytoskeleton, not the microtubule counterpart, is responsible for the asymmetric movement of the nucleus. Time-lapse live imaging indicates that mitosis-specific F-actin cables interact with the nuclear membrane, thereby possibly generating an asymmetrical pushing force. In addition, constriction of the actomyosin ring further promotes nuclear displacement. This nuclear movement is beneficial, because if the nuclei were retained in the cell center, subsequent cell division would impose the lethal cut phenotype (Hirano et al., 1986; Yanagida, 1998), in which chromosomes are intersected by the contractile actin ring and the septum. Thus, fission yeast escapes from mitotic catastrophe by means of actin-dependent nuclear movement.