Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. While the mechanisms coordinating the segregation of the genome are well known, the processes which ensure organelle segregation between daughter cells remain less well-understood 1 . Here, we identify multiple actin assemblies that play distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria through mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (K ≈ 10 to 10 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (K ≈ 10 M), but those retaining exon 10 (ABR+10) did so only weakly (K ≈ 10 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.

Mitochondrial homeostasis requires a dynamic balance of fission and fusion. The actin cytoskeleton promotes fission; we find that the mitochondrially-localized myosin, Myosin 19 (Myo19), is integral to this process. Myo19 knock-down induces mitochondrial elongation, while Myo19 overexpression induces fragmentation. This mitochondrial fragmentation is blocked by a Myo19 mutation predicted to inhibit ATPase activity and strong actin binding but not by mutations predicted to affect the motor's working stroke that preserve ATPase activity. Super-resolution imaging indicates a dispersed localization of Myo19 on mitochondria, which we find to be dependent on metaxins. These observations suggest that Myo19 acts as a dynamic actin-binding tether that facilitates mitochondrial fragmentation. Myo19-driven fragmentation is blocked by depletion of either the endoplasmic reticulum (ER)-anchored formin INF2-CAAX or the mitochondrially-localized F-actin nucleator Spire1C, which together polymerize actin at sites of mito-ER contact for fission. These observations imply that Myo19 promotes fission by stabilizing mito-ER contacts; we used a split-luciferase system to demonstrate a reduction in these contacts following Myo19 depletion. Our data support a model in which Myo19 tethers mitochondria to ER-associated actin to promote mitochondrial fission.

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