Regulation of mitotic spindle orientation: an integrated view

Abstract

Mitotic spindle orientation is essential for cell fate decisions, epithelial maintenance, and tissue morphogenesis. In most animal cell types, the dynein motor complex is anchored at the cell cortex and exerts pulling forces on astral microtubules to position the spindle. Early studies identified the evolutionarily conserved Gαi/LGN/NuMA complex as a key regulator that polarizes cortical force generators. In recent years, a combination of genetics, biochemistry, modeling, and live imaging has contributed to decipher the mechanisms of spindle orientation. Here, we highlight the dynamic nature of the assembly of this complex and discuss the molecular regulation of its localization. Remarkably, a number of LGN-independent mechanisms were described recently, whereas NuMA remains central in most pathways involved in recruiting force generators at the cell cortex. We also describe the emerging role of the actin cortex in spindle orientation and discuss how dynamic astral microtubule formation is involved. We further give an overview on instructive external signals that control spindle orientation in tissues. Finally, we discuss the influence of cell geometry and mechanical forces on spindle orientation.

Keywords: NuMA; actin cortex; astral microtubules; cell geometry; spindle orientation.

Figure 1. The LGN complex

(A) The scheme shows the LGN domains and its interactions with Gαi membrane‐anchored subunits, and with NuMA, as well as the interaction with cortical proteins (Dlg, Afadin) that regulate LGN cortical localization. (B) LGN complex localization in different systems, showing the polarity proteins regulating this specific localization when applicable. (i) Drosophila embryonic neuroblasts, (ii) C. elegans zygote, (iii) neural progenitors in the vertebrate neuroepithelium, and (iv) mammalian cell lines.

Figure 2. Temporal–spatial regulation of LGN complex localization

Left: Scheme of interphase and mitotic phases indicating the distribution of LGN, NuMA, and dynein, as well as the localization of specific molecules (Ran‐GTP, PLK1, and centralspindlin proteins) that are involved in controlling this distribution in cultured HeLa cells. Note that NuMA and dynein cortical levels increase in anaphase. Right: Detail of the molecular mechanisms involved in the control of LGN/NuMA localization and of dynein by Ran‐GTP/centralspindlin and PLK1, respectively. The control of NuMA cortical levels by CDK1 activity is also indicated.

Figure 3. The role of actin in spindle orientation

(A) In Drosophila SOP, dishevelled localizes to the posterior cortex activating two parallel pathways required for spindle orientation: i) The recruitment of NuMA via the DEP domain allows dynein enrichment at this site, ii) a molecular cascade involving the tail domain of dishevelled, and the Canoe and RhoA molecules leads to the activation of the actin nucleator diaphanous at this cortical site. (B) RhoA and the anthrax receptor 2a (Antxr2a) orient the spindle along the animal–vegetal axis in zebrafish epiblast. Activation of Fzz promotes RhoA recruitment to the “animal cortex”. In turn, RhoA induces actin nucleation leading to the formation of an actin cap, and together with the anthrax receptor activates the downstream effector zDia. (C, D) Involvement of different actin‐related molecules in xy spindle orientation in single cells cultured on fibronectin micropatterns. In this context, the distribution of actin retraction fibers dictates the orientation of the spindle. (C) Polarized actin subcortical clouds make the link between the distribution of retraction fibers and spindle orientation. Myosin 10 mediates the link between actin and microtubules in this context. The classical LGN/dynein complexes are proposed to act in parallel to this pathway, leading to robust spindle orientation. (D) The ezrin–radixin–moesin proteins are enriched in the adhesive cortex in cells cultured in L patterns. These proteins control the initial distribution of LGN and NuMA, during prometaphase, which favor spindle rotation along the depicted axis.

Figure 4. Modulation of spindle orientation through regulation of astral microtubules

(A, B) Schema illustrating the centrosome and astral microtubules as well as generic proteins localized on these structures. Cortically recruited dynein is believed to walk on the minus‐end direction of astral MT, generating the force that orients the spindle. (C) Regulation of different processes (i–iv) controls the density, length, and behavior of astral microtubules, and thus spindle orientation. The process and cellular structure concerned are indicated in red. Loss of function of specific proteins (in light blue) results in defects in the indicated processes and spindle misorientation. In (iii), MISP acts from the cellular cortex regulating cortex–MT interaction.

Figure 5. Extracellular stimuli influencing spindle orientation

(A) The Wnt/Fz pathway controls spindle orientation in diverse contexts including (i) the 4‐cell C. elegans embryo, (ii) zebrafish epiblast cells, and (iii) embryonic stem cells. (B) Involvement of integrins–ECM in spindle orientation: I) In mouse skin progenitors during stratification, β1 integrin is necessary for correct cell polarity (PKC), LGN apical localization and thus spindle orientation; II) in cultured cells, interaction of β1 integrin with extracellular matrix components is necessary for spindle orientation in parallel to the substrate. (C) Semaphorins control planar spindle orientation (i) in the context of epithelial morphogenesis in MDCK cysts and (ii) in neural progenitors of the mouse spinal cord.

Figure 6. Role of cell geometry and external forces

(A) Cells in the basal layer of the developing mouse epidermis adopt a binary orientation: Symmetric divisions occur in the plane of the epithelium, and asymmetric divisions divide along the apico‐basal axis in an Insc/Gαi/LGN/NuMA/dynein manner, with one daughter cell delaminating into the suprabasal cell layer. Upon NuMA or p150 depletion, cortical force generators are not functioning and most divisions now take place in the plane of the epithelium, suggesting that the “default” planar orientation may be dictated by the flat cell shape in this tissue. Green lines: Insc and Gαi3 apical accumulation; orange lines: force generators (dynein). See Williams et al (2011) 39. (B) In single cells cultured on fibronectin micropatterns (light blue), a field of maximal force is associated with polarized retraction fibers (blue lines). Cells cultured on “cross”‐shaped patterns orient their spindle along the long arms of the cross, where maximal forces are observed. Laser ablation of retraction fibers on the long arms induces a 90° spindle rotation and alignment to face the “new” maximal forces. See Fink et al (2011) 67. (C) In the fly notum epithelium, NuMA accumulates at tricellular junctions in the G2 phase. Left panel: A vector corresponding to the cells long axis (gray bar) or to the geometry of tricellular junctions (or Mud accumulation, red dots) can be drawn (blue bar). In elongated cells, both vectors are aligned (top light red cell), whereas they do not always align in cells with an isotropic shape (bottom light red cell). Middle panel: The “Mud accumulation” vector predicts the orientation of cell divisions more accurately than the long axis. Right panel: Position and shape of the daughter cells after division. See Bosveld et al (2016) 140.