Lissencephaly (smooth brain) is a brain malformation disorder associated with haploinsufficiency of Lissencephaly-1 (LIS1), also called PAFAH1B1 (Platelet-activating factor acetylhydrolase IB subunit alpha) (Dobyns et al., 1993; Hattori et al., 1994; Reiner et al., 1993; Moon and Wynshaw-Boris, 2013). At the molecular level, LIS1 is an important regulatoratory protein controlling cytoplasmic dynein and microtubules (MTs), that is also known to have dual roles coordinating both MTs and the actin cytoskeleton (Kholmanskikh et al., 2003; Kholmanskikh et al., 2006; Jheng et al., 2018). Besides pivotal roles of LIS1 in post-mitotic neurononal migration, Pafah1b1 mouse mutant studies suggest additional cellular functions of LIS1 in neocortical neural progenitor cell (NPC) division by regulating mitotic spindle orientation and cell fate (Yingling et al., 2008; Youn et al., 2009; Hippenmeyer et al., 2010; Bershteyn et al., 2017; Moon et al., 2014). The mitotic phenotypes of Pafah1b1 mutants are closely related and consistent with those of other mutants of MT/dynein-associated proteins such as LGN, NDE1, and NDEL1 (Bradshaw and Hayashi, 2017; Doobin et al., 2016; Wynne and Vallee, 2018). However, unlike these other mouse mutants of LIS1-interacting proteins, Pafah1b1 mutants displayed a significant decrease in neuroepithelial stem cells in the neocortex and subsequent neonatal death compared with a less catastrophic phenotype seen in radial glial (RG) progenitors (Yingling et al., 2008). Our recent studies with human-induced pluripotent stem cells (iPSCs) of Miller-Dieker syndrome, a severe form of lissencephaly caused by heterozygosity of more than 20 genes including PAFAH1B1, demonstrated a prolongation of mitotic division time of oRG progenitors but not ventricular zone radial glial (vRG) progenitors (Bershteyn et al., 2017). All of these previous studies suggest that cellular functions of LIS1 other than MT/dynein-mediated mitotic spindle regulation dictate further mitotic progression from mitotic (M) phase to anaphase/telophase in late stages of mitosis and daughter cell separation.

Although the functions of LIS1 in F-actin regulation have been studied previously, its downstream cellular signaling pathways that regulate actomyosin during late mitosis and cytokinesis has not been explored. When cytokinesis is initiated, MTs provide positional cues for the cleavage furrow location and membrane ingression at the equatorial cortex by inducing central spindle assembly (Bement et al., 2005; von Dassow, 2009; Werner et al., 2007). At this point, dynamic crosstalk between astral MTs and cortical filamentous actin (F-actin) transmit inhibitory signals at the polar cortex, which indirectly instructs cleavage furrow positioning to the equatorial cortex (Canman et al., 2003; Foe and von Dassow, 2008; Murthy and Wadsworth, 2008). During cleavage plane formation, a small GTPase RhoA functions as a master regulator of cytokinetic furrow ingression (Bement et al., 2005; Nishimura and Yonemura, 2006) that recruits contractile ring components, including the scaffold protein Anillin (D'Avino et al., 2008; Gregory et al., 2008; Oegema et al., 2000; Piekny and Glotzer, 2008) and Septin (Kinoshita et al., 2002; Maddox et al., 2007; Oegema et al., 2000). At the same time, Myosin II accumulates at the equatorial cortex and serves as a constriction force generator by interacting with F-actin (Glotzer, 2005; Straight et al., 2003; Zhou and Wang, 2008). However, when coordination between MTs and F-actin is perturbed, abnormally fewer or elongated astral MTs destabilize the cleavage furrow. This MT dysfunction induces actomyosin dysregulation, leading to cell membrane hyper-contractility, RhoA and F-actin mislocalization outside of the midzone/cleavage furrow, cell shape oscillation, and subsequent mitotic failure (Canman et al., 2003; Murthy and Wadsworth, 2008; Rankin and Wordeman, 2010; Watanabe et al., 2008). It is important to note that LIS1 is involved in astral MT movements and F-actin polymerization, so it is possible that LIS1 contributes to important cellular processes during cytokinesis by regulating actomyosin and other cleavage furrow-associated proteins during late stages of mitosis and cytokinesis.

We hypothesized that the LIS1-dynein-MT network may be essential for cytokinesis, perhaps by fine-tuning actomyosin and cell membrane contractility. Therefore, we assessed the mitotic phenotypes of neocortical from Pafah1b1-deficient mouse embryos, and monitored the entire duration of mitoses of Pafah1b1-deficient mouse embryonic fibroblasts (MEFs), using time-lapse live-cell imaging. Through imaging of Pafah1b1-deficient MEFs, we found severe defects in cleavage plane positioning and dysregulation of cell membrane contractility. Here we demonstrate that LIS1 determines cleavage plane positioning through a RhoA-actomyosin signaling pathway, defining a novel molecular mechanism of LIS1 underlying spatiotemporal regulation of cell membrane contractility during mitotic cell division.

A number of previous studies have demonstrated that LIS1 is required for neuronal migration and the production of the appropriate numbers of post-mitotic neurons that migrate into their functional cortical layers during embryonic brain development (Gambello et al., 2003; Hirotsune et al., 1998; Sasaki et al., 2000; Tsai et al., 2005; Youn et al., 2009; Hippenmeyer et al., 2010). In addition, LIS1 plays an important role in mitotic spindle orientation by controlling MTs and dynein complex in the interphase and metaphase of MEFs and neocortical NPCs (Faulkner et al., 2000; Yingling et al., 2008; Moon et al., 2014). However, during late stages of mitosis including cytokinesis, the cellular functions of LIS1 have not been clearly elucidated. Here we provide new evidence that LIS1 plays a crucial role in regulating cleavage plane positioning at late stages of mitosis and cytokinesis during early mouse development. We demonstrated the instructive functions of LIS1 in spindle orientation segregating the cell fate determinants into two daughter cells during mitosis, and LIS1 also regulates RhoA and Anillin-ring localization during cytokinesis. Collectively, this study provides key insights into underlying cellular mechanisms of which Pafah1b1 deficiency leads to impairments in neurogenesis as well as defects in neuronal migration.

Since Pafah1b1^hc/ko NPCs display severe mitotic progression perturbation causing cell cycle arrest at prometaphase and metaphase and frequent apoptosis in the neocortex (Gambello et al., 2003; Yingling et al., 2008), we needed to employ genetically manipulated MADM mouse lines to identify the sparsely labeled apical NPCs and daughter cells in Pafah1b1 mutant embryonic brains in vivo. Pafah1b1^hc/ko NPCs also exhibited severe mitotic defects and it was not possible to culture viable NPCs in vitro. Thus, it was unclear that Pafah1b1 deficiency in NPCs leads to the same or similar cytokinetic phenotypes seen in MEFs in vitro. In the developing mouse brains, neocortical NPCs express specific nuclear transcription factors (such as PAX6, SOX2) to maintain its proliferative capacity and potency to promote neurogenesis. Neocortical NPCs undergo complex basal-to-apical abcission-mediated cytokinetic process in vivo and they also generate one daughter cell with a different fate, a neuron with different lineage depending on the birth date and subtypes of NPCs in regionally specified neocortical domain. By contrast, MEFs only generate two MEFs with symmetric divisions. Despite the different tissue origins of these two distinct cell types (MEFs and NPCs), both MEF and NPC populations share cellular and cytoarchitectural features during cytokinesis. We confirmed that NPCs retain apical RhoA and Anillin localization when they undergo cytokinesis at the ventricular surface where F-actin polarization occurred in vivo. RhoA and Anillin play crucial roles in determining cytokinetic furrow localization by cross-interacting with F-actin and Myosin II at the equatorial cell membrane and the midbody. It appears that RhoA and Anillin-mediated cytokinetic furrow formation is controlled in a similar manner in both MEFs in vitro and apical NPCs in vivo (vRGs). Multiple in vitro studies have highlighted that genetic mutant MEFs are versatile and reliable cell types to investigate cellular mechanisms underlying cytokinetic defects, including those in the developing brain (Janisch et al., 2013; Rosario et al., 2010; Cook et al., 2011; Serres et al., 2012). In genetically targeted MEFs with reduced protein dose (e.g. Kif20b, Plk4, Ect2, and P27), cytokinesis-associated phenotypes were found, including multi-nucleation and centrosome amplification. Therefore, the use of MEFs enabled us to perform long-term live-cell imaging for various analyses that were not possible in NPCs in vitro or in mouse brains in vivo.

These Pafah1b1 mutant MEF studies of late mitosis and cytokinesis using live-cell imaging revealed that LIS1 restricts the cleavage furrow location at the equatorial cortex by inhibiting cell membrane hyper-contractility, demonstrating that LIS1 plays a critical role in cleavage plane specification as well as in actomyosin-mediated cell membrane contractility. We discovered that Pafah1b1 mutant MEFs displayed abnormal cellular phenotypes at late stages of mitosis and cytokinesis, with increased incidence of binucleation and cell death resulted from the failure in cell separation. Cell shape oscillations and chromosome rocking along the mitotic spindles were also evident in Pafah1b1 mutant MEFs, surprisingly similar to those of Anillin-depleted cells (Echard et al., 2004; Kechad et al., 2012; Piekny and Glotzer, 2008; Straight et al., 2003; Zhao and Fang, 2005). Together, these results suggest that Pafah1b1 deficiency in MEFs induces cleavage furrow mispositioning and failure in the recruitment of contractile ring components to the equatorial cortex. Intriguingly, cell shape oscillation and cytokinesis defects were also previously reported in nocodazole-treated cells (Canman et al., 2003), suggesting that misregulation of either MT or the MT-actomyosin interface at the cell membrane may induce hyper-contractility leading to abnormal membrane contraction in Pafah1b1 mutant MEFs. During cytokinesis, Pafah1b1-dependent cell membrane contractility may be mediated indirectly by the dyregulation of astral MTs reaching to the cell membrane (Moon et al., 2014).

We aimed to identify the molecular mechanisms underlying Pafah1b1-dependent cell membrane contractility of MEFs. Therefore, we examined the distribution of the other proteins defining cleavage furrow formation including RhoA and its downstream effectors, like contractile ring components and actomyosin. We found that RhoA and Anillin were mislocalized in Pafah1b1 mutant MEFs. For instance, RhoA was found in ectopically localized in large polar blebs in Pafah1b1 mutant MEFs. The Myosin II-positive and F-actin focus zone were dispersed at the equatorial cortex or aberrantly located at the polar blebs in Pafah1b1 mutant MEFs. By alternatively tracing MRLC1 and SEPT6, we confirmed that LIS1 defines cleavage plane positioning by dual regulation of actomyosin and contractile ring. Finally, RhoA hyper-activation in WT MEFs phenocopied the cytokinesis defects seen in Pafah1b1 mutant MEFs, and inhibition of RhoA in Pafah1b1 mutant MEFs decreased the cytokinesis defects observed in these MEFs. Thus, these results suggest that LIS1 exerts its function to restrict the cleavage furrow at the equatorial cortex to finely modulate RhoA-Anillin-actomyosin signaling.

Previous Pafah1b1 mutant mouse studies demonstrated that LIS1 modulates GTPase-mediated F-actin dynamics in interphase cells and post-mitotic neurons. For example, Pafah1b1 heterozygous (Pafah1b1^ko/+) post-mitotic neurons displayed F-actin dysregulation at the leading processes and aberrant GTPase activities of RhoA-Rac1-Cdc42. Similarly, Pafah1b1^ko/+ MEFs exhibited abnormal hyper-activation of RhoA GTPase (Kholmanskikh et al., 2003). A recent Pafah1b1 knockdown fibroblast study also unraveled F-actin dysfunction and focal adhesion disruption during traction-dependent migration (Jheng et al., 2018). Together, these previous findings indicate that LIS1 may regulate not only MTs but F-actin in migrating phases of multiple cell types. Based on the aberrant cytokinetic phenotypes seen in Pafah1b1 mutant NPCs and MEFs, we now demonstrate that loss of LIS1 results in hyper-activation of RhoA signaling and mislocalization of F-actin and Myosin II that generates hyper-contractility forces ectopically at the polar cortex, which is an abnormal site of cleavage furrow ingression.

Multiple mouse studies have suggested that RhoA GTPase regulates the integrity of the apical NPC niche, through controlling F-actin at the adherens junction (Cappello et al., 2012; Herzog et al., 2011; Katayama et al., 2011). In accordance with this finding, a study of chick neural tube development indicated that RhoA activity modulation and overexpression of a DN-RhoA similarly altered mitotic spindle orientation of apical NPCs (Roszko et al., 2006). In the present study, we demonstrated that neocortical LIS1 is an important upstream mediator of signaling cascades of RhoA-actomyosin-contractile ring components during cytokinesis of apical NPCs. In mouse apical NPCs, Anillin-ring constriction from the basal to apical side determines the cleavage furrow ingression site in mouse apical NPCs (Kosodo et al., 2008). During cytokinesis of neocortical NPCs, Citron kinase and RhoA colocalize at the apical membrane of NPCs where the cleavage furrow and the midbody are positioned (Di Cunto et al., 2000; Sarkisian et al., 2002). In mice, mutations in Citron kinase alter mitotic spindles and RhoA-mediated neurogenic cytokinesis of apical NPCs. In humans, neocortical NPC phenotypes lead to primary microcephaly and microlissencephaly (Basit et al., 2016; Li et al., 2016; Shaheen et al., 2016; Harding et al., 2016; Gai and Di Cunto, 2017). Furthermore, in addition to Citron kinase and RhoA, three members of Kinesin family, Kif20a, Kif20b, and Kif4 regulate neocortical NPC cytokinesis and mutations of these genes result in reduced cortical size with microcephaly (Janisch et al., 2013; Moawia et al., 2017; Geng et al., 2018). Together, these previous studies suggest that dynamic movements of RhoA and cleavage furrow-associated proteins to the midbody is the key cellular process regulating NPC cytokinesis by precisely segregating cell determinants along the cell cleavage axis.

In the developing neocortex, apical NPCs self-renew via symmetric divisions, and produce the major populations of post-mitotic neurons via asymmetric divisions. The orientiation of the cleavage plane is thought to be a major regulator of symmetric versus asymmetric cleavage. Our present study suggests that the LIS1-RhoA-actomyosin pathway sets the apical NPC cleavage plane by enrichment of cytokinetic furrow proteins to the ventricular surface, contributing to the later mitotic events when cell fate determinants separate into the daughter cells. Immunohistochemical analyses of the mouse neocortex uncovered that Pafah1b1 mutant NPCs displayed mislocalization of RhoA and Anillin skewed to only one daughter cell, indicating unequal inheritance and this may impact the segregation of cell fate determinants by inducing an imbalance of symmetric versus asymmetric divisions. Consistent with this data, we previously reported that Pafah1b1-deficient E14.5 neocortex had an increase in TUJ1-positive neurons and cleaved caspase-3-positive apoptotic cells (Gambello et al., 2003). This suggests that neocortical daughter cell fates and survival/death are sensitive to LIS1 expression levels in neocortical NPCs. We also demonstrated that Pafah1b1-deficient E15.5 neocortex displayed partial diffuseness and thinning of cortical plate with weak formation of the subplate. In the cortex of postnatal day 5 NPC-specific Pafah1b1 CKO mice (GFAP-Cre; Pafah1b1^hc/hc) also had reduced numbers of upper-layer neurons marked by FOXP1 (Layer III/VI) and CUX1 (Layer II/III) (Youn et al., 2009), indicating that Pafah1b1 deficiency in NPCs leads to impairments in neuronal subtype specification and total neurogenesis accompanied with cortical patterning defects.

In the present study, we observed mitotic phenotypes from apical NPCs, the vRGs, in the E14 neocortex. Pafah1b1 deficiency at earlier time points (E9.5~E11.5) resulted in neural epithelial stem cell death via apoptotis, due to disruption of symmetric mitotic division with vertical cleavage plane, so these earlier stem cells could not be studied. At mid-gestation, we were able to utilize conventional hypomorphic mutants and NPC-specific CKO mutants and circumbented mitotic arrest and death mechanisms. E14 vRGs had a sufficient amount of LIS1 to avoid severe mitotic catastrophe and still displayed robust cytokinetic defects with distorted cleavage plane and mitotic spindles.

Our novel findings of LIS1 functions in cytokinesis may explain the mechanism of the recent studies with induced pluripotent stem cells (iPSCs) of Miller Dieker syndrome, a severe form of lissencephaly with haploinsufficiency of PAFAH1B1 as well as about 20 other genes. We demonstrated a prolongation of mitosis of oRG progenitors but not ventricular zone radial glial (vRG) progenitors (Bershteyn et al., 2017). The specificity of mitotic effects in the oRGs and lack of its effects in the vRGs may be the result of different dose dependencies of LIS1 in mouse vs. human, such that LIS1 deficiency results in more prolonged mitotic time specifically in oRGs rather than vRGs. Compared with vRGs, oRG populations lack their endfeet attachment to the apical VZ surface. Intriguingly, human oRGs exhibit rapid mitotic somal translocation toward the cortical plate right before cytokinesis (Hansen et al., 2010). These abrupt movements of human oRGs suggest dynamic cytoskeletal rearrangements during mitotic phases of oRGs. In our current mouse studies, we could not investigate oRG phenotypes due to the low number oRGs present in the neocortices of mice. By contrast, the neocortices of gyrencephalic mammals (from the ferret to primates) contain substantial number of oRGs that exhibit unique mitotic somal translocation, unrestricted mitotic spindle angles, multiple rounds NPC self-renewal and promote expansion of progenitors, neocortical folding and increased topology. Since oRGs highly express a specialized transcriptional factor, HOPX (Pollen et al., 2015) on top of the pan-RG markers PAX6 and SOX2, it would be crucial to investigate the orchestrated crosstalk between HOPX-dependent transcriptional/epigenetic network and actomyosin-mediated cytokinesis in oRGs. In the future, characterization of the localization of diverse protein markers enriched to the cleavage furrow in human or primate iPSC-derived NPCs and fetal neocortical NPCs, such as RhoA, Anillin, and Citron kinase of NPCs, will need to be analyzed to better understand the oRG-specific sensitivity to LIS1 dosage especially in human. It would also be interesting to determine whether other lissencephaly or brain malformation-associated genes modulate RhoA-Anillin-actomyosin-pathways, and to test whether these genes, like LIS1, also possess dual roles in actomyosin and MT regulation important for apical NPCs by controlling key cellular processes not only mitosis but cytokinesis, a critical final step of cell determinant segregation.

All data analyzed during this study and its analysis has been described in the manuscript.

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Author details

1. Hyang Mi Moon

   Department of Pediatrics, Institute for Human Genetics, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States

   Present address

   Department of Neurosurgery, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Project administration

   For correspondence

   chorong@stanford.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2755-3767

2. Simon Hippenmeyer

   Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, United States

   Present address

   IST Austria (Institute of Science and Technology Austria), Klosterneuburg, Austria

   Contribution

   Resources, Visualization, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2279-1061

3. Liqun Luo

   Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, United States

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5467-9264

4. Anthony Wynshaw-Boris

   1. Department of Pediatrics, Institute for Human Genetics, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
   2. Department of Genetics and Genome Sciences, Case Western Reserve University, School of Medicine, Cleveland, United States

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Validation, Investigation, Project administration

   For correspondence

   ajw168@case.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2780-1540

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