Abstract
Unequal cell division (UCD) is a fundamental process responsible for creating sibling cell size asymmetry; however, how microtubules are specifically depolymerized on one aster of the mitotic spindle creating a smaller sibling cell during UCD has remained elusive. Using invertebrate chordate embryos (ascidian) that possess a large cortical structure (CAB) that causes UCD, we identified a microtubule depolymerase (Kif2) involved in creating cell size asymmetry. Kif2 localizes to the cortical subdomain of endoplasmic reticulum in the CAB. During three successive UCDs, Kif2 protein accumulates at the CAB during interphase and is delocalized from the CAB in mid mitosis. Rapid imaging of microtubule dynamics at the cortex revealed that microtubules do not penetrate the CAB during interphase. Inhibition of Kif2 function prevents the development of mitotic aster asymmetry and centrosome movement towards the CAB thereby blocking UCD, whereas locally increasing microtubule depolymerization causes exaggerated asymmetric spindle positioning. This study provides insights into the fundamental process of UCD and for the first time shows that a microtubule depolymerase is localized to a cortical site controlling UCD.
Control of microtubule dynamics at the cell cortex is important for a myriad of processes including spindle positioning at the cell center during cell division (Garzon-Coral et al., 2016; Kern et al., 2016; Minc and Piel, 2012), asymmetric spindle positioning during unequal cell division (UCD) in embryos (Cowan and Hyman, 2004; Kotak and Gönczy, 2013), and for axonal pruning during nervous system development in mammals (Homma et al., 2003; Maor-Nof et al., 2013). Microtubule dynamics have been intensively studied during asymmetric cell division (ACD) which is sometimes coupled with UCD creating one large and one small daughter cell that have different fates, as in Drosophila neuroblasts (Siller and Doe, 2009), sea urchin micromeres (Wessel et al., 2014) and C. elegans 1-cell zygotes (Galli and van den Heuvel, 2008). Such UCD relies on the development of unbalanced forces that has two components: cortical pulling force acting on astral microtubule plus ends (Grill et al., 2001) and depolymerization of microtubule plus ends as they encounter the cortex (Kozlowski et al., 2007; Labbé et al., 2003) which create unbalanced forces to position the mitotic spindle asymmetrically. These two processes acting together overcome the forces that cause mitotic spindles to move to the center of the cell, a process that senses and integrates force over the length of microtubules (Minc et al., 2011). However, one key piece missing from this model of UCD is the identity of the protein(s) that cause astral microtubule plus end depolymerization at the cortex, which is important not only for UCD but also for the mitotic spindle centering mechanism based on astral microtubule length that operates during symmetric cell division.
Microtubule plus ends can be induced to depolymerize via different mechanisms. In vitro experiments indicate that dynein can cause catastrophe of microtubule plus ends (Laan et al., 2012), raising the possibility that in intact cells dynein couples pulling with depolymerization. A different mechanism regulates microtubule plus end depolymerization in developing mammalian neurites which is dependent on the cortically-localized microtubule depolymerase Kif2A (Homma et al., 2003). Kif2A is a member of the kinesin-13 family of microtubule depolymerases (Lawrence et al., 2004) which includes MCAK/Kif2C that causes microtubule plus end depolymerization at kinetochores during anaphase (Wordeman et al., 2007). However, in cells that divide unequally it is still not known what causes astral microtubule plus end depolymerization at the cortex, and the only protein known to limit microtubule growth at the cortex during UCD in one cell C.elegans embryos does not affect astral microtubules (O’Rourke et al., 2010).
C.elegans embryos have provided a wealth of knowledge about the cortical pulling forces that act upon astral microtubules of the spindle during UCD. For example, following fertilization and symmetry breaking in C. elegans, the Par polarity complexes are partitioned to distinct cortical subdomains (Gönczy, 2008). Anterior Par3/Par6/aPKC (PKC-3) phosphorylates LIN-5 (NuMA) at the anterior cortex inhibiting the cortical anterior spindle pulling forces (Galli et al., 2011), while NuMAs binding partner GPR-1/2 (Pins/LGN) becomes enriched at the posterior cortex during mitosis (Gotta et al., 2003). The Dynactin/Dynein complex protein DRBY-1 interacts with Pins and NuMA thus providing a cortical link to microtubules (Couwenbergs et al., 2007). During metaphase and anaphase the mitotic spindle is pulled towards the posterior cortex causing UCD (Grill et al., 2001). Late in mitosis the posterior centrosome has changed from a spherical shape to a flattened and disc-shaped structure (Severson and Bowerman, 2003). Symmetric cell divisions in somatic cells also depend upon cortical dynein to center the mitotic spindle (Collins et al., 2012; Kern et al., 2016; Kiyomitsu and Cheeseman, 2013). In addition to dynein pulling forces, it has been shown that astral microtubule depolymerization also plays a role in posterior spindle displacement. For example, pulling forces are lacking when microtubules are stabilized by taxol, while in embryos carrying a temperature-sensitive mutation in a β-tubulin gene the posterior displacement distance of the spindle is enhanced (Nguyen-Ngoc et al., 2007). Based on these and other data a dual force-generation mechanism has been proposed that relies on microtubule pulling forces (dynein-dependent) combined with microtubule depolymerization (Kozlowski et al., 2007; Nguyen-Ngoc et al., 2007). Thus in C.elegans, although cortical microtubule depolymerization is thought to be part of the mechanism for posterior spindle displacement, the mechanism regulating cortical microtubule depolymerization is not known (Gönczy, 2008).
Many embryos provide more extreme examples of UCD whereby the two asters of the mitotic spindle become highly asymmetric in size and shape with the smaller of the two asters being inherited by the smaller of the two daughter cells. Similar to the flattened posterior centrosome in C.elegans one-cell embryos (Severson and Bowerman, 2003), such mitotic aster asymmetry has also been observed during UCD in spiralian (Lambert, 2010; Rabinowitz and Lambert, 2010), echinoderm (Holy and Schatten, 1991; Schroeder, 1987), and ascidian (Hibino et al., 1998; Prodon et al., 2010) embryos. Mitotic aster asymmetry commonly occurs at the third cleavage in spiralian embryos with the smaller aster associating with an animal cortical domain at the 4-cell stage leading to UCD (Rabinowitz and Lambert, 2010). A similar phenomenon has been documented for sea urchin embryos where one aster in each future micromere associates with a cortical domain situated at the vegetal pole of the 8-cell stage embryo creating aster asymmetry during UCD (Schroeder, 1987; Holy and Schatten, 1991). The clearest example of a cortical domain that causes aster asymmetry and UCD comes from ascidian embryos. Here a large cortical structure termed the CAB (centrosome-attracting body) causes three successive rounds of UCD accompanied by the CAB-proximal aster becoming smaller (Hibino et al., 1998; Nishikata et al., 1999; Prodon et al., 2010). Although the Par complex (aPKC, Par3 and Par6) is localized to the CAB (Patalano et al., 2006), we do not currently know how the CAB affects microtubule dynamics leading to aster asymmetry. Due the large dimensions of the CAB (circa 20µm at the 8-cell stage) we wondered whether it would be possible to identify proteins involved in regulating microtubule dynamics at the cortex via live cell imaging during UCD in the early ascidian embryo.
We have developed the optically transparent ascidian species Phallusia mammillata as a system to perform live cell imaging to study microtubule dynamics during UCD (McDougall et al., 2015; Prodon et al., 2010). By analyzing microtubule dynamics at the cortex we discovered that a microtubule depolymerase (Kif2) localizes to the cortical CAB in a cell-cycle dependent manner. Through live cell imaging of Kif2::Venus/mCherry/Tomato and immunofluorescence we demonstrate that exogenous and endogenous Kif2 localizes to the cortical CAB. In particular, Kif2 accumulates on a domain of cortical endoplasmic reticulum (cER) concentrated at the CAB during interphase and leaves the CAB cER during mid mitosis when CAB proximal microtubules become short. In addition, we show that microtubules do not penetrate the cortical CAB during interphase. Finally, we found that inhibiting endogenous Kif2 protein function prevents the establishment of mitotic aster asymmetry and UCD, and conversely that increasing depolymerization of microtubules near the CAB causes the CAB proximal spindle pole to move nearer to the CAB.
RESULTS
Unequal cell division and the CAB
The early embryo of the European ascidian Phallusia mammillata is favorable for live-cell imaging and functional studies because its cells are transparent (see Movie S1) and readily translate exogenous mRNAs such as those encoding GFP fusions and dominant negative constructs (McDougall et al., 2015). Ascidian embryos display three rounds of UCD that depend upon the centrosome-attracting body/CAB (Hibino et al., 1998; Nishikata et al., 1999; Prodon et al., 2010). The mitotic spindle aligns relative to the cellular polarity with one spindle pole pointed towards the CAB (Figure 1A). During these three rounds of UCD, astral microtubules emanating from the centrosome that polymerize towards the CAB and midline are shorter than those microtubules that do not polymerize towards the CAB or microtubules originating from the more distant centrosome as the mitotic spindle approaches the CAB and midline (Figure 1 A). A smaller and flattened aster thus forms nearest the CAB and midline during each round of UCD (Figure 1 and Movies S2, S3 and S4 with a 3D rendering of an 8-cell stage embryo shown in Movie S5). In Phallusia embryos both centrosomes appeared similar for γ-tubulin staining (sFig. 1) indicating that aster size is not a function of γ-tubulin loss from one centrosome as has been observed in leech zygotes (Ren and Weisblat, 2006).
The CAB is a multilayer structure and can be visualized in several ways (Paix et al., 2011a; Patalano et al., 2006). Because the CAB creates a protrusion it can be visualized with plasma membrane markers such as PH::Tom (Figure 1 B and Movie S6). Also, because the CAB is rich in cortical ER and excludes mitochondria it can be visualized by specific lipophilic dyes that label either the mitochondria or cER in the CAB (Figure 1 B). Finally, antibodies to aPKC label the cortical surface of the CAB but do not label the cER which appears as a dark zone surrounded by mitochondria labelled with anti mitochondrial antibody-NN18 (Figure 1 B).
Characterization of the microtubule depolymerase Kif2 in ascidian embryos
In order to understand how the CAB may be involved in creating aster asymmetry we searched for CAB-resident proteins by screening likely candidates by either probing with antibody for immunofluorescence or localization of expressed fluorescently tagged proteins. We identified a number of proteins including Kif2, a member of the kinesin-13 family of proteins that instead of possessing motor activity displays microtubule depolymerization activity (Hirokawa and Takemura, 2004). Vertebrates contain three members of the Kif2/kinesin-13 family: Kif2a, Kif2b and MCAK (Kif2c) (Hirokawa and Tanaka, 2015). There is only one member of the Kif2 family in the ascidian (P. mammillata: PmKif2 and C. intestinalis: CiKif2) and other non-vertebrate deuterostomes (sFig 2), suggesting that the vertebrate family of proteins evolved from a non-vertebrate deuterostome Kif2a/b/c (henceforth Kif2). Consistent with this ascidian Kif2 also localizes to spindle poles (like Kif2a/Kif2b) and chromosomes (like Kif2c/MCAK) (Figure 3) although CAB localization was strongest. Overexpressing Kif2 to high levels shortened microtubules consistent with it acting as a microtubule depolymerase (sFig 3). In two species of ascidian (Phallusia mammillata and Ciona intestinalis) Kif2 is a CAB-resident protein (Figure 2 A and sFig 4). By Western blot Kif2 antibody recognizes both endogenous Kif2 and injected Kif2 coupled to GFP (Figure 2 B). Finally, exogenous Kif2::Tomato localized to the CAB during UCD at the 8-cell, 16-cell, and 32-cell stage in Phallusia embryos (Figure 2 C).
Next we wished to pinpoint which domain of Kif2 protein was required for CAB localization. For this we injected mRNAs encoding fusions between Venus tag and various portions of Kif2 and evaluated CAB localization of each construct. Our results show that the N-terminal 72 amino acids of Kif2 was capable of driving localization to the CAB, and conversely removing the N-terminal domain abolished CAB localization of the Venus-tagged constructs (Figure 2 D).
Kif2 cycles onto and off of the CAB
We noticed that Kif2 protein appeared to accumulate on the CAB during interphase and leave the CAB during mitosis. In order to confirm that Kif2 cycled onto and off of the CAB we performed live cell ratiometric imaging of Kif2::Tom levels relative to Plk1::Ve or other fluorescent constructs (Par6::Venus, H2B::GFP or PH::GFP). Our analysis revealed that Kif2 protein accumulates at the CAB during interphase peaking at early mitosis and that Kif2 protein begins to leave the CAB during prometaphase with levels reaching a minimum at late anaphase/cytokinesis onset (Figure 3 A and Movie S7). Careful examination of endogenous Kif2 levels at the CAB in either Phallusia or Ciona embryos revealed a similar dynamic delocalization of Kif2 during mitosis, whereby endogenous KIF2 protein was delocalized from the CAB by late anaphase (Figure 3 B and sFig 4). Other CAB-resident proteins such as PEM1 or aPKC do not leave the CAB during mitosis (Figure 3 B). In order to determine whether loss of signal at the CAB was due to delocalization or degradation we used the photo-convertible construct Kif2::Dendra to follow a specific pool of Kif2 protein in vivo. UV illumination converts the green Kif2::Dendra fusion protein into a red one. UV illumination of Kif2::Dendra in a region within one CAB caused just one CAB to become red (Figure 3 C). Since Kif2 is also a kinetochore localized protein, we reasoned if we could detect red Kif2::Dendra on chromosomes that would indicate that the red version of Kif2 protein left the CAB following photo conversion to become captured by adjacent chromosomes (it is important to note that this does not rule-out destruction but does show that red Kif2 protein leaves the CAB). Figure 3 C shows that we were able to detect red Kif2::Dendra on chromosomes in the blastomere containing the photo-converted red Kif2::Dendra. Thus the red Kif2 protein we detected on the chromosomes came from the CAB (Figure 3 C), indicating that some Kif2 protein leaves the CAB in mitosis.
Kif2 localizes to cortical ER in the CAB
As noted previously, the CAB is a multilayered structure, comprised of a thick layer of cortical endoplasmic reticulum (cER) protruding into the cytoplasm which adheres to a specialized region of actin-rich cortex (Patalano et al., 2006). By co-staining for mitochondria which surround and outline the cortical ER mass, and for pMNK, a cER resident protein (Paix et al., 2011b), the surface and deeper cER can be visualized (Figure 4 B). Unlike aPKC protein which is contiguous with the actin layer, Kif2 protein occupies the thicker cER layer (Figure 2). To determine the precise localization of Kif2 protein in the CAB we prepared isolated cortices. By sticking 8-cell stage embryos to coverslips followed by shearing using an isotonic buffer, the cortex and its associated cER is retained on the cover slip (Figure 4 A). Probing these cortical preparations with anti-Kif2 revealed a concentration of Kif2 in the cER of the CAB (Figure 4 A, and inset for higher definition). In live embryos we were able to observe dynamic finger-like projections from the surface of the CAB labelled with Kif2::Tom (Figure 4 C), consistent with the notion that Kif2 was localized to the tubes of cER in the CAB.
Microtubule dynamics at the CAB
To visualize the rapid dynamics of microtubule at the cortex embryos have to be immobilized close to a flat surface, here provided by the coverslip. In one cell C.elegans zygotes this permitted the accurate determination of plus end dynamics leading to the development of the “touch and pull” mechanism of ACD (Kozlowski et al., 2007). In ascidians, measurement of microtubule plus end dynamics in a cortical slice containing the CAB and non-CAB cortex is complicated by both the movement and the geometry of the embryo: since the CAB is an apical cortical structure close to the midline it invariably curves away from the coverslip. In order to overcome these problems we dissociated blastomeres with calcium-free seawater and used protamine-coated coverslips to immobilize them (Figure 5 A). Blastomere isolation does not perturb UCD in Phallusia embryos (Prodon et al., 2010). Ens::3GFP fluorescence was then measured in a cortical slice within 1µm of the coverslip every 0.8 seconds (Figure 5).
Microtubules are present as a dense network on the cortex during interphase but in the CAB domain they are absent (Figure 5 A, B, see Movie S8). In Movie S8, microtubules can be observed polymerizing in the direction of the CAB (labelled red with Kif2::Tom) but they fail to penetrate the CAB. Occasionally during interphase the CAB has a CAB-free zone at its center and microtubules can be seen to reach the cortex in the hole in the center of the CAB but are absent from the Kif2-labelled CAB (Figure 5 A, B). Microtubules also avoid the CAB in the intact embryo at the 32-cell stage even though they can be seen growing around the CAB (Figure 5 C and Movie S9) as in isolated blastomeres (Figure 5 A), however since the embryo moves in the imaging plane many z sections were acquired so the time resolution in one z plane is greatly reduced.
Microtubule depolymerization and aster asymmetry
In order to determine the role of Kif2 in the CAB we generated a mutant form of Kif2 protein (DN-Kif2) based on the construct which behaves as a dominant negative in mammalian cells (Wordeman et al., 2007). DN-Kif2 contains the N-terminal domain which targets it to the CAB and the C-terminal domain which permits dimerization, but lacks the catalytic domain (Figure 6). mRNA encoding the DN Kif2 and H2B::Rfp1 constructs were injected into one blastomere of a two cell stage embryo previously injected with Ens::3GFP and Kif2-Nter::Venus to monitor microtubules and the CAB in both halves of the embryo (Figure 6). DN Kif2 prevented the development of aster asymmetry and abolished UCD (Figure 6Ai). We measured the distance between the nearest spindle pole and the CAB in both halves of the embryo at the 8-cell stage and found that DN Kif2 significantly increased the spindle pole to CAB distance (Figure 6 Ai). Since taxol stabilizes microtubules and reduces depolymerization we reasoned that taxol should have a similar effect to DN Kif2. Treating embryos in mitosis with taxol also prevented the development of aster asymmetry (Figure 6 Aii). These data suggest that aster asymmetry and asymmetric spindle positioning require the activity of Kif2 and microtubule depolymerization.
If aster reduction facilitates the migration of one spindle pole towards the cortex, we reasoned that increasing microtubule depolymerization near the CAB would enhance spindle pole movement towards the CAB, since microtubules would become shorter. We employed a method to depolymerize microtubules locally by using a micro-pipette as a spatially-confined source of nocodazole (see materials and methods) (Figure 6 Bi and Movie S10 and S11). Since it was not possible to perform these experiments on our confocal microscope we used an epifluorescence system. We bathed embryos in Cell Mask orange which labels both the plasma membrane and vesicles that accumulate around spindle poles during mitosis (McDougall et al., 2015). This was more convenient that using Ens::3GFP and also gave better visualization of both spindle poles in each blastomere. However, to confirm that these nocodazole micropipettes caused localized microtubule depolymerization we imaged microtubules in zygotes (Figure 6 B iv). We applied different diameter pipettes with the same nocodazole concentration near the CAB in one blastomere at the 8-cell stage and measured the effect on the position of the proximal spindle pole. Relatively small diameter nocodazole pipettes caused the proximal spindle pole to migrate closer toward the CAB and midline than normally observed (Figure 6 Bi and Movie S10). Importantly, the blastomeres far from the source of nocodazole were unaffected and divided normally (Figure 6 Bi and Movie S10). By using a larger bore pipette we could affect both CAB-containing blastomeres so that both CAB-proximal spindle poles moved closer to the CAB and midline while blastomeres farther from the pipette behaved normally and divided (Figure 6 Bii). Control DMSO-containing pipettes had absolutely no effect on spindle positioning (Figure 6 Biii). To depolymerize microtubules via a second method we used photo-activation of caged Combretastatin while imaging microtubules with Ens::3GFP (Figure 6 C). Activation of caged Combretastatin during mitosis caused astral microtubule depolymerization while leaving spindle microtubules intact. As noted with nocodazole pipettes, the whole spindle migrated even closer towards the CAB (Figure 6 C). These data revealed that reducing aster size could augment the asymmetric positioning of the spindle, supporting the hypothesis that astral microtubule length sets the distance of the spindle pole to the CAB.
DISCUSSION
Here we present evidence for the localization and function of a microtubule depolymerase (Kif2) at a cortical site that causes UCD. Precise control of microtubule dynamics at the cortex is a fundamental cellular mechanism that is involved in diverse biological processes ranging from the control of axon morphology (Homma et al., 2003; Maor-Nof et al., 2013; Noda et al., 2012) to the unequal division of cells (Cowan and Hyman, 2004; Siller and Doe, 2009). UCD is an extremely widespread process in biology occurring in bacteria (Adams and Errington, 2009; Aldridge et al., 2012), yeast (Carminati and Stearns, 1997), and many different types of embryo. Amongst embryos, UCD has been observed in ctenophores (Martindale and Henry, 1999), chaetognaths (Carré et al., 2002), spiralians (Lambert, 2010), echinoderms (Schroeder, 1987; Holy and Schatten, 1991), and invertebrate chordate embryos of the ascidian (Hibino et al., 1998). A common theme among several embryos that display UCD is the development of aster asymmetry. Unequally cleaving spiralian embryos (Tubifex, and the leech Helobdella) display aster asymmetry at the 1 to 2 -cell stage leading to eccentric positioning of the mitotic spindle (Weisblat, 2007). Sea urchin embryos also display UCD starting at the 8 to 16-cell stage culminating in the formation of four micromeres (Schroeder, 1987; Holy and Schatten, 1991) at the vegetal pole where aPKC is absent (Prulière et al., 2011). Again the mechanism underlying this UCD is not known in these embryos, however, observations indicate that one centrosome becomes disc-shaped and closely apposed to the cortex before UCD (Schroeder, 1987; Holy and Schatten, 1991). In ascidians three successive rounds of UCD occur starting at the 8 to 16-cell stage, resulting in the formation of two small blastomeres at the 64-cell stage that are germ cell precursors (Hibino et al., 1998). We showed previously that the CAB causes one pole of the mitotic spindle to approach the CAB during prometaphase through anaphase which is accompanied by the shrinking of the CAB-proximal aster (Prodon et al., 2010).
One central unresolved question therefore is how specialized cortical sites affect astral microtubules leading to the development of aster asymmetry. Here we show that in ascidian embryos the microtubule depolymerase Kif2 plays a key role in promoting aster asymmetry which in turn is required for UCD. Kif2 is concentrated on a subdomain of cortical endoplasmic reticulum (cER) found at the centrosome-attracting body or CAB during interphase and leaves the CAB during mitosis (Figure 4). We propose that Kif2 affects local microtubule dynamics at the cortex in the vicinity of its location, causing depolymerization of the nearest microtubules during mitosis thus leading to the development of aster asymmetry. By artificially increasing the amount of local depolymerization of astral microtubules in the proximity of the CAB we found that the spindle pole moved even closer to the CAB (Figure 6).
Our results have led us to the conclusion that polymerization of astral microtubules opposes the pulling forces that likely displace the mitotic spindle towards the CAB. We therefore propose that the diffusion of Kif2 from the CAB in mitosis causes the local depolymerization of those astral microtubules nearest the CAB thus facilitating the eccentric positioning of the mitotic spindle near the CAB cortex.
The mechanism we have discovered here in a chordate deuterostome embryo extends our understanding of UCD, which so far has been heavily studied in two protostomes, C.elegans and Drosophila. In Drosophila larval neuroblasts, even though the apically localized Gα/Pins/NuMA complex aligns the mitotic spindle (Nipper et al., 2007; Siller et al., 2006; Siller and Doe, 2009), UCD can be driven by a cortical subdomain of Myosin II via a mechanism which is independent of spindle position (Cabernard et al., 2010). Work in C. elegans zygotes has demonstrated the fundamental importance of the conserved Gαi/Pins/NuMA complex, which accumulates at the posterior cortex and creates a pulling force on astral microtubules that is greater at the posterior cortex, thus causing unequal cleavage (Grill et al., 2001; Redemann et al., 2010). Intriguingly, microtubule depolymerization is thought to be required for UCD in C. elegans (Gönczy et al., 2001; Kozlowski et al., 2007; Nguyen-Ngoc et al., 2007) although the mechanism responsible is currently unknown. It appears that this is not Kif2-dependent, since inhibition of MCAK with RNAi does not prevent eccentric spindle positioning (Grill et al., 2001).
The control of microtubule plus end dynamics during UCD by Kif2 is remarkably similar to the axonal pruning function of Kif2 in mammalian post-mitotic neurons. For example, in developing neurites Kif2A is thought to bind to the plasma membrane associated protein phosphatidylinositol 4-phosphate 5-kinase (Noda et al., 2012). Kif2A localizes to the tips of developing neurites in vivo and functions to suppress collateral branch extension since the knockout of Kif2A in mice causes increased microtubule stability at the cell edge in growth cones creating more growth of collaterals (Homma et al., 2003).
Our results presented here indicate that the flattened aster with short microtubules and movement of the spindle toward the CAB are both mediated by the action of a cortically-localized microtubule depolymerase. Given that Kif2 controls UCD in invertebrate chordate embryos and morphogenesis of developing mammalian neurites, it may be worthwhile investigating the role played by kinesin-13 family members such as Kif2 and other microtubule depolymerases during UCD as well as other biological processes where microtubule dynamics alter when they encounter the cortex.
METHODS
Origin of the animals
Phallusia mammillata were collected at Sète (Etang de Tau, Mediterranean coast, France) and Ciona intestinalis at Roscoff. Ascidian gamete collection, dechorionation, fertilization and embryo cultures were as described previously (McDougall et al., 2014).
Antibodies, fixation and reagents
Embryos were fixed in −20° methanol containing 5uM EGTA and immunolabelled as previously described (Sardet et al 2011). We used anti aPKC (Santa Cruz 216) and anti PEM1 (Paix et al., 2011b) to label the CAB, anti-tubulin (YL1/2 and DM1a, Sigma) for microtubules, anti Kif2 (Ganem and Compton, 2004), γ-tubulin (Sigma GTU88) for centrosomes. DiI and DiO (Invitrogen) were used to label ER and mitochondria respectively, Paclitaxel (Sigma) to stabilize microtubules and nocodazole (Sigma), caged Combretastatin (provided by D. Burgess) to depolymerize microtubules.
For Western blot samples were prepared in Laemmli sample buffer and migrated on 10 % polyacrylamide gels by SDS-PAGE using standard procedures. Two lanes were loaded containing either 40 uninjected eggs or 40 eggs which had been injected with mRNA encoding full-length Kif2::GFP. After transfer to nitrocellulose, each lane was cut into two strips lengthwise which were incubated overnight with either anti-Kif2 or anti-GFP at a dilution of 1:1000 in TBS + 5% dry milk. The 4 strip blots were then washed in TBS + 0.1% tween, incubated with anti-rabbit secondary at 1:10000, and washed in TBS-tween. The signal was detected using West Pico chemiluminescent substrate (Fisher) at an exposure of 2 minutes.
Preparation of isolated cortices
Isolated cortices were prepared following the methods previously established in the laboratory (Patalano et al., 2006; Sardet et al., 2011). Briefly, embryos at the desired stage are transferred to calcium free seawater then placed at high density on cover slips coated with protamine (1 mg/ml) to which they adhere within 1 minute. After two washes with cortex isolation medium (CIM (cortex isolation medium): 0.8 M glucose, 0.1 M KC1, 2 mM MgCl2, 5mM EGTA, 10 mM MOPS buffer, pH 7), a stream of CIM is sprayed gently with a Pasteur pipette, shearing off the embryos but leaving attached to the glass imprints of the adherent membrane and associated cortical structures. The coverslips are washed rapidly in CIM then placed in cold methanol for fixation, then rehydrated in PBS and processed for immunofluorescence by standard procedures used for whole embryos.
Microinjection and imaging
Microinjection was performed as previously described (McDougall et al., 2014). Briefly, dechorionated oocytes were mounted in glass wedges and injected with mRNA (1–2 µg/µl pipette concentration/ ~2% injection volume) using a high pressure system (Narishige IM300). mRNA-injected oocytes were left for 2–5 hours or overnight before fertilization and imaging of fluorescent fusion protein constructs. The lipophilic dye Cell Mask Orange (Molecular Probes) was prepared at a concentration of 10 mg/ml in DMSO and diluted in sea water at 20 µg/ml then mixed 1:1 with the embryos just prior to imaging. Epifluorescence imaging was performed with an Olympus IX70, Zeiss Axiovert 100 or Axiovert 200 equipped with cooled CCD cameras and controlled with MetaMorph software package as previously described. Confocal microscopy was performed using a Leica SP5 or SP8 fitted with 40x/1.3na oil objective lens and 40x/1.1na water objective lens. All live imaging experiments were performed at 18–19°C. For fast imaging of cortical preparations a rectangular image section of the imaging array was selected to increase the temporal resolution to 0.8 images/sec. Image analysis was performed using Image J, ICY and MetaMorph software packages. Calcium-free sea water: 450 mM NaCl, 9 mM KCl, 33 mM Na2SO4, 2.15 mM NaHCO3, 10 mM Tris pH 8, 2.5 mM EGTA.
Micromanipulation
All manual micromanipulation experiments were performed on an Olympus IX70 microscope using a 20x objective lens and Metamorph acquisition software. Embryos at the 4-cell stage were incubated with Cell Mask Orange diluted in sea water (1/1000) for 90 sec. then washed with sea water. Cell Mask labelled embryos were mounted at the 8-cell stage for observation. To prepare the nocodazole pipettes, nocodazole was added to liquid 1% low melt agarose in sea water giving a final concentration of 50µM nocodazole. Using a Narishige PN30 puller microinjection pipettes were pulled from GC100-T glass (filament-free) capillary tubes. The tips of the microinjection pipettes were broken and calibrated by microscopic observation. These micropipettes were dipped into the liquid nocodazole/agarose solution, placed at room temperature which caused the agarose containing nocodazole to solidify. These prepared microinjection needles were stored in humid chambers and used the same day. The nocodazole/agarose needles were advanced towards the 8-cell stage embryos and placed on the surface of one B4.1 blastomere near the CAB starting at nuclear envelope breakdown. Bright-field and fluorescence images were acquired every 10 seconds using MetaMorph software package.
Synthesis of RNAs
We used the Gateway system (Invitrogen) to prepare N and C terminal fusion constructs using pSPE3::Venus (a gift from P. Lemaire), pSPE3::Rfp1, pSPE3::Cherry, pSPE3::tomato for all constructs except PH::GFP which was cloned into pRN3. For construct details please refer to our previous methods publication (McDougall et al., 2015) All synthetic mRNAs were transcribed and capped with mMessage mMachine kit (Ambion).
Bioinformatics
We created a database of animal protein sequences derived from the complete genomes of various metazoan lineages: Amphimedon queenslandica, Mnemiopsis leidyi, Trichoplax adhaerens, Nematostella vectensis, Acropora digitifera, Hydra vulgaris, Crassostrea gigas, Aplysia californica, Capitella teleta, Lingula anatina, Caenorhabditis elegans, Drosophila melanogaster, Strongylocentrotus purpuratus, Branchiostoma floridae, Ciona intestinalis, Phallusia mammillata and Homo sapiens. All data was retrieved from the NCBI genomes portal: https://www.ncbi.nlm.nih.gov/genome/browse/
We searched this database with the PFAM Kinesin hidden Markov model [PMID: 26673716] using the global local strategy implemented in HMMER2 [http://hmmer.org/download.html] and the model specific 'gathering threshold' bit scores as a cutoff. Kinesin regions (649 sequences), as defined by HMMER2 alignments, were extracted from full length sequences and aligned using the MAFFT software package with default parameters [PMID: 12136088].
This alignment was used to create a phylogeny of all Kinesin domains, using the Bayesian approach implemented in phylobayes, with an LG+G model of sequence evolution [PMID: 19535536]. Two chains were run for 3500 generations. 700 generations were discarded as burnin. Although the chains had not converged, the region of the phylogeny around the human Kif2 proteins revealed a stable clade composed of orthologs of the human KIF19, KIF18, KIF24 and KIF2A/B/C genes with full support from posterior probabilities. The sequences representing this clade of orthologous groups were extracted. The Phallusia mammillata Kif13 protein sequence was added to the alignment, and another phylogeny reconstructed using the Phyml package with an LG+G evolutionary model and 100 bootstrap replicates [PMID: 20525638]. This tree is shown in sFig. 2.
AUTHOR CONTRIBUTIONS
A.McD, J.C. and V.C conceived project and conducted experiments.
A.McD and J.C. wrote the ms.
V.C, G.P, D.B, and M.F performed experiments.
R.C performed bioinformatics analysis.
C.H and L.B provided molecular tools.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Supplementary Movies (please go to: http://movincell.org/)
Movie S1. Unequal cell division from 8 to 16-cell stage
Bright-field images of 8-cell stage Phallusia mammillata embryo viewed from the vegetal side from interphase until cell division. The two vegetal posterior blastomeres that divide unequally (B4.1 pair) are at the bottom. Following nuclear envelope breakdown it is possible to discern the movement of the mitotic spindle in the two bottom blastomeres. Time in min:sec.
Movie S2. Unequal Cell Division 1. 8-16-cell
Unfertilized Phallusia eggs injected with Ens::3GFP (green). Confocal images of 8-cell stage embryos showing microtubule behavior during late interphase and mitosis. B4.1 pair of blastomeres are at the bottom. Note that the aster is asymmetric in both bottom blastomeres. Time in min. Scale bar = 30µm.
Movie S3. Unequal Cell Division 2. 16 to 32-cell
Unfertilized Phallusia eggs injected with Ens::3GFP (green). Confocal images of 16-cell stage embryo showing microtubule behavior during interphase and mitosis. B5.2 pair of blastomeres are at the bottom. Note that the aster becomes asymmetric in both bottom blastomeres. Time in min. Scale bar = 30µm.
Movie S4. Unequal Cell Division 3. 32 to 64-cell
Unfertilized Phallusia eggs injected with Ens::3GFP (green) and also with Kif2::Venus (also appears green) to label the CAB. Confocal images of 32-cell stage embryo showing microtubule behavior during interphase and mitosis. B6.3 pair of blastomeres are at the bottom. The CAB can be seen centered between the two pairs of centrosomes. Note that the upper spindle pole in each B6.3 blastomere approaches the CAB and midline and that the asters nearest the CAB become progressively smaller. Time min:sec. Scale bar = 30µm.
Movie S5. 3D rendering of microtubules during UCD
Unfertilized Phallusia eggs injected with Ens::3GFP (green). 3D-renedered confocal z-planes showing astral microtubule behavior merged with brightfiled images from an 8-cell stage embryo. Note the size and behavior of the asters nearest the CAB (bottom right blastomeres) versus those far from the CAB in the B4.1 blastomeres.
Movie S6. CAB visualization
Unfertilized Phallusia eggs injected with PH::Tom (red) to label the plasma membrane and also with EB3::GFP (green) to label microtubules. Embryo at the 8-cell stage with the two CAB blastomeres at the bottom. Several z-planes from a confocal time-series rendered in 3D to better visualize the CAB protrusion. Note the CAB can be seen as a protrusion in both blastomeres (red).
Movie S7. Kif2 protein leaves the CAB during mitosis
Unfertilized eggs injected with EB3::GFP and Kif2::Tom then imaged using a confocal microscope during the 8 to 16-cell stage. Bright-field images are overlayed with the EB3::GFP and Kif2::Tom fluorescence images. Kif2 protein can be seen to leave the CAB during mitosis. Scale bar = 30 µm. Time in sec.
Movie S8. Microtubule polymerization around the CAB
Fast confocal imaging of a cortical z-plane containing the CAB in isolated B5.2 blastomeres. Microtubules labelled with Ens::3GFP and the CAB with Kif2::Tom. Note that microtubules polymerize around the CAB and also occasionally in the hole in the center of the CAB. Scale bar = 5µm. Time in sec.
Movie S9. Microtubule growth around the CAB in an intact embryo
Confocal images of Ens::3GFP and Kif2::Ven fluorescence during interphase of a 32 cell stage embryo. Microtubules can be seen to grow around the CAB but they do not penetrate the CAB. Scale bar = 20µm. Time in sec.
Movie S10. Nocodazole pipette to depolymerize microtubules at the 8-cell stage
Embryos at the 8-cell stage (interphase) were bathed in Cell Mask orange to label the mitotic spindle poles and the plasma membrane and during mitosis a nocodazole pipette was applied to the surface of one B4.1 blastomere. Note the exaggerated movement of the spindle pole nearest the nocodazole pipette towards the surface of the blastomere and CAB. Time in min:sec.
Movie S11. Nocodazole pipette to depolymerise CAB proximal microtubules in zygote
Zygote with microtubules labelled with Ens::3GFP. A nocodazole pipette was advanced towards one aster and led to the loss of microtubules on one side of the zygote. Time in min:sec.
ACKNOWLEDGEMENTS
We thank Duane Compton and Bernardo Orr for the generous gift of anti Kif2 antibody, Linda Wordermann for useful advice, Remi Dumollard for thoughtful insights, and members of the LBDV for helpful discussions. We thank Mafalda Loreti and Paul Stolz for preliminary experiments that do not appear in the article. This work was supported by the Agence National de la Recherche (ANR-12-BSV2-0005-01), and in part by support from Sorbonne Universities ANR-11-IDEX-0004-02 to the Picard Network, and by NSF grant 124425 to DB.