Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport

The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin's microtubule binding activity, located in the p150 CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Analysis of p150 mutants engineered by genome editing suggests that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating robust cortical pulling forces. Instead, mutations in p150's CAP-Gly domain inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of α-tubulin's C-terminal tyrosine, and both p150 CAP-Gly and tubulin tyrosine mutants decrease the frequency of early endosome transport from the cell periphery towards centrosomes during centration. Our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing one-cell embryo, and that this function of p150 is critical for generating cytoplasmic pulling forces for centrosome centration.

The microtubule-based motor dynein generates pulling forces for centrosome centration and mitotic spindle positioning in animal cells. How the essential dynein activator dynactin regulates these functions of the motor is incompletely understood. Here, we dissect the role of dynactin's microtubule binding activity, located in the p150 CAP-Gly domain and an adjacent basic patch, in the C. elegans zygote. Analysis of p150 mutants engineered by genome editing suggests that microtubule tip tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex and for generating robust cortical pulling forces. Instead, mutations in p150's CAP-Gly domain inhibit cytoplasmic pulling forces responsible for centration of centrosomes and attached pronuclei. The centration defects are mimicked by mutations of α-tubulin's C-terminal tyrosine, and both p150 CAP-Gly and tubulin tyrosine mutants decrease the frequency of early endosome transport from the cell periphery towards centrosomes during centration. Our results suggest that p150 GAP-Gly domain binding to tyrosinated microtubules promotes initiation of dynein-mediated organelle transport in the dividing one-cell embryo, and that this function of p150 is critical for generating cytoplasmic pulling forces for centrosome centration.

Author summary
Animal cells rely on molecular motor proteins to distribute intracellular components and organize their cytoplasmic content. The motor cytoplasmic dynein 1 (dynein) uses microtubule filaments as tracks to transport cargo from the cell periphery to the cell center, where the microtubule minus ends are embedded at the centrosome. Conversely, when dynein is anchored at the cell cortex or on organelles in the cytoplasm, the motor can pull on microtubules to position centrosomes within the cell. The intracellular location of centrosomes determines cell geometry and cell fate, and studying the underlying mechanisms

Introduction
Cytoplasmic dynein 1 (dynein) is the major microtubule (MT) minus-end directed motor in animals and transports various cargo from the cell periphery to the cell interior. The motor also moves and positions intracellular structures such as nuclei and centrosomes by pulling on the MTs to which they are connected. To generate pulling force, dynein is either attached to anchor proteins fixed at the cell cortex (cortical pulling) [1][2][3][4], or dynein is anchored on organelles in the cytoplasm (cytoplasmic pulling) [5,6]. In the latter instance, dynein generates MT length-dependent pulling forces by working against viscous drag as it transports organelles along MTs toward centrosomes.
Dynactin is an essential multi-subunit activator of dynein that forms a tripartite complex with the motor and cargo-specific adaptors proteins [7][8][9][10][11], but how dynactin supports the diverse functions of dynein remains incompletely understood. Dynactin is built around a short actin-like Arp1 filament and has its own MT binding activity, which resides at the end of a long projection formed by the largest subunit p150 [12]. p150 has a tandem arrangement of MT binding regions consisting of an N-terminal cytoskeleton-associated protein glycine-rich (CAP-Gly) domain and an adjacent patch rich in basic residues [13,14]. The CAP-Gly domain binds to MTs and to the MT plus-end tracking proteins (+TIPs) CLIP-170 and end-binding (EB) protein. In animal cells, +TIP binding of dynactin recruits dynein to growing MT ends [9,[15][16][17][18].
The p150 CAP-Gly domain recognizes the C-terminal EEY/F motif present in α-tubulin and EB/CLIP-170 [19][20][21][22]. The C-terminal tyrosine of α-tubulin can be removed and religated in a tyrosination-detyrosination cycle and is proposed to regulate the interactions with molecular motors and other MT binding proteins [23,24]. Tubulin tyrosination is required in mouse fibroblasts to localize CAP-Gly proteins, including p150, to MT plus ends [25], and recent work in vitro demonstrated that the interaction between p150's CAP-Gly domain and tyrosinated MTs enhances the initiation of processive dynein motility [26].
The functional significance of MT binding by p150 in animals is best understood in neurons. Single point mutations in the CAP-Gly domain cause the ALS-like motor neuron degenerative disease HMN7B and a form of parkinsonism known as Perry syndrome [27][28][29]. Cellular and in vivo studies addressing the underlying molecular defects revealed that p150 CAP-Gly domain-dependent binding of dynactin to dynamic MTs in the distal axon enhances the recruitment of dynein, which in turn facilitates efficient initiation of retrograde transport [30][31][32].
While the critical role of p150's CAP-Gly domain in neuronal trafficking is firmly established, little is known about how MT binding by dynactin regulates dynein functions in other cellular contexts. A study in D. melanogaster S2 cells reported multipolar spindles with a p150 Glued construct lacking the CAP-Gly domain, suggesting a role in organizing MT arrays [33]. In budding yeast, introduction of the motor neuron disease mutation into p150 Nip100 inhibited the initial movement of the spindle and nucleus into the bud neck during mitosis [34], suggesting that dynactin binding to MTs helps dynein generate pulling forces under load. In budding and fission yeast, dynein is off-loaded to cortical anchors via MTs for subsequent force production, and in budding yeast this requires MT tip tracking of dynein [35][36][37][38][39]. Whether MT tip tracking of dynein plays a role in delivering the motor to the cortex in animal cells remains to be determined.
MT binding of dynactin is significantly enhanced by electrostatic interactions between the p150 basic patch and the acidic tails of tubulins [14,40,41]. In the filamentous fungus A. nidulans, deletion of the basic patch in p150 NUDM diminishes the accumulation of dynactin and dynein at MT tips and partially impairs nuclear migration and early endosome distribution [42]. Interestingly, humans express tissue-specific splice isoforms of p150 that lack the basic patch [43,44], but the implications for dynactin function are unclear.
In the C. elegans one-cell embryo, dynein and dynactin are essential for centrosome separation, migration of the maternal and paternal pronucleus, centration and rotation of the two pronuclei and the associated centrosomes (the nucleus-centrosome complex, NCC), assembly and asymmetric positioning of the mitotic spindle, chromosome congression, and transversal spindle oscillations in anaphase [45][46][47][48][49]. Here, we use a set of p150 dnc-1 and α-tubulin mutants constructed by genome editing to define the role of dynactin's MT binding activity in this system. Our results uncover a functional link between the efficient initiation of dynein-mediated organelle transport, which requires dynactin binding to tyrosinated MTs, and the cytoplasmic pulling forces responsible for centration of centrosomes.

Results
Identification of +TIPs required for microtubule plus-end targeting of dynein-dynactin in the C. elegans early embryo To investigate whether dynactin's MT binding activity contributes to dynein function in the early C. elegans embryo, we first asked whether dynactin is present at MT plus ends at this developmental stage. Live confocal imaging in the central plane of metaphase one-cell embryos co-expressing endogenous GFP::p50 DNC-2 and transgene-encoded EBP-2::mKate2 revealed that dynactin travelled on growing MT tips from mitotic spindle poles to the cell cortex ( Fig  1A, S1 Movie). Imaging of the cortical plane allowed end-on visualization of MT tips as they arrived at the cortex (Fig 1B and 1C), which facilitated quantification of dynactin levels at plus ends. Measurements of fluorescence intensity revealed the expected positive correlation between GFP::p50 DNC-2 and EBP-2::mKate2 levels, but also showed that there is considerable variation in the amount of GFP::p50 DNC-2 at MT plus ends (S1A Fig). Cortical residency times for EBP-2::mKate2 and GFP::p50 DNC-2 were nearly identical (1.67 ± 0.03 s and 1.50 ± 0.05 s, respectively) and agreed with previously published measurements for cortical residency times of GFP::EBP-2 (S1C and S1D Fig) [50]. We also generated a dynein heavy chain dhc-1 ::gfp knock-in allele to assess the localization of endogenous dynein. DHC-1::GFP was readily detectable on growing MT plus ends in early embryos (S1B Fig, S2A Fig, S5A Fig, S2 Movie), although the signal appeared weaker than that of GFP::p50 DNC-2 . We conclude that a pool of dynein-dynactin tracks with growing MT plus ends in the early C. elegans embryo.
We conclude that in the C. elegans early embryo, dynein and dynactin are interdependent for targeting to growing MT plus ends and require EBP-2 and LIS-1, but not EBP-1, EBP-3, or the CLIP-170 homolog CLIP-1 ( Fig 1E).
Splice isoforms of the p150 DNC-1 basic patch modulate microtubule plusend targeting of dynactin Having established that dynein and dynactin require the EB homolog EBP-2 for targeting to MT tips, we next examined the role of the dynactin subunit p150 DNC-1 , whose N-terminal CAP-Gly domain (residues 1-69) mediates binding to EB and MTs (Fig 2A). In addition, p150 DNC-1 contains a~200-residue basic-serine rich region between the CAP-Gly domain and the first coiled-coil (CC1A), which has been proposed to regulate p150 DNC-1 association with MTs [54,55] (Fig 2A). The highest density of basic residues is found between residues 140-169 (30% K or R, pI = 12.02). This region is encoded by exon 4 and part of exon 5, which are subject to alternative splicing ( Fig 2B, S3A Fig). This is similar to human p150, which contains an alternatively-spliced basic patch of 28 residues (43% K or R, pI = 12.7) adjacent to the CAP-Gly domain [43]. We detected four splice isoforms of p150 dnc-1 by reverse transcription PCR of RNA isolated from adult animals (S3B Fig): full-length p150 dnc-1 including exons 4 and 5, p150 dnc-1 without exon 4 (Δexon 4), p150 dnc-1 without exon 5 (Δexon 5), and p150 dnc-1 lacking exons 4 and 5 (Δexon 4-5). To define the function of individual splice isoforms, we edited the p150 dnc-1 locus to generate animals in which p150 dnc-1 expression was restricted to one of the four isoforms ( Fig 2B, S3A Fig). Reverse transcription PCR confirmed that animals expressed single p150 dnc-1 isoforms corresponding to full length, Δexon 4, Δexon 5, or Δexon 4-5 (S3B Fig). All mutant animals were homozygous viable and fertile (S3C Fig), demonstrating that none of the p150 DNC-1 isoforms is essential. Despite differences in predicted molecular weight of only a few kDa (S3A Fig), single isoforms expressed in mutant animals were distinguishable by size on immunoblots with an antibody raised against a C-terminal region of p150 DNC-1 ( Fig  2C). Side-by-side comparison of isoform mutants and wild-type animals on the same immunoblot revealed that neither full-length p150 DNC-1 nor p150 DNC-1 (Δexon 4-5) is prevalent in wild-type adults ( Fig 2C). Instead, immunoblotting, together with reverse transcription PCR data (S3B and S3D Fig), suggested that p150 DNC-1 (Δexon 4) is the predominant isoform.
Humans express the neuron-specific splice variant p135, which lacks the entire N-terminal MT binding region [56]. In C. elegans hermaphrodite adults, 302 out of 959 somatic cells are neurons, yet we did not find evidence for a p135 dnc-1 isoform at the mRNA level (S3D Fig), nor chain DYCI-1 or LIS-1 decreases dynactin levels at MT plus ends. Images correspond to maximum intensity projections over time (12 frames acquired every 5 s). Scale bar, 5 μm; insets, 2 μm. (D) Quantification of dynactin levels at MT plus ends using fluorescence intensity measurements of GFP::p50 DNC-2 at the cortex in the conditions shown in (B) and (C). For DYCI-1 and LIS-1 partial depletions were performed (shorter RNAi treatment, see methods), because penetrant depletions resulted in sterility of the mother. Error bars represent the SEM with a 95% confidence interval. For each condition, n indicates the total number of individual measurements from 7-11 embryos. The t-test was used to determine statistical significance. ****P < 0.0001; *P < 0.05; ns = not significant, P > 0.05. (E) Requirements for MT tip tracking by dynein-dynactin in the early C. elegans embryo.
https://doi.org/10.1371/journal.pgen.1006941.g001  (EB). The N-terminal region of the p150 DNC-1 subunit contains a did our p150 DNC-1 antibody detect any protein below~150 kDa in wild-type animals ( Fig 2C). We also generated a p150 dnc-1 ::3xflag knock-in allele, and immunoblotting with antibody against 3xFLAG similarly failed to detect a p135 isoform (S3E Fig). We speculated that specifically suppressing the expression of p150 DNC-1 isoforms might facilitate the detection of p135 and engineered a null allele of p150 dnc-1 by inserting a stop codon in exon 1 immediately following the start codon (S3A Fig). The null mutation did not affect splicing of p150 dnc-1 mRNA (S3B Fig) and therefore should permit expression of p135 from an alternative start codon, as is the case in humans [56]. However, immunoblotting produced no evidence of p135 expression in the absence of p150 DNC-1 isoforms (Fig 2C). We conclude that C. elegans does not express significant amounts of a p135 isoform.
Point mutations in p150 DNC-1 's CAP-Gly domain that cause neurodegenerative disease in humans displace dynactin and dynein from microtubule plus ends Our analysis of p150 DNC-1 isoforms suggested that the basic region had a relatively minor role in targeting dynactin to MT tips. To examine the role of the CAP-Gly domain, we used genome editing to separately introduce three point mutations into p150 DNC-1 that compromise CAP-Gly domain function and cause neurodegenerative disease in humans ( Fig 2E): G33S corresponds to human G59S, which causes motor neuropathy 7B [27]; G45R corresponds to human G71R, which causes Perry Syndrome [28]; and F26L corresponds to human F52L, which was recently identified in a patient with Perry Syndrome-like symptoms [29]. The conserved CAP-Gly domain (residues 1-69) predicted to bind the end-binding homology (EBH) domain of EB and the EEY/F motifs present at the C-termini of EB and α-tubulin. It also contains a basic patch (residues 140-169) predicted to interact with the negatively charged C-terminal tails (E-Hooks) of α-and β-tubulin. (B) Schematic of the four p150 dnc-1 N-terminal splice isoforms identified by reverse transcription PCR and strategy for CRISPR-Cas9-based genome editing to restrict p150 dnc-1 expression to single isoforms. (C) Immunoblot of adult worms with an antibody against a C-terminal region of p150 DNC-1 , showing that engineered p150 dnc-1 mutants express single isoforms that are distinguishable by size. α-Tubulin was used as the loading control. Molecular mass is indicated in kilodaltons. (D) Quantification of GFP::p50 DNC-2 levels at MT plus ends in p150 dnc-1 isoform mutants using fluorescence intensity measurements at the cortex. Error bars represent the SEM with a 95% confidence interval, and n indicates the total number of individual measurements from 5-8 different embryos per condition. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; *P < 0.05; ns = not significant, P > 0.05. (E) Sequence alignment of the p150 CAP-Gly domain region where point mutations have been identified that cause neurodegenerative disease in humans. Analogous point mutations were introduced into the C. elegans p150 dnc-1 locus using CRISPR-Cas9-based genome editing. (F) Immunoblots of adult worms, showing that protein levels of p150 DNC-1 are decreased in the G33S mutant but not in the G45R and F26L mutants. α-Tubulin served as the loading control. (G) Quantification of p150 DNC-1 protein levels using intensity measurements from immunoblots as in (F). Three independent immunoblots were performed for each condition. (H) Central confocal section in metaphase one-cell embryos expressing GFP::p50 DNC-2 and wild-type p150 DNC-1 or p150 DNC-1 CAP-Gly mutants. Images correspond to maximum intensity projections over time (10 frames acquired every 200 ms). Scale bar, 5 μm. (I) Cortical confocal section of embryos as in (H), additionally expressing EBP-2::mKate2 as a marker for MT plus ends. Images correspond to maximum intensity projections over time (12 images acquired every 5 s). Scale bar, 5 μm. (J) Quantification of dynactin levels at MT plus ends using fluorescence intensity measurements of GFP::p50 DNC-2 at the cortex, as shown in (I). Error bars represent the SEM with a 95% confidence interval, and n indicates the number of individual measurements from 8 different embryos per condition. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; **P < 0.01. F26L and G45R mutants could be propagated as homozygotes with high embryonic viability (99 ± 1% and 90 ± 2%, respectively), whereas the G33S mutant was lethal in the F2 generation (1 ± 1% embryonic viability) (S4A Fig). Immunoblotting of homozygous F1 adults showed that G33S animals had decreased levels of p150 DNC-1 , indicating that the mutation destabilized the protein (Fig 2F and 2G). By contrast, total levels of p150 DNC-1 were not affected in the F26L or G45R mutant. Central plane imaging in one-cell embryos expressing GFP::p50 DNC-2 showed that dynactin containing the F26L or G45R mutation was present on the mitotic spindle and prometaphase kinetochores but displaced from MT tips (Fig 2H, S3 Movie). Cortical imaging after introduction of the EBP-2::mKate2 marker revealed that GFP::p50 DNC-2 levels at MT tips were reduced to 34 ± 4% and 27 ± 4% of controls in the F26L and G45R mutant, respectively ( p150 dnc-1 CAP-Gly mutants exhibit defects in centration/rotation of the nucleus-centrosome complex, chromosome congression, and spindle rocking Next, we asked whether the p150 dnc-1 mutants affected dynein-dynactin function in the onecell embryo. We crossed the mutants with animals co-expressing GFP::histone H2B and GFP:: γ-tubulin, which allowed precise tracking of pronuclei and centrosomes, respectively ( Fig 3A). None of the mutants exhibited defects in centrosome separation, and pronuclear migration along the anterior-posterior axis proceeded with normal kinetics until pronuclear meeting, which occurred at the correct position in the posterior half of the embryo ( In all mutants, spindle orientation recovered during prometaphase, so that the spindle axis was largely aligned with the anterior-posterior axis of the embryo at the time of anaphase onset ( Fig 3D, S6C Fig).
In controls, the mitotic spindle was displaced from the embryo center toward the posterior in preparation for asymmetric division (Fig 3C). By contrast, spindle assembly in p150 dnc-1 CAP-Gly mutants already occurred in the posterior half of the embryo, and the spindle had to be moved only slightly to the posterior to be correctly positioned. In controls, the regular and vigorous oscillations of spindle rocking began at anaphase onset and lasted for approximately 100 s (Fig 3E, S7 Movie). By contrast, spindle rocking in p150 dnc-1 CAP-Gly mutants was irregular and significantly dampened.
In addition to defects in NCC centration/rotation and spindle rocking, we observed a slight but consistent delay in chromosome congression in p150 dnc-1 CAP-Gly mutants, indicating problems with the interactions between chromosomes and spindle MTs (S7A and S7B Fig, S6 Movie, S7 Movie). This did not result in obvious chromosome mis-segregation in the first We conclude that mutations in the p150 DNC-1 CAP-Gly domain perturb a specific subset of dynein-dynactin functions in the one-cell embryo.

Inhibition of cortical dynein and p150 dnc-1 CAP-Gly mutants cause distinct defects during centration of the nucleus-centrosome complex
Anaphase spindle rocking requires cortical dynein pulling on astral MTs [2,57]. Since spindle rocking was affected in p150 dnc-1 CAP-Gly mutants, we sought to assess the extent of phenotypic overlap between p150 dnc-1 CAP-Gly mutants and inhibition of dynein-dependent cortical pulling. We tracked centrosomes and pronuclei after co-depleting GPR-1 and GPR-2, which are required for cortical anchoring of dynein-dynactin [2,57]. In contrast to p150 dnc-1 CAP-Gly mutants, gpr-1/2(RNAi) delayed the initial separation of centrosomes and the onset of pronuclear migration (Fig 4A-4C). Pronuclear migration and NCC centration subsequently occurred at a slightly faster rate than in controls, so that the NCC achieved near-normal centration by nuclear envelope breakdown (NEBD) (Fig 4A and 4C). These results are consistent with slowed centrosome separation and faster centering reported after co-depletion of GOA-1 and GPA-16, the Gα proteins acting upstream of GPR-1/2 [58,59]. Thus, the kinetics of pronuclear migration and NCC centration differ between gpr-1/2(RNAi) and p150 dnc-1 CAP-Gly mutants. NCC rotation, by contrast, was affected in both perturbations ( Fig 4D). Importantly, gpr-1/2(RNAi) in the p150 dnc-1 (G45R) mutant enhanced the rotation defect of gpr-1/2(RNAi) on its own, arguing that GPR-1/2 and the p150 DNC-1 CAP-Gly domain contribute to NCC rotation through parallel pathways. After NEBD, depletion of GPR-1/2 prevented posterior displacement of the spindle and the lack of cortical pulling was especially evident in the track of the posterior centrosome ( Fig 4C). In addition, the mitotic spindle was shorter than controls during metaphase and failed to elongate properly in anaphase ( Fig 4B). By contrast, posterior centrosome movement towards the cortex in p150 dnc-1 CAP-Gly mutants was indistinguishable from controls ( Fig 3C, Fig 4C), and spindle length was normal throughout metaphase and anaphase ( Fig 4B). These results argue that, although spindle rocking is compromised in p150 dnc-1 CAP-Gly mutants, cortical dynein is still able to generate robust pulling forces on astral MTs. We also tracked centrosomes after depletion of EBP-2, which, just like p150 dnc-1 CAP-Gly mutants, delocalized dynein-dynactin from MT tips (Fig 1B and 1D). Strikingly, controls and p150 dnc-1 mutants. Chromosomes and centrosomes are marked with GFP::histone H2B and GFP::γtubulin, respectively. Scale bar, 5 μm. (B) Migration kinetics of the male pronucleus in the embryos shown in (A). The position of the male pronucleus along the anterior-posterior axis was determined in images captured every 10 s. Individual traces were normalized to embryo length, time-aligned relative to pronuclear meeting, averaged for the indicated number (n) of embryos, and plotted against time. Error bars represent the SEM with a 95% confidence interval. (C) Kinetics of centrosome positioning along the anterior-posterior axis, determined as described for (B) and plotted relative to nuclear envelope breakdown. Solid lines indicate the midpoint between the two centrosomes (spindle position). Anaphase begins at 200 s. Error bars represent the SEM with a 95% confidence interval. (D) Angle between the centrosome-centrosome (C-C) axis and the anterior-posterior (A-P) axis in one-cell embryos at nuclear envelope breakdown (NEBD) and anaphase onset. Circles correspond to measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; **P < 0.01; ns = not significant, P > 0.05.  posterior spindle displacement was exaggerated in ebp-2(RNAi) embryos compared with controls ( Fig 4C). These results suggest that cortical pulling forces used for asymmetric spindle positioning can be generated in the absence of MT tip-localized dynein-dynactin.
MT plus-end tracking of dynein-dynactin is dispensable for targeting the motor to the cell cortex The robust dynein-dependent cortical pulling observed in one-cell embryos depleted of EBP-2 and in embryos of p150 dnc-1 CAP-Gly mutants implied that the motor was able to target to the cortex under these conditions. To test this directly, we measured the intensity of the DHC-1:: GFP signal in line scans drawn across the cortex in one-cell embryos at anaphase. This revealed a cortically enriched pool of DHC-1::GFP that was dependent on GPR-1/2, as expected ( Fig  4E). Cortical dynein in the one-cell embryo was unaffected in the p150 dnc-1 (G45R) mutant and after ebp-2(RNAi). We also imaged the 4-cell embryo, in which dynactin and dynein become prominently enriched at the EMS-P2 cell border prior to EMS and P2 spindle rotation [60,61]. Quantification of GFP::p50 DNC-2 and DHC-1::GFP levels at the EMS-P2 cell border revealed that cortical levels of dynein-dynactin were unchanged in the p150 dnc-1 (G45R) mutant and after ebp-2(RNAi) (Fig 4F). We conclude that MT tip tracking of dynein-dynactin is dispensable for cortical targeting of the motor in the early embryo.
Mutation of α-tubulin's C-terminal tyrosine causes defects in NCC centration/rotation that resemble those of p150 dnc-1 CAP-Gly mutants CAP-Gly domains bind the C-terminal EEY/F motif of α-tubulin, and the tyrosine residue is critical for the interaction (Fig 2A) [22]. We therefore asked whether decreased affinity of dynactin for tyrosinated MTs could be contributing to the defects observed in p150 dnc-1 CAP-Gly mutants. Of the 9 α-tubulin isoforms in C. elegans, TBA-1 and TBA-2 are the major α-tubulin isotypes expressed during early embryogenesis [62]. We mutated the C-terminal tyrosine of showing that inhibition of cortical pulling forces and p150 dnc-1 CAP-Gly mutants cause distinct defects in centration. The position of the male pronucleus, marked by GFP::histone H2B, was determined along the anterior-posterior axis in images captured every 10 s. Individual traces were normalized to embryo length, time-aligned relative to pronuclear meeting (left) or nuclear envelope breakdown (NEBD) (right), averaged for the indicated number (n) of embryos, and plotted against time. Arrows point to differences in migration kinetics between the p150 dnc-1 (G45R) mutant and gpr-1/2 (RNAi). Error bars represent the SEM with a 95% confidence interval. (B) Plot of centrosome-centrosome distance over time in one-cell embryos expressing GFP::γ-tubulin. Measurements were made in images captured every 10 s. Individual traces were time-aligned relative to NEBD, averaged for the indicated number (n) of embryos, and plotted against time. Error bars represent the SEM with a 95% confidence interval. Arrows points to delays in centrosome separation (-300 s) and to defective mitotic spindle elongation (200 s) in embryos depleted of GPR-1/2. (C) Positioning of centrosomes, marked by GFP::γ-tubulin, measured in time-lapse sequences as described for (A) and plotted relative to NEBD. Solid lines indicate the midpoint between the two centrosomes (spindle position). Error bars represent the SEM with a 95% confidence interval. Arrows highlight the difference in posterior centrosome displacement between the p150 dnc-1 (G45R) mutant, gpr-1/2(RNAi), and ebp-2(RNAi). (D) Angle between the centrosome-centrosome (C-C) axis and the anterior-posterior (A-P) axis in one-cell embryos at NEBD. Circles correspond to measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by oneway ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; **P < 0.01; ns = not significant, TBA-1 and TBA-2 to alanine and obtained animals homozygous for either mutation alone (YA) or both mutations combined (YA/YA) (Fig 5A). Immunoblotting of adult animals with the monoclonal antibody YL1/2, which is specific for tyrosinated tubulin, revealed that levels of tubulin tyrosination were decreased in tba-1(YA) and tba-2(YA) single mutants, with tba-2 (YA) having a more pronounced effect (Fig 5B). Combining the two mutations dramatically decreased total levels of tubulin tyrosination. Importantly, immunoblotting with an antibody insensitive to tubulin tyrosination confirmed that total α-tubulin levels were not affected in the three mutants (Fig 5B). We then used immunofluorescence to directly assess tyrosinated tubulin levels in the early embryo. In controls, the mitotic spindle of the one-cell embryo was prominently stained with the antibody against tyrosinated tubulin (Fig 5C). By contrast, the tubulin tyrosination signal was undetectable in the tba-1/2(YA/YA) double mutant, despite normal spindle assembly. Thus, we were able to generate animals without detectable tubulin tyrosination in early embryos.
We next addressed the functional significance of tubulin tyrosination in the one-cell embryo. Strikingly, we found that the tba-1/2(YA/YA) mutant exhibited NCC centration/rotation defects reminiscent of those observed in p150 dnc-1 CAP-Gly mutants (Fig 5D-5G, S8 Movie). Combining the tba-1/2(YA/YA) mutant with the p150 dnc-1 (G45R) mutant did not significantly exacerbate the centration/rotation defects of the either mutant on its own, indicating that both mutants act in the same pathway (Fig 5D-5G, S8 Movie). Interestingly, in contrast to p150 dnc-1 CAP-Gly mutants, anaphase spindle rocking was not affected in the tba-1/2(YA/YA) mutant (Fig 5H).
We also examined the effect of the tba-1/2(YA/YA) mutant on GFP::p50 DNC-2 localization and found that dynactin levels at MT tips were identical to controls (Fig 5I). Thus, in contrast to mouse fibroblasts [25], tubulin tyrosination in the C. elegans embryo is not required to target dynactin to MT tips. p150 dnc-1 CAP-Gly mutants and α-tubulin tyrosine mutants decrease the frequency of centrosome-directed early endosome movements Dynein-mediated transport of small organelles along MTs towards centrosomes is proposed to generate the cytoplasmic pulling forces for centration (the centrosome-organelle mutual pulling model) [5,63,64]. To ask whether the centration defects in our mutants correlate with defects in MT minus end-directed organelle transport, we monitored the movement of early endosomes, labelled with mCherry::RAB-5, from pronuclear meeting until NEBD. Time-lapse sequences recorded in a focal plane that included the NCC were used for semi-automated tracking of early endosomes that moved from the cell periphery towards centrosomes ( Fig  6A). In control embryos, we counted 16.3 ± 3.5 tracks/min during the~6 min centration interval (Fig 6B). This was reduced to 0.8 ± 0.6 tracks/min in embryos depleted of p150 DNC-1 by RNAi, confirming that dynactin is required for early endosome movement directed towards centrosomes. The p150 dnc-1 (G45R + Δexon 4-5) mutant also strongly reduced the number of observed tracks to 5.3 ± 1.1 tracks/min (Fig 6B, S9 Movie). The tba-1/2(YA/YA) mutant had a less severe effect but still substantially reduced the number of tracks to 10.6 ± 2.2 per min. We also determined the maximal velocity in each track (determining the mean speed was complicated by frequent pausing of particles) and the total track displacement. This revealed only minor differences between controls and either mutant (Fig 6B). We conclude that p150 dnc-1 CAP-Gly and α-tubulin tyrosine mutants reduce the frequency with which early endosomes move towards centrosomes during the centration phase. These results are consistent with the idea that dynactin binding to tyrosinated MTs enhances the efficiency of transport initiation by dynein, as recently documented in vitro [26]. In the distal axon of neuronal cells, EB-dependent recruitment of dynactin to dynamic MT plus ends is proposed to ensure efficient initiation of retrograde transport by dynein [32]. To test whether EBP-2 plays a role in the initiation of centrosome-directed organelle transport in the one-cell C. elegans embryo, we tracked early endosomes after ebp-2(RNAi). The number of early endosome tracks was reduced from 16.3 ± 3.5 to 6.9 ± 1.7 per minute after EBP-2 depletion (Fig 6B). Strikingly, ebp-2(RNAi) in the tba-1/2(YA/YA) mutant further reduced the number of early endosome tracks to 2.7 ± 1.0 per minute ( Fig 6B) and enhanced the NCC centration defect compared to the individual perturbations (Fig 6C, S8 Movie). This suggests that EBP-2 is able to promote the initiation of dynein-mediated transport from MT tips even in the absence of tubulin tyrosination, consistent with the observation that dynactin targeting to MT tips is unaffected in the tubulin tyrosine mutant (Fig 5I). We conclude that EBP-2 and tubulin tyrosination independently contribute to the initiation of dynein-mediated organelle transport and NCC centration.

Discussion
Dynactin's MT binding activity is crucial in neurons, as illustrated by single point mutations that compromise the function of the p150 CAP-Gly domain and cause neurodegenerative disease [30,31,65,66]. Here, we introduced these CAP-Gly mutations into C. elegans p150 DNC-1 to investigate how dynactin's interaction with MTs and +TIPs contributes to dynein function in early embryogenesis. Together with the analysis of engineered p150 dnc-1 splice isoform and tubulin tyrosine mutants, our work provides insight into the regulation and function of MT tip tracking by dynein-dynactin in animals and uncovers a link between dynactin's role in initiating dynein-mediated transport of small organelles and the generation of cytoplasmic pulling forces.
Dynein accumulates at and tracks with growing MT plus ends in species ranging from fungi to mammals, but requirements for MT tip tracking differ. In the C. elegans early embryo, MT tip recruitment of dynein-dynactin shares similarity with the pathway in budding yeast (dynactin depends on dynein and LIS-1) and mammalian cells/filamentous fungi (dynein depends on dynactin). Surprisingly, similar to what was reported for the fungus U. maydis [65], accumulation of dynein-dynactin at MT tips does not require a CLIP-170-like protein in C. elegans. Instead, dynactin is likely directly recruited by EBP-2, one of the three EB homologs. Work in mouse fibroblasts knocked out for tubulin tyrosine ligase showed that decreased tyrosinated tubulin levels displaced CLIP-170 and p150 from MT tips [25]. By contrast, we show that MT tip targeting of C. elegans dynactin is independent of tubulin tyrosination, residues were mutated to alanine using CRISPR-Cas9-based genome editing. (B) Immunoblot of adult worms showing decreased levels of tubulin tyrosination in α-tubulin YA mutants. The same membrane was probed sequentially with an antibody against tyrosinated α-tubulin (antibody YL1/2) and against total α-tubulin (antibody B512). GAPDH served as a loading control. Molecular mass is indicated in kilodaltons. (C) Immunofluorescence images showing lack of tubulin tyrosination in tba-1/2(YA/YA) embryos at the one-cell stage (anaphase). Embryos were co-stained with antibodies against tyrosinated α-tubulin and total α-tubulin. Image with adjusted intensity levels shows unspecific background signal in the α-tubulin tyrosine mutant. Scale bars, 5 μm. (D)-(H) Still images from time-lapse sequences (D) and analysis of male pronuclear migration (E), centrosome positioning (F), NCC/spindle orientation (G), and anaphase spindle oscillations (H) during the first embryonic division for controls and α-tubulin tyrosine mutants. Data is displayed as described for Fig 3. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; **P < 0.01; ns = not significant, P > 0.05. Scale bar, 5 μm. (I) (left) Cortical confocal section of a control and tba-1/2(YA/YA) one-cell embryo in metaphase expressing GFP::p50 DNC-2 . Images correspond to maximum intensity projections over time (12 images acquired every 5 s). Scale bar, 5 μm. (right) Quantification of dynactin levels at MT plus ends using fluorescence intensity measurements of GFP::p50 DNC-2 at the cortex. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of individual measurements from 8-9 different embryos per condition. The t-test was used to determine statistical significance. ns = not significant, P > 0.05. https://doi.org/10.1371/journal.pgen.1006941.g005

Fig 6. Dynactin's MT binding activity, tyrosinated α-tubulin, and EBP-2 are required for efficient initiation of centrosome-directed organelle transport. (A)
Selected image from a time-lapse sequence recorded during centration of the nucleus-centrosome complex in an embryo expressing the early endosome marker mCherry::RAB-5. Arrows point to early endosomes that are about to move from the cell periphery towards the centrosomes along the tracks shown in blue. Scale bar, 5 μm. (B) Quantification of the number of tracks per min, the maximal track velocities, and the total track displacement for mCherry::RAB-5-labelled particles travelling towards centrosomes during the centration/rotation phase. Particles were tracked in time-lapse sequences as shown in (A) with images captured every 400 ms. Circles correspond to data points of individual embryos. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of individual tracks analyzed from 8-9 embryos per condition. Statistical significance was determined by oneway ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns = not significant, P > 0.05. (C) Migration kinetics of the male pronucleus in one-cell embryos, showing that depletion of EBP-2 in the tba-1/2(YA/YA) mutant results in enhanced centration defects relative to either perturbation alone. The position of the male pronucleus (marked by GFP::histone H2B as in Figs 3A and 5D) along the anteriorposterior axis was determined in images captured every 10 s. Individual traces were normalized to embryo length, time-aligned relative to pronuclear meeting, averaged for the indicated number (n) of embryos, and plotted against time. Error bars represent the SEM with a 95% confidence interval. (D) Model summarizing the functional significance of dynactin's MT binding activity in the one-cell embryo. The CAP-Gly domain of p150 DNC-1 binds to MTs and MT tips through interactions involving the C-terminal tails of α-tubulin and EBP-2, respectively. This promotes the initiation of organelle transport by dynein along the length of MTs, as well as from MT tips. Dynein-dependent movement of organelles along MTs towards centrosomes generates cytoplasmic pulling forces required for centration of the nucleus-centrosome complex. Tracking of dynein-dynactin with MT tips is not required for the delivery of the motor to cortical anchors. Instead, dynein-dynactin bind to the cortex directly from the cytoplasm and subsequently capture MT tips for generation of cortical pulling forces.
possibly because there is no requirement for a CLIP-170 homolog. Overall, our analysis of dynein-dynactin targeting to MT tips in C. elegans highlights the evolutionary plasticity of +TIP networks.
Our characterization of engineered p150 dnc-1 mutants establishes the functional hierarchy among p150 DNC-1 's tandem arrangement of MT binding regions: the CAP-Gly domain clearly provides the main activity, while the adjacent basic region plays an auxiliary role. Together with previous work in human cells [43], our results support the idea that alternative splicing of p150's basic region constitutes a conserved mechanism in animals for fine-tuning dynactin's affinity for MTs.
In budding yeast, dynein must first be targeted to MT tips prior to associating with cortical anchors [35,67]. Our results indicate that this pathway may not be used in C. elegans, as cortical accumulation of dynein-dynactin in the early embryo was unaffected in the p150 dnc-1 (G45R) mutant and after depletion of EBP-2, which displaced the majority of dynactin and dynein from MT tips. In agreement with normal cortical targeting of the motor, dynein-dependent cortical pulling forces remained robust in p150 dnc-1 CAP-Gly mutants, although defects in spindle rocking indicate that the p150 DNC-1 CAP-Gly domain does contribute to proper cortical force generation in anaphase. Importantly, depletion of EBP-2 even appeared to enhance cortical pulling during posterior spindle displacement. Thus, our results argue that dynein is recruited by its cortical anchors directly from the cytoplasm, and that dynein-dependent cortical pulling is therefore mechanistically uncoupled from prior MT tip tracking of the motor (Fig 6D). Surprisingly, even the p150 dnc-1 (G45R + Δexon 4-5) mutant, which shows the most severe reduction in dynactin levels at MT tips (15 ± 4% of controls), is viable and fertile, suggesting that MT tip tracking of dynein-dynactin is by and large dispensable for development.
If not delivery of dynein to the cell cortex via MTs, what is the purpose of dynactin's MT binding activity? We found that p150 dnc-1 CAP-Gly mutants have defects in the centration and rotation of the NCC, which consists of the two centrosomes and the associated female and male pronucleus. Experimental work and biophysical modelling support the idea that centration forces in the one-cell embryo are generated by dynein-mediated cytoplasmic pulling [5,63,64], although a centration/rotation model based on cortical pulling has also been proposed [68]. In the cytoplasmic pulling model, dynein works against viscous drag as it transports small organelles (e.g. endosomes, lysosomes, yolk granules) along MTs towards centrosomes, which generates pulling forces on MTs that move the NCC. Prior work showed that movements of early endosomes and centrosomes are correlated, and RNAi-mediated depletion of adaptor proteins that tether dynein to early endosomes and lysosomes inhibited centration, indicating that there is a functional link between organelle transport and cytoplasmic pulling forces [5]. In agreement with this idea, the p150 dnc-1 (G45R + Δexon 4-5) mutant not only inhibited centration but also significantly decreased the number of early endosomes that displayed directed movement toward centrosomes. This effect on early endosome transport is consistent with the p150 CAP-Gly domain's role in initiating dynein-mediated transport, which is well-established in the context of retrograde axonal transport in neurons [30][31][32]. Compromising the efficiency with which organelle transport is initiated is predicted to decrease cytoplasmic pulling forces, because the magnitude of the net pulling force acting on centrosomes is proportional to the number of organelles travelling along MTs.
The frequency of centrosome-directed early endosome movement was also decreased in the tba-1/2(YA/YA) mutant, which severely reduced the levels of tubulin tyrosination in the early embryo. This fits well with recent work in vitro demonstrating that the interaction between the p150 CAP-Gly domain and tyrosinated MTs enhances the efficiency with which processive motility of dynein-dynactin is initiated [26]. Furthermore, a recent study in neurons provided evidence that initiation of retrograde transport in the distal axon is regulated by tubulin tyrosination [69]. Interestingly, depletion of EBP-2, both on its own and in the tba-1/2(YA/YA) mutant, also decreased early endosome transport. This suggests that EBP-2 promotes dyneinmediated transport initiation from MT tips, presumably through its interaction with the p150 DNC-1 CAP-Gly domain, and that it can do so even in the absence of tyrosinated tubulin. In agreement with this idea, we observed that dynactin was still recruited to MT tips in the tubulin tyrosine mutant. Importantly, in addition to lowering the frequency of early endosome transport, the tba-1/2(YA/YA) mutant also affected centration of the NCC, and depletion of EBP-2 in the tba-1/2(YA/YA) mutant exacerbated the centration defect, as predicted by the centrosome-organelle mutual pulling model (Fig 6D).
Why do the p150 dnc-1 CAP-Gly and tubulin tyrosine mutants affect centration of the NCC, but not pronuclear migration until pronuclear meeting? One plausible explanation is that during pronuclear migration the male and female pronuclei, which are large (~10 μm diameter) and equal in size, assist each other's movement as dyneins anchored on the female pronucleus walk along MTs nucleated by the centrosomes attached to the male pronucleus [70]. By contrast, during centration, the two pronuclei must be moved in the same direction, which might render cytoplasmic pulling forces more sensitive to changes in centrosome-directed transport of small (~1 μm diameter) organelles.
Finally, our data suggest that MT binding by dynactin contributes to chromosome congression. The effect is unlikely an indirect consequence of the delay in spindle orientation along the A-P axis, as chromosome congression problems were not observed after gpr-1/2(RNAi), which also causes spindle orientation defects. Likewise, normal chromosome congression after ebp-2 (RNAi) suggests that the defect in p150 dnc-1 CAP-Gly mutants is not due to delocalization of dynactin from MT tips. Therefore, it is likely that the contribution to chromosome congression comes from the p150 DNC-1 CAP-Gly domain pool at kinetochores, where it could aid in the capture of MTs. The decrease in embryonic viability in p150 dnc-1 CAP-Gly mutants after inhibition of the SAC indicates that the chromosome congression defects persist in later embryonic divisions.
In summary, our work demonstrates that dynactin's MT binding activity is functionally relevant in the context of embryonic cell division. Unlike previous work that addressed p150 CAP-Gly domain function in D. melanogaster S2 cells [33], we do not observe defects in bipolar spindle formation in p150 dnc-1 CAP-Gly mutants. Instead, the most striking consequence of inhibiting p150 DNC-1 CAP-Gly function or tubulin tyrosination is defective centrosome centration, which we propose is a consequence of defective initiation of dynein-mediated organelle transport, in agreement with the centrosome-organelle mutual pulling model [5]. The transport initiation function of p150's CAP-Gly domain is likely generally relevant in circumstances where positioning of subcellular structures depends on dynein-mediated cytoplasmic pulling, for example the centration of sperm asters in the large eggs of amphibians and sea urchins [6,[71][72][73].

RNA interference
For production of double-stranded RNA (dsRNA), oligos with tails containing T3 and T7 promoters were used to amplify regions from N2 genomic DNA or cDNA. Primers used for dsRNA production are listed in S3 Table. PCR reactions were cleaned (NucleoSpin Clean-up, Macherey-Nagel) and used as templates for T3 and T7 transcription reactions (MEGAscript, Invitrogen). Transcription reactions were cleaned (NucleoSpin RNA Clean-up, Macherey-Nagel) and complementary single-stranded RNAs were annealed in soaking buffer (3x soaking buffer is 32.7 mM Na 2 HPO 4 , 16.5 mM KH 2 PO 4 , 6.3 mM NaCl, 14.1 mM NH 4 Cl). dsRNAs were delivered by injecting L4 hermaphrodites, and animals were processed for live imaging after incubation at 20˚C for 24 h or 48 h for partial and penetrant depletions, respectively.
Images were recorded on an inverted Zeiss Axio Observer microscope at 1 x 1 binning with a 100x NA 1.46 Plan-Apochromat objective and an Orca Flash 4.0 camera (Hamamatsu). Image files were imported into Fiji for further processing.

Reverse transcription PCR
Total RNA was isolated from adult hermaphrodites using the TRIzol Plus RNA Purification Kit (Invitrogen). After 3 washes with M9, pelleted worms were homogenized in 200 μL of TRIzol reagent with a pellet pestle homogenizer and incubated at room temperature for 5 min. After addition of 40 μL chloroform, samples were shaken vigorously by hand, incubated at room temperature for 3 min, and centrifuged at 12000 x g for 15 min at 4˚C. The upper phase containing the RNA was transferred to an RNase-free tube and an equal volume of 70% ethanol was added. Further RNA purification steps were performed according to the manufacturer's instructions. Purified RNA was treated with DNase I (Thermo Scientific), and cDNA was synthesized with the iScript Select cDNA Synthesis Kit (Bio-Rad Laboratories). The following oligos were used for the PCR reactions in S3B Fig: forward oligo on p150 dnc-1 exon 3 (GAATGTCACCTGCTGCTT); forward oligo on p150 dnc-1 exon 4 (AAAGCGGTCTACAA CTCC); reverse oligo on p150 dnc-1 exon 5 (GATTGCGATAAGTTGGAGA); reverse oligo on p150 dnc-1 exon 6 (AGTAGTCGTGGACGCTTT). For the SL1 PCR shown in S3D Fig, the following oligos were used: forward oligo on SL1 (GGTTTAATTACCCAAGTTTGA); reverse oligo on p150 dnc-1 exon 6 (TCCAGTATCATCAATCTTCTT).

Embryonic viability
Embryonic viability tests were performed at 20˚C. L4 hermaphrodites were grown on NGM plates with OP50 bacteria for 40 h at 20˚C, then singled-out to mating plates (NGM plates with a small amount of OP50 bacteria). After 8 h, mothers were removed and the number of hatched and unhatched embryos on each plate was determined 16 h later.

Imaging conditions and image analysis
All imaging was performed in one-cell embryos unless otherwise indicated. Image analysis was performed using Fiji software (Image J version 2.0.0-rc-56/1.51h).
Pronuclear migration, centrosome positioning, centrosome-centrosome distance, and orientation of centrosome-centrosome axis. Time-lapse sequences of GFP::histone H2B and GFP::γ-tubulin, consisting of 7 x 1 μm z-stacks for GFP fluorescence and one central differential interference contrast (DIC) image captured every 10 s, were recorded at 2 x 2 binning with a 63x oil immersion objective from the start of pronuclear migration until the onset of cytokinesis. Embryo length was defined as the distance between the outermost points of the egg shell visible in DIC. After maximum intensity projection of GFP z-stacks, the x and y coordinates of pronuclei and centrosomes were recorded over time using the MTrackJ plugin by manually clicking in the center of the centrosome or nucleus. The position of centrosomes and pronuclei along the anterior-posterior axis was then calculated relative to embryo length, with the anterior reference point set to 0%. Tracks from individual embryos were aligned relative to pronuclear meeting or NEBD.
Transversal oscillations of the mitotic spindle. Time-lapse sequences of GFP::γ-tubulin, consisting of 12 x 1 μm z-stacks captured every 2 s, were recorded at 2 x 2 binning with a 63x oil immersion objective from the beginning of metaphase until the end of anaphase. After maximum intensity projection of z-stacks, the x and y coordinates for centrosomes were determined over time with MTrackJ and the transversal distance of each centrosome to a line bisecting the embryo along the anterior-posterior axis was calculated.
Levels of GFP::p50 DNC-2 and dynein heavy chain DHC-1 ::GFP at the nuclear envelope, kinetochores, and on the mitotic spindle. Time-lapse sequences, consisting of 8 x 1 μm zstacks captured every 10 s, were recorded at 1 x 1 binning with a 60× NA 1.4 oil immersion objective from 40-50 s prior to pronuclear meeting until the onset of cytokinesis. Nuclear envelope (NE) signal was quantified 3 frames prior to pronuclear meeting using a maximum intensity projection of the 3 z-sections representing the best in-focus images of the NE. A 2 pixelwide line was drawn on top of the NE along its entire circumference, and a similar line was drawn next to the NE on the cytoplasmic side around the nucleus. The mean fluorescence signal of the cytoplasmic line was then subtracted from the mean fluorescence signal of the NE line.
Kinetochore signal was measured 7-8 frames before the onset of sister chromatid separation using a maximum intensity projection of the z-stack. The top 10 local maxima intensities on kinetochores were identified using the "Find Maxima" function, and the 10 values were averaged. The mean fluorescence intensity of the spindle background close to the kinetochore region was measured and subtracted from the kinetochore signal.
Mitotic spindle signal was measured 2 frames after the onset of chromosome segregation using a maximum intensity projection of the z-stack. The mean intensity in 3 separate 10 x 10 pixel squares on the spindle was determined and the three values were averaged. The mean intensity of three equivalent squares in the cytoplasm adjacent to the spindle served as background signal and was subtracted from the spindle signal. Levels of GFP::p50 DNC-2 and dynein heavy chain DHC-1 ::GFP at MT plus ends. Timelapse sequences, consisting of a single cortical confocal section captured every 5 s, were recorded at 1 x 1 binning with a 100× NA 1.4 oil immersion objective for 1 min beginning at metaphase. 3 images that were at least 3 frames apart from each other were used for quantifications. A circle with a 5-pixel radius was drawn around individual MT plus ends (marked by EBP-2::mKate2 or mCherry::β-tubulin) and the integrated fluorescence intensity was measured in the GFP channel. The circle was then expanded by increasing the radius by 2 pixels, and the integrated intensity of this larger circle was measured. Background was defined as the difference in integrated intensities between the larger and the smaller circle. The background value was scaled in proportion to the smaller circle and then subtracted from the integrated intensity of the smaller circle to obtain a final value for the GFP signal. For presentation in graphs, all values were normalized to the mean value of the control. For quantification of the GFP::p50 DNC-2 signal at MT plus ends after ebp-2(RNAi) (Fig 1D), embryos co-expressing GFP::p50 DNC-2 and mCherry::β-tubulin were used to locate MT plus ends at the cortex.
Cortical residency times of GFP::p50 DNC-2 , dynein heavy chain DHC-1 ::GFP, and EBP-2:: mKate2. Time-lapse sequences, consisting of a single cortical confocal section captured every 200 ms, were recorded at 1 x 1 binning with a 100× NA 1.4 oil immersion objective for 1 min beginning at metaphase. Image sequences were analyzed using the LoG Detector (estimated blob diameter 10 pixels) and the Simple LAP Tracker (linking max distance 5 pixels; gap-closing max distance 5 pixels; gap-close max frame gap 0 frames) in the TrackMate plugin. The values for track duration were considered the cortical residency times for GFP and mKate2 puncta.
Levels of GFP::p50 DNC-2 and dynein heavy chain DHC-1 ::GFP at the EMS-P2 cell border. Time-lapse sequences, consisting of 8 x 1 μm z-stacks captured every 30 s, were recorded at 1 x 1 binning with 60× NA 1.4 oil immersion objective. Four-cell embryos were imaged from the beginning of nuclear envelope breakdown in AB cells until cytokinesis of the P2 cell. The signal at the EMS-P2 cell border was measured at the time of EMS spindle rotation in maximum intensity projections of the z-stacks. The top 10 local maxima intensities on the EMS-P2 cell border were identified using the "Find Maxima" function, and the 10 values were averaged. The mean fluorescence intensity of an adjacent area (20 x 20 pixels) in the EMS cytoplasm served as background and was subtracted from the EMS-P2 cell border signal.
Tracking of early endosomes marked with mCherry::RAB-5. Time-lapse sequences, consisting of a single confocal section captured every 400 ms, were recorded at 1 x 1 binning with a 60× NA 1.4 oil immersion objective for 6 min beginning at pronuclear meeting. Image sequences were analyzed using the LoG Detector (estimated blob diameter 6 pixels) and the Simple LAP Tracker (linking max distance 8 pixels; gap-closing max distance 16 pixels; gapclose max frame gap 2 frames) in the TrackMate plugin. All tracks whose particles showed directed movement towards centrosomes and had a track displacement of at least 0.9 μm (5 pixels) were considered.

Statistical analysis
Values in figures and text are reported as mean ± SEM with a 95% confidence interval. Statistical analyses was performed with GraphPad Prism 7.0 software. The type of statistical analysis performed is indicated in the figure legends. Differences were considered significant at P values below 0.05. , as indicated on the right. Introduction of a frameshift mutation after the p150 dnc-1 start codon generated a null allele. (B) Results of reverse transcription PCRs using RNA isolated from adult worms and primer pairs that allow detection of the four splice isoforms. Primer locations and predicted sizes of PCR products for the different isoforms are indicated. Crosses (x) indicate that the PCR will not amplify any product. All four N-terminal splice isoforms are detected in wild-type and p150 dnc-1 null mutant adults, whereas only one isoform is detected in each p150 dnc-1 isoform mutant. Asterisks next to gel bands denote unspecific PCR products. M, DNA size marker. (C) Embryonic viability assay for p150 dnc-1 isoform mutants. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of hermaphrodite mothers whose progeny was counted (> 500 total progeny per condition). Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ns = not significant, P > 0.05. (D) Result of a reverse transcription PCR using RNA isolated from adult wild-type worms with one of the primers recognizing the spliced leader sequence 1 (SL1) and the other located in exon 6 of p150 dnc-1 . In C. elegans, about 70% of mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders, SL1 or SL2, which replace the 5' ends of pre-mRNAs. One major product was amplified in the SL1 PCR and identified as the Δexon 4 isoform by sequencing. A PCR reaction with a primer recognizing SL2 did not amplify any product. M, DNA size marker. (E) Immunoblot of wild-type or p150 dnc-1 ::3xflag adult worms with an antibody against the 3xFLAG tag, showing that there is no detectable p150 DNC-1 isoform corresponding to human p135. α-Tubulin was used as the loading control. Molecular mass is indicated in kilodaltons. (TIF)

S4 Fig. Embryonic viability and localization of p150 dnc-1 CAP-Gly mutants. (A)
Embryonic viability assay for p150 dnc-1 CAP-Gly mutants. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of hermaphrodite mothers whose progeny was counted (> 500 total progeny per condition). Statistical significance was determined by oneway ANOVA followed by Bonferroni's multiple comparison test. ÃÃÃÃ P < 0.0001; ns = not significant, P > 0.05. (B) Quantification of dynactin levels at the nuclear envelope, kinetochores, and the mitotic spindle for the p150 dnc-1 mutants G45R and F26L, using fluorescence intensity measurements of GFP::p50 DNC-2 . Circles represent measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ÃÃÃÃ P < 0.0001; ns = not significant, P > 0.05. (TIF) S5 Fig. p150 dnc-1 CAP-Gly mutants delocalize dynein from MT tips. (A) Stills from time-lapse sequences in 2-cell embryos expressing dynein heavy chain DHC-1 ::GFP, demonstrating that dynein is delocalized from MT plus ends in the p150 dnc-1 (G45R) mutant. Scale bar, 5 μm. (B) Quantification of dynein levels at MT plus ends using fluorescence intensity measurements of dynein heavy chain DHC-1 ::GFP at the cortex of metaphase one-cell embryos. Error bars represent the SEM with a 95% confidence interval, and n indicates the total number of individual measurements from 6-7 embryos per condition. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ÃÃÃÃ P < 0.0001. (C) Stills from time-lapse sequences in one-cell embryos expressing dynein heavy chain DHC-1 :: GFP, showing that the p150 dnc-1 (G45R) mutant reduces dynein levels on the mitotic spindle and at centrosomes, but not at the nuclear envelope and kinetochores. Scale bar, 5 μm. (D) Quantification of dynein levels at the nuclear envelope, kinetochores, and the mitotic spindle for the p150 dnc-1 mutants G45R and F26L, using fluorescence intensity measurements for dynein heavy chain DHC-1 ::GFP in images as shown in (C). Circles represent measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ÃÃÃ P < 0.001; ns = not significant, P > 0.05. (TIF) S6 Fig. Functional analysis of p150 dnc-1 isoform mutants. (A) Migration kinetics of the male pronucleus in one-cell embryos expressing single isoforms of p150 DNC-1 . The position of the male pronucleus, marked by GFP::histone H2B, was determined along the anterior-posterior axis in images captured every 10 s. Individual traces were normalized to embryo length, timealigned relative to pronuclear meeting, averaged for the indicated number (n) of embryos, and plotted against time. Error bars represent the SEM with a 95% confidence interval. (B) Positioning of centrosomes, marked by GFP::γ-tubulin, measured in time-lapse sequences as described for (A) and plotted relative to nuclear envelope breakdown. Solid lines indicate the midpoint between the two centrosomes (spindle position). Anaphase begins at 200 s. Error bars represent the SEM with a 95% confidence interval. (C) Angle between the centrosome-centrosome (C-C) axis and the anterior-posterior (A-P) axis in one-cell embryos at nuclear envelope breakdown (NEBD) and anaphase onset. Circles correspond to measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ÃÃ P < 0.01; ns = not significant, P > 0.05. (TIF) S7 Fig. p150 dnc-1 CAP-Gly mutants delay chromosome congression. (A) Selected frames from time-lapse sequences in one-cell embryos co-expressing GFP::histone H2B and GFP::γtubulin, showing that congression of chromosomes is delayed in the p150 dnc-1 (F26L) mutant. Time is relative to nuclear envelope breakdown. Scale bar, 5 μm. (B) Interval duration for nuclear envelope breakdown (NEBD) to full alignment of chromosomes and full alignment of chromosomes to anaphase (onset of sister chromatid separation). Error bars represent the SEM with a 95% confidence interval, and n indicates the number of embryos analyzed. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ÃÃÃ P < 0.001. (C) Embryonic viability assay for p150 dnc-1 CAP-Gly mutants with and without depletion of the spindle assembly checkpoint component Mad1 MDF-1 . Error bars represent the SEM with a 95% confidence interval, and n indicates the number of hermaphrodite mothers whose progeny was counted (> 500 total progeny per condition). Statistical significance was determined by two-way ANOVA followed by Bonferroni's multiple comparison test. ÃÃÃ P < 0.001; Ã P < 0.05; #### P < 0.0001; ### P < 0.001. (TIF)

S8 Fig. Defects in the p150 dnc-1 (G45R) mutant are not exacerbated by the tba-1/2(YA/YA) mutant. (A)
Migration kinetics of the male pronucleus, marked by GFP::histone H2B, along the anterior-posterior axis. Nuclear position was determined in images captured every 10 s, individual traces were normalized to embryo length, time-aligned relative to pronuclear meeting, averaged for the indicated number (n) of embryos, and plotted against time. Error bars represent the SEM with a 95% confidence interval. (B) Angle between the centrosome-centrosome (C-C) axis and the anterior-posterior (A-P) axis in one-cell embryos at nuclear envelope breakdown (NEBD) and anaphase onset. Circles correspond to measurements in individual embryos. Error bars represent the SEM with a 95% confidence interval. The t-test was used to determine statistical significance. ns = not significant, P > 0.05. (TIF) S1 Movie. Microtubule tip tracking of dynactin in the C. elegans early embryo. One-cell embryo in metaphase co-expressing GFP::p50 DNC-2 and EBP-2::mKate2. The anterior side is to the left. A single confocal section in the embryo center was acquired every 0.4 s. Playback speed is 30 frames per second. Scale bar, 10 μm. (AVI) S2 Movie. Microtubule tip tracking of dynein in the C. elegans early embryo. Two-cell embryo expressing dynein heavy chain DHC-1 ::GFP. The anterior side is to the left. A single confocal section near the cortex was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm. (AVI) S3 Movie. The p150 dnc-1 CAP-Gly mutants F26L and G45R delocalize dynactin from microtubule tips-center view. One-cell embryos in metaphase expressing GFP::p50 DNC-2 . The anterior side is to the left. A single confocal section in the embryo center was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm. (AVI) S4 Movie. The p150 dnc-1 CAP-Gly mutant F26L delocalizes dynactin from microtubule tips -cortical view. One-cell embryos in metaphase expressing GFP::p50 DNC-2 . The anterior side is to the left. A single confocal section at the cortex was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm. (AVI) S5 Movie. The p150 dnc-1 CAP-Gly mutant G45R delocalizes dynactin from microtubule tips-cortical view. One-cell embryos in metaphase expressing GFP::p50 DNC-2 . The anterior side of the embryo is to the left. A single confocal section at the cortex was acquired every 0.2 s. Playback speed is 30 frames per second. Scale bar, 10 μm. (AVI) S6 Movie. Centration/rotation defects in p150 dnc-1 CAP-Gly mutants. One-cell embryos expressing GFP-labelled histone H2B and γ-tubulin to mark chromosomes and centrosomes, respectively. The anterior side is to the left. Time lapse is 10 s and playback speed is 6 frames per second. Scale bar, 10 μm. (AVI) S7 Movie. Delayed chromosome congression and dampened anaphase spindle oscillations in p150 dnc-1 CAP-Gly mutants. One-cell embryos expressing GFP-labelled histone H2B and γ-tubulin to mark chromosomes and centrosomes, respectively. The anterior side is to the left. Time lapse is 2 s and playback speed is 12 frames per second. Scale bar, 10 μm. (AVI) S8 Movie. Centration/rotation defects in α-tubulin tyrosine mutants. One-cell embryos expressing GFP-labelled histone H2B and γ-tubulin to mark chromosomes and centrosomes, respectively. The anterior side is to the left. Time lapse is 10 s and playback speed is 6 frames per second. Scale bar, 10 μm.