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Vol. 19, Issue 6, 2553-2565, June 2008

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RAB-11 Permissively Regulates Spindle Alignment by Modulating Metaphase Microtubule Dynamics in Caenorhabditis elegans Early Embryos

Haining Zhang^*,, Jayne M. Squirrell, and John G. White^,

*Laboratory of Genetics, Laboratory of Molecular Biology and Department of Anatomy, University of Wisconsin, Madison, WI 53706

Submitted September 6, 2007; Revised March 18, 2008; Accepted March 24, 2008
Monitoring Editor: Sean Munro

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Alignment of the mitotic spindle along a preformed axis of polarity is crucial for generating cell diversity in many organisms, yet little is known about the role of the endomembrane system in this process. RAB-11 is a small GTPase enriched in recycling endosomes. When we depleted RAB-11 by RNAi in Caenorhabditis elegans, the spindle of the one-cell embryo failed to align along the axis of polarity in metaphase and underwent violent movements in anaphase. The distance between astral microtubules ends and the anterior cortex was significantly increased in rab-11(RNAi) embryos specifically during metaphase, possibly accounting for the observed spindle alignment defects. Additionally, we found that normal ER morphology requires functional RAB-11, particularly during metaphase. We hypothesize that RAB-11, in conjunction with the ER, acts to regulate cell cycle–specific changes in astral microtubule length to ensure proper spindle alignment in Caenorhabditis elegans early embryos.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The first cell division of the Caenorhabditis elegans zygote (P0) is asymmetric, giving rise to two daughter cells with different sizes and fates: the smaller P1 cell inherits germline determinants such as PIE-1 (Mello et al., 1996) and the larger AB cell develops into most of the somatic tissues (Sulston et al., 1983). This asymmetric division requires two well-regulated spindle alignment phases. First, before metaphase, the centrosomal-nuclear complex migrates to the center of the embryo while undergoing a 90° rotation to align the mitotic spindle onto a preformed A-P axis defined by the axially segregated PAR proteins (Cowan and Hyman, 2004a). This rotational alignment depends on the interactions of astral microtubules (MTs) with cortical regulators such as dynein-dynactin (Skop and White, 1998; Gonczy et al., 1999a) and the DEP domain protein LET-99 (Tsou et al., 2002). In the second phase, the redundant Gproteins, GOA-1 and GPA-16, transduce the polarity cues of the PAR proteins into forces that displace the spindle posteriorly during anaphase (Gotta and Ahringer, 2001; Tsou et al., 2003). Although Gdistributes uniformly around the cortex (Gotta and Ahringer, 2001), its upstream activator, GPR-1/2, localizes asymmetrically in response to the PARs: it is enriched on the posterior cortex where PAR-2 is localized and reduced on the anterior cortex where PAR-3 is localized (Colombo et al., 2003; Gotta et al., 2003). The activity of G is down-regulated by Gβ and LET-99, possibly by reducing the level of GPR-1/2 on the cortex (Tsou et al., 2003). Although little is known about the downstream effectors of G, it has been hypothesized that the minus-end MT motor dynein-dynactin might be the force generator activated by G (Grill et al., 2003). Both the rotation of the spindle and the posterior spindle displacement at anaphase are dependent on the cortical PAR polarity (Colombo et al., 2003), together with region-specific cortical interactions with astral MTs. Mutations in genes that result in short astral MTs, such as tbb-2 (tubulin β-subunit; Wright and Hunter, 2003), zyg-8 (a doublecortin-related kinase; Gonczy et al., 2001), and zyg-9 (the XMAP215 ortholog; Matthews et al., 1998), all fail to align the mitotic spindle properly. How MT length is regulated during the cell cycle in order to execute different spindle alignment processes is not well understood.

A factor that might contribute to the regulation of spindle alignment is membrane trafficking. Studies in Fucus (Shaw and Quatrano, 1996) and the EMS cell of C. elegans (Skop et al., 2001) have shown that treating the embryos with the secretion inhibitor brefeldin A (BFA) inhibits rotational alignment of the spindle. In the case of C. elegans, it is not clear whether BFA prevents spindle rotation in the EMS cell by perturbing the P2/EMS signaling (such as Wnt and MES-1/SRC-1 pathways) that act to polarize EMS (Walston and Hardin, 2006) or by affecting the spindle alignment process directly. Furthermore, when either of two C. elegans ER proteins, OOC-3 (a putative transmembrane protein) and OOC-5 (a Torsin-related AAA ATPase), are mutated, the majority of the embryos exhibit P1 spindle rotation defect, caused by either disrupting the polarization of the P1 cell or the organization of actin cytoskeleton at the midbody remnant (Pichler et al., 2000; Basham and Rose, 2001).

To understand further how membrane trafficking may affect spindle alignment, we examined the functions of the Rab family proteins in C. elegans one-cell embryos (P0) in which spindle alignment is cell autonomous (Goldstein, 2000) and is well studied (Cowan and Hyman, 2004a). Rab proteins regulate the specificity of membrane trafficking by localizing to the cytosolic surface of distinct membrane compartments and facilitating all stages of membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion (Zerial and McBride, 2001). In this report, we focus on Rab11, which localizes to recycling endosomes (RE) and is required for both constitutive and regulated protein recycling from RE to the plasma membrane (PM), as well as transporting de novo synthesized proteins from the trans-Golgi network (TGN) to the PM in mammalian cells (Prekeris, 2003). Rab11 achieves its function by interacting with Rab11-FIPs (Rab11 family of interacting proteins; Prekeris, 2003), which colocalize to RE with Rab11 (Hales et al., 2001; Lindsay et al., 2002; Wallace et al., 2002; Horgan et al., 2004). In addition to regulating endocytic recycling, Rab11 is involved in several cellular functions, such as cell migration and cytokinesis (Skop et al., 2001; Pelissier et al., 2003; Jones et al., 2006). Both events require coordination between targeted delivery and fusion of membranes as well as cytoskeletal rearrangements. Rab11 has been shown to function in membrane trafficking but whether it is involved directly or indirectly in the cytoskeletal rearrangements in these processes is not known. Recent studies have shown that Rab11 can indeed affect the cytoskeleton. In Drosophila, Rab11 organizes MT plus ends during oogenesis (Dollar et al., 2002) and remodels the actin cytoskeleton during cellularization (Riggs et al., 2003), although the detailed mechanisms are unknown. Our study identifies a new role for RAB-11 (the C. elegans ortholog of mammalian Rab11a) in regulating the cytoskeleton, namely to facilitate astral MT elongation during metaphase to ensure proper spindle alignment in the first cell division. Also, we show that RAB-11 is required for the normal endoplasmic reticulum (ER) morphology during metaphase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
C. elegans Strains
All worm strains were maintained as described (Brenner, 1974). The following strains were used: N2: wild type (WT), WH0204: unc-119(ed3); ojIs1[beta- tubulin::GFP unc-119(+)], WH0327: unc-119(ed3); ojIs23[SP12::GFP unc-119(+)], WH0347: unc-119(ed3); ojIs35 [rab-11.1:: GFP unc-119(+)], TH66: EBP-2::GFP, WH0258: unc-119(ed3); ojIs5[dnc-2::GFP unc-119(+)], DH235: zyg-8 (b235)III, SP432: unc-4(e120) ooc-3(mn241)/mnC1 dpy-10(e128) unc-52(e444)II (Kemphues et al., 1988), and KK696: ooc-5(it145) unc-4(e120)/mnC1 dpy-10(e128) unc-52(e444) II. We grew SP432 (ooc-3(mn241)) and KK696 (ooc-5(it145)) worms at 16°C and used the uncoordinated worms, which are homozygous for the mutations for analysis. DH235 (zyg-8 (b235)) was also maintained at 16°C and L4s shifted to 25°C overnight before analysis. TH66 (EBP-2::GFP) was maintained at 20°C and L4s were shifted to 25°C for both control and RNAi treatment for 30–32 h before imaging.

RNA Interference Treatment
The rab-11 feeding vector pRrab-11 was constructed by cloning the full-length rab-11 cDNA (F53G12.1/yk1108c6) into the feeding vector L4440 and then transformed into HT115 bacteria (Timmons and Fire, 1998). RNA interference (RNAi) experiments were performed as described (Fire et al., 1998; Timmons and Fire, 1998). Fifteen N2 L4 worms were put on each 30-mm plate and fed for at least 44 h at 20°C before analysis. Green fluorescent protein (GFP)-expressing worms were fed for 30–40 h before analysis as they became sterile after 42 h of feeding. To get same amount of RNAs for RNAi against two genes, we constructed double feeding vectors with both genes between the T7 promoter sites of L4440. The double feeding vectors, pRrab-11&par-2, pRrab-11&par-3, pRrab-11&gpr-1/2, pRrab-11&dnc-2, pRrab-11&let-99, and pRrab-11&gpb-1 were made by amplifying each individual gene with the SpeI site added to the primer ends. These PCR fragments were inserted into the pRrab-11 feeding vector cut with the SpeI site. The following cDNAs or genomic DNA were used: par-2: coding region from 1 to 1200 base pairs from yK325e4; par-3: coding region from 650 to 2030 base pairs from yk552e12; gpr-1/2: full-length yk645d1; dnc-2: full-length genomic DNA; let-99: full-length yk262g2; and gpb-1: coding region from 1 to 820 base pairs from yk325g7. Except for rab-11&par-2 RNAi, all the RNAi experiments were carried out by feeding 10 or 15 N2 L4s at least 40 h before analysis. For rab-11&par-2 RNAi, double-strand RNA (dsRNA) was produced using the in vitro T7 transcription Kit (Ambion, Austin, TX). 1 mg/ml dsRNA was injected into N2 young adults and analyzed 36 h later. Full-length rab-11 3' untranslated region (UTR) was amplified and cloned into the L4440 vector. Both N2 and WH347 (RAB-11::GFP) strains were fed at the same time for at least 40 h before imaging or counting dead embryos. The zyg-9 feeding vector was from Ahringer's feeding library (Kamath et al., 2003).

Live Imaging
Because rab-11(RNAi) embryos were sensitive to pressure and osmotic strength (data not shown), embryos were mounted in a hanging-drop blastomere culture medium (Shelton and Bowerman, 1996) for imaging. Worms were cut open in 3 µl blastomere culture medium on the coverslip. A slide with a circle of Vaseline was then pressed onto the coverslip to form a sealed chamber. Four-dimensional Nomarski imaging was performed as described previously (Skop and White, 1998). We used a Nikon Optiphot-2 upright microscope with a Nikon PlanApo 60 x 1.4 NA differential interference contrast (DIC) lens (Melville, NY) and a Hamamatsu C2400 CCD cameras (Hamamatsu Photonics, Hamamatsu City, Japan) or a Nikon Diaphot300 inverted microscope with a 60 x 1.4 NA DIC lens (Melville, NY) and a Sony XC-75 CCD camera (Tokyo, Japan). All GFP images were collected using multiphoton excitation on an optical workstation (Wokosin et al., 2003), which consists of a Nikon Eclipse TE300DV inverted microscope with a Nikon Super Fluor 100 x 1.3 NA lens. The excitation source is a Ti:sapphire laser (Spectra Physics, Mountain View, CA) tuned to 890 nm. The detector is a high quantum efficiency Hamamatsu H7422-40 detector. Except for EBP-2::GFP, images were collected at 512 x 512-pixel resolution at 4.5-s intervals and analyzed with ImageJ v. 1.34s (http://rsb.info.nih.gov/ij/). Images for TH66 (EBP-2::GFP) were collected at 256 x 256-pixel resolution at 0.89-s interval with room temperature at 18°C. The posterior part of the embryos was zoomed in for a better visualization.

Immunohistochemistry
rab-11(RNAi) and WT worms were cut open in blastomere culture medium. Embryos labeled for membrane structures (anti-RAB-11 and anti-HDEL) were prepared as previously described with slight modification (Gonczy et al., 1999b). Fifteen worms were cut open in 15 µl H₂O on a subbed slide. An 18-mm coverslip was placed onto the drop and excess fluid was wicked away with 3MM Whatman paper (Clifton, NJ). The slides were frozen on a metal block in a –80°C freezer for 5 min. After removing the coverslips, the slides were fixed in 100% methanol at –20°C for 15 min. Slides were rehydrated in 1x phosphate-buffered saline (PBS) for 5 min and incubated with 50 µl of primary antibody in PBS for 45 min at room temperature. After the incubation, slides were washed for 5 min in PBT (PBS-0.05% Tween 20), 5 min in PBS, and incubated as described above for 45 min with the secondary antibodies. Slides were washed twice with PBS for 5 min before mounting in 7 µl mounting media (Vectashield; Vector Laboratories, Burlingame, CA). Embryos labeled with the anti-ZYG-8 antibody were fixed in 100% methanol at –20°C for 1 min (P. Gonczy, personal communication). All other antibody labeling was performed using another published protocol (Skop and White, 1998). Staining of WT and rab-11(RNAi) embryos was carried out under the same conditions for each antibody. Antibodies were diluted as follows: DM1, mouse anti--tubulin, 1:100 (Sigma, St. Louis, MO); rabbit anti-PIE-1, 1:100; rabbit anti-GPR-1/2, 1:200; rabbit anti-ZYG-8, 1:200; rabbit anti-PAR-2 (e3), 1:5; and mouse anti-PAR-3 (P4A1), 1:5 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA); mouse anti-HDEL, 1:20; rabbit anti-RAB-11, 1:200; and rabbit anti-ZYG-9, 1:40. Secondary antibodies were as follows: goat anti-mouse Alexa 568, 1:200 and goat anti-rabbit Alexa 488, 1:200 (Molecular Probes, Eugene, OR). DNA was labeled using Topro3 (1 mM, 1:500; Molecular Probes, Eugene, OR) and DAPI (1.5 µg/ml, Vectashield). Slides were viewed on a Bio-Rad MRC1024 confocal microscope (Hercules, CA); instrument settings were the same for both WT and experimental embryos within each staining procedure. Images were prepared for publication with Adobe Photoshop (version 8.0, San Jose, CA).

Measure MT Dynamics in rab-11(RNAi) and zyg-8(b235) Embryos
MT length during metaphase in fixed WT, zyg-8(b235) and rab-11(RNAi) embryos labeled with the anti--tubulin antibody was measured as previously described (Gonczy et al., 2001). Namely, the three longest MTs projecting toward the anterior cortex in one optical section from each of five embryos were chosen to measure their length. "MT-cortex gap" refers to the mean distance between the anterior cortex and the plus ends of the longest astral MTs. The posterior astral MTs were adjacent to the posterior cortex in both WT and rab-11(RNAi) embryos. The MT growth rate and nucleation rate in rab-11(RNAi) embryos were calculated as described (Srayko et al., 2005). Because of the violent movements of both centrosomes during anaphase in rab-11(RNAi) embryos, all the measurements were performed with the metaphase MTs and centrosomes. Tracking of EBP-2::GFP was performed manually in ImageJ v. 1.34s. Because some portion of the astral MTs were attached by extra chromosomes because of the polar body extrusion defect, these MTs no longer underwent active growing in rab-11(RNAi) embryos. To make more accurate measurements, a one-quarter circle (instead of a half-circle) was drawn 3.8 µm away from the posterior centrosomes to calculate the MT nucleation rate in movies of 100 frames (89 s). ImageJ v. 1.34s was used to generate the kymograph. The same 89-s movies were also projected into a single image using ImageJ v. 1.34s to reveal the paths of many EBP-2::GFP dots. Student's t test with two-tailed unequal variance was used to determine whether the differences of the MT length, MT growth, and nucleation rate between WT and rab-11(RNAi) embryos were statistically significant.

RAB-11::GFP
Full-length RAB-11 was amplified from genomic DNA and cloned into the plasmid pFJ1.1 (Squirrell et al., 2006). The plasmid was then bombarded into unc-119 worms as described (Praitis et al., 2001). Worms were allowed to grow for at least 2 wk. The rescued worms were examined for GFP expression.

Drug Treatments
Nocodazole treatment was carried out as described (Encalada et al., 2005). β-tubulin::GFP or WT worms were cut open on the gasket slide in 5 µl 50 µg/ml to 100 µg/ml nocodazole (Invitrogen, Carlsbad, CA) in egg buffer (Skop and White, 1998). The same dilution of DMSO was used as a control. The coverslip was immediately put on the top of the gasket slide and sealed with petroleum jelly. Embryos undergoing pronuclear migration or centration were imaged either on the multiphoton workstation (β-tubulin::GFP) or with Nomarski optics (N2 embryos). BFA (Invitrogen) treatment was carried out by soaking young embryos, before eggshell formation (usually during meiosis II) in hanging drops containing 150 µg/ml BFA diluted in egg buffer. The same dilution of DMSO was used as control.

Supplemental Data
Supplemental Data including Figures S1 and S2, Tables S1 and S2, and Supplemental Videos are available online.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
rab-11(RNAi) Embryos Exhibit Spindle Alignment Defects
It has been previously reported that partial knockdown of RAB-11 by RNAi causes cytokinesis failure (Skop et al., 2001). We found that prolonged RNAi treatment (>44 h) also caused additional abnormalities evident during the first cell cycle, including osmotic sensitivity, failure to extrude polar bodies (52%, n = 23), no or minimal pseudocleavage (83%, n = 12), and failure of the centrosomal-nuclear complex to migrate to the center of the embryos (57%, n = 23). Most strikingly, the P0 spindle failed to rotate to the A-P axis (the angle of the spindle to the A-P axis was 45–90°; 73.9%, n = 23), and the spindle exhibited abnormal displacement during anaphase (100%, n = 23). In WT embryos, the anaphase spindle elongates and moves smoothly toward posterior, with a slight rocking of the posterior centrosome (Figures 1A and 3A; Video 1). By contrast, during the metaphase to anaphase transition, we found that the mitotic spindle underwent violent movements in 82.6% of the rab-11(RNAi) embryos (n = 23): the spindle migrated to the posterior pole and then rebounded to the A-P axis (Figures 1B and 3B; Video 2, top). In the remaining embryos (17.4%), both centrosomes rocked extensively and the spindle was displaced further toward the posterior pole (see Figure 3C; Video 2, bottom). The violent spindle movements are specific to RAB-11 depletion, because disruption of other RAB genes (Table S1), known endosome regulators such as RME-1 (Grant et al., 2001) or other osmotic sensitive mutants such as pod-1 and pod-2 (Tagawa et al., 2001) did not exhibit similar phenotypes.

View larger version (54K): [in this window] [in a new window]   Figure 1. rab-11(RNAi) embryos undergo violent spindle movements during one-cell anaphase. Time-lapse differential interference contrast (DIC) microscopy recordings of the first cell division of WT embryo (A, Video 1), and rab-11(RNAi) embryo (B, Video 2) after 44 h of feeding from prometaphase (t = 0) to the end of the first division. Blue dots, anterior centrosomes; red dots, posterior centrosomes. In the rab-11(RNAi) embryo, the P0 spindle did not align along the anterior-posterior axis of polarity and the posteriorly positioned centrosome (labeled with red dot) ended up in an anterior location at the end of anaphase due to the violent spindle movements. The anterior positioned centrosome was out of focus at some time points. Cytokinesis of the first division failed to complete in the rab-11(RNAi) embryo. Scale bar, 10 µm.

View larger version (61K): [in this window] [in a new window]   Figure 2. Polarity defects in rab-11(RNAi) embryos. WT (A, C, E, and G) and rab-11(RNAi) (B, D, F, and H) one-cell embryos stained with anti-PAR-3 (A and B), anti-PAR-2 (C and D) antibodies and Topro3 (E and F) to reveal DNA. Arrowheads mark the boundary of the PAR-2 domain. In the merged images (G and H), PAR-3 is red, PAR-2 is green, and DNA is blue. Arrows indicate the boundary of the overlapping region of PAR-2 and -3 in rab-11(RNAi) embryos. Note the extra pronucleus indicating the polar body was not extruded in the rab-11(RNAi) embryo. (I) Quantification of the egg length of PAR-2 and -3 domains before pronuclear centration in WT and rab-11(RNAi) embryos. n = 6 embryos for each measurement. Error bars, SD. Statistically significant difference between WT and rab-11(RNAi) embryos; *p < 0.05; Student's t test, two-tailed unequal variance. For PAR-2, p = 0.00016; PAR-3, p = 0.011. (J and K) Immunofluorescence staining of PIE-1 (red) in WT (J) and rab-11(RNAi) (K) embryos during pronuclear migration. DNA (blue) is labeled with Topro3. Scale bar, 10 µm.

View larger version (37K): [in this window] [in a new window]   Figure 3. G/GPR-1/2 activity may not be up-regulated in rab-11(RNAi) embryos. Schematic representations of the movements of one or both centrosomes in WT (A, Video 1), rab-11(RNAi) (B and C, Video 2), rab-11; par-3(RNAi) (D, Video 3, top), rab-11; let-99(RNAi) (E, Video 4, top), rab-11; gpb-1(RNAi) (F, Video 4, bottom), rab-11; par-2(RNAi) (G, Video 3, bottom), rab-11; gpr-1/2(RNAi) (H, Video 5, top), and rab-11; dnc-2(RNAi) embryo (I, Video 5, bottom). Each drawing is from a single representative embryo. These trajectories describe the path of centrosome movement from prometaphase to the end of anaphase (dots). The dots are not at the same time intervals. For rab-11; let-99(RNAi) and rab-11; gpb-1(RNAi) embryos (E and F), because the violent movements of the centrosomes begin before that observed in rab-11(RNAi), the traces of movements start from pronuclear centration (asterisks). In embryos whose spindles underwent violent movements, only the posterior centrosomes are shown as the anterior centrosomes were frequently out of the plane of focus. In the rab-11; par-2(RNAi) (G) and rab-11; dnc-2(RNAi) (I) embryos, the P0 spindles first set up perpendicular to the longitudinal axis. In rab-11; dnc-2(RNAi) embryo, the spindle failed to centrate due to disruption of DNC-2 activity. During anaphase, the spindles elongated and flipped to the longitudinal axis due to the constraints of the eggshell. (J and K) Immunofluorescence staining of GPR-1/2 (red) in WT (J) and rab-11(RNAi) (K) embryos during one-cell metaphase. DNA (blue) is labeled with Topro3. Arrows delineate regions of GPR-1/2 enrichment at posterior cortex. In the rab-11(RNAi) embryo, the P0 spindle did not rotate and the chromosomes did not align properly along the metaphase plate. Immunolabeled rab-11(RNAi) embryos are larger because they are pressure sensitive and flattened more than WT embryos during fixation. Scale bar, 10 µm.

Violent Spindle Movements in rab-11(RNAi) Embryos Are Subjected to G/GPR-1/2 Regulation

MT Dynamics during Metaphase Are Altered in rab-11(RNAi) Embryos
Because MT dynamics are likely to play a crucial role in spindle orientation and movements, we examined the MTs in rab-11(RNAi) embryos. Immunofluorescence staining of MTs and imaging of EBP-2::GFP (the worm EB1 homolog that labels the growing MT plus ends) revealed that astral MT organization was altered in rab-11(RNAi) embryos. Although the anaphase MTs were normal in rab-11(RNAi) embryos, during metaphase aspects of MT dynamics, such as the distance between the plus ends of the MTs and the anterior cortex, MT growth, and nucleation rates (Table 1 and Figure 4, B and E), were altered compared with WT. During metaphase the distance between MT plus ends and the cortex was significantly greater in rab-11(RNAi) embryos than WT, probably because of the reduced MT length: 14.0 ± 1.6 µm in rab-11(RNAi) embryos compared with 22.1 ± 4.4 µm in WT (p < 0.001), although at anaphase, the MT ends contacted the cortex in both cases (Table 1). In addition, fewer growing astral MTs reached to the cortex during metaphase in rab-11(RNAi) embryos (n = 3/3) than in WT (Figure 4H, Video 6), suggesting the catastrophe rate may also be higher in rab-11(RNAi) embryos, although this rate cannot be measured directly using the EBP-2::GFP marker. This observation indicates that the inability of MT to reach the cortex in metaphase in these rab-11(RNAi) embryos may result from both slow growth and increased MT depolymerization.

View larger version (101K): [in this window] [in a new window]   Figure 4. rab-11(RNAi) and zyg-8(b235) embryos exhibit different MT length defects during one-cell division. (A–C) Metaphase MTs stained with anti--tubulin antibody in WT (A), rab-11(RNAi) (B), and zyg-8(b235) (C) embryos. (D--F) Anaphase MTs stained with anti--tubulin antibody in WT (D), rab-11(RNAi) (E), and zyg-8(b235) (F) embryos. (G and H) Projections of 100-frame EBP-2::GFP movies (89 s in length) in WT (G, Video 6, left) and rab-11(RNAi) (H, Video 6, right) embryos during metaphase (see Materials and Methods). Very few MTs were seen to grow out to the cortex in the rab-11(RNAi) embryos (H). Notice that EBP-2::GFP accumulation at the kinetochore–MT interface was also reduced in rab-11(RNAi) embryos. The significance of this defect is not known. (I and J) Immunofluorescence staining of ZYG-8 in WT (I) and rab-11(RNAi) (J) embryos. ZYG-8 localization to the spindle and centrosomes was normal in the rab-11(RNAi) embryos, although the cytoplasmic staining seemed to be reduced in rab-11(RNAi) embryo. Scale bar, 10 µm.

We investigated next whether short metaphase MTs were sufficient to drive the violent spindle movements seen in the rab-11(RNAi) embryos by using the MT-depolymerizing drug nocodazole. The nocodazole dose and time of application were adjusted such that astral MTs in treated embryos were shortened during metaphase, yet the drug effect wore off during anaphase so that the astral MTs resumed elongation. We found that although the spindle movements in control embryos were normal (Figure 5A; Video 7), in embryos treated with nocodazole in this manner, the spindles underwent "rab-11-like" movements: either moving to the posterior (n = 3/7) or with both centrosomes rocking extensively (n = 4/7; Figure 5, B and C; Videos 8 and 9). This observation further supports the notion that a polarity defect in rab-11(RNAi) embryos does not necessarily contribute to the violent spindle movements because nocodazole treatment does not affect polarity (Cowan and Hyman, 2004b) yet is sufficient to generate the violent spindle movements.

View larger version (32K): [in this window] [in a new window]   Figure 5. Nocodazole treatment can phenocopy the violent spindle movements in rab-11(RNAi) embryos. Multiphoton time series of embryos expressing β-tubulin::GFP show the following developmental stages: metaphase, early anaphase, late anaphase, and telophase. (A) β-tubulin::GFP embryos treated with the same dilution of DMSO (Video 7). (B and C) β-tubulin::GFP embryos treated with 50 µg/ml nocodazole during pronuclear migration or centration (Videos 8 and 9). Note that the metaphase MTs were shortened by nocodazole, but they resumed elongation during anaphase. Centrosomal positions that best describe the trace of the movements from metaphase to telophase are shown in the schematic drawings (blue dots, anterior centrosomes; red dots, posterior centrosomes; dots are not at the same time intervals). Scale bar, 10 µm.

View larger version (70K): [in this window] [in a new window]   Figure 6. RAB-11 colocalizes extensively with the ER. Metaphase (A) and anaphase (B) localization of RAB-11::GFP. One-cell anaphase embryo labeled with anti-RAB-11 (C), anti-HDEL (F), and merged (H). Four-cell embryo with ABa and ABp at anaphase, P2 and EMS at metaphase labeled with anti-RAB-11 (D), anti-HDEL (G), and merged (I). Arrow points to a structure unique to anti-RAB-11 labeling, and arrowhead to a structure unique to anti-HDEL labeling. In rab-11(RNAi) embryo (E), the anti-RAB-11 labeling is greatly reduced. Scale bar, 10 µm.

View larger version (81K): [in this window] [in a new window]   Figure 7. Metaphase ER morphology is disrupted in rab-11(RNAi) embryos. Immunofluorescence staining of the ER (anti-HDEL, red) and Topro3 staining of DNA (blue) in metaphase and anaphase WT embryos (A and B), rab-11(RNAi) embryo (C and D), and zyg-8(b235) mutant embryos (E and F). Arrows mark the positions of the mitotic spindle poles. Arrowhead shows one of the ER clumps. (G and H) Multiphoton time series of embryos expressing SP12::GFP show the following developmental stages: prometaphase (t = 0), metaphase, early anaphase, and late anaphase. Arrowhead shows one of the ER clumps in metaphase. Scale bar, 10 µm.

View larger version (92K): [in this window] [in a new window]   Figure 8. Microtubule and ER morphologies for gene disruptions and BFA treatment listed in Table 2. (A and B) Immunofluorescence staining of the ER (anti-HDEL, red) and Topro3 staining of DNA (blue) in ooc-3(mn241) mutant embryos during metaphase (A) and anaphase (B). Although previous work indicated that that the ER structure was less affected in ooc-5(it145) mutant embryos than in ooc-3(mn241) embryos (Basham and Rose, 2001), we observed a similar defect of ER morphology in these embryos (data not shown). Arrows mark the position of the mitotic spindle poles. Arrowheads show one of the ER clumps. (C and D) Immunofluorescence staining of the microtubules (red) and Topro3 staining of DNA (blue) in ooc-3(mn241) mutant embryos during one-cell metaphase (C) and anaphase (D). ooc-5(it145) mutant embryos showed similar phenotype (data not shown). ooc-3(mn241) and ooc-5(it145) mutant embryos are often smaller than the WT embryos (Basham and Rose, 1999). Scale bar, 10 µm. (E and F) SP12::GFP embryos treated with let-99 RNAi showed that ER morphology was not affected during either metaphase (E) or anaphase (F). (G and H) SP12::GFP embryos treated with zyg-9 RNAi showed that ER morphology was similar to that observed with nocodazole or tba-2(RNAi) treatment (Poteryaev et al., 2005). The accumulation of ER structure at the anterior during anaphase may be due to the exaggerated cytoplasmic flow resulting from short microtubules. (I and J) BFA treatment caused nuclear-centrosomal complex rocking during pronuclear centration. Nomarski images show the centrosome movements during pronuclear centration and the final stage of the first division (the last time point). (I, Video 10, top) WT meiosis II embryos treated with same dilution of DMSO as control (n = 15). (J, Video 10, bottom) WT meiosis II embryos treated with 150 µg/ml BFA (n = 14). Arrows mark the positions of the two centrosomes. Schematic representations of the movements of the posterior centrosome during pronuclear centration are drawn. Scale bar, 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Short Astral MTs at Metaphase Contribute to Violent Spindle Movements during Anaphase
Both rab-11(RNAi) and zyg-8 mutant embryos exhibit violent spindle movements during anaphase in the P0 cell. In zyg-8(t1650) animals this violent spindle movement is caused by the failure of astral MT elongation during anaphase (Gonczy et al., 2001), whereas in rab-11(RNAi) embryos the lengths of the anaphase astral MTs were normal and the metaphase astral MTs were much shorter compared with those in WT embryos. How do short astral MTs at metaphase cause violent spindle alignment during anaphase?

In WT embryos a posterior pulling force acts on the spindle during late prophase and prometaphase but is balanced by the tethering of astral MTs at the anterior cortex (Labbe et al., 2004; Figure 9A). We propose that the short astral MTs observed during metaphase in rab-11(RNAi) embryos cannot be tethered by the anterior cortex; thus the spindle becomes prematurely subjected to the posterior pulling force. This posterior pulling force may be exerted on the spindle by the few astral MTs that do manage to reach to the posterior cortex and can be captured and shortened by the active force generators (e.g., dynein-dynactin; Figure 9B). Indeed, the number of the active force generators that displace the anaphase spindle in the WT embryo may be as few as 50 throughout the embryo, with more being present on the posterior cortex (Grill et al., 2003). We hypothesize that interactions between cortex and the many anterior astral MTs may function to counter the posterior pulling force in normal situations. Both reduction of the astral MT length and number are necessary to reach a certain threshold in order to destabilize the balance and generate the violent spindle movements that we observed. The observation that nocodazole-treated embryos showed a similar range of defects supports this hypothesis.

View larger version (21K): [in this window] [in a new window]   Figure 9. Model for the violent spindle movements in rab-11(RNAi) embryos. In the WT embryo (A), the posterior pulling force at metaphase is balanced by the tethering of astral MTs at the anterior cortex (Labbe et al., 2004). In the rab-11(RNAi) embryo (B), the metaphase MTs are short and no longer stabilized at the anterior, subjecting the spindle only to the pulling force from the posterior. The longer MT represents the few that do manage to reach to the posterior cortex where more abundant GPR-1/2 localizes, so that dynein-dynactin (shown in green dots) can exert pulling forces on the spindle. In the rab-11; par-3(RNAi) embryo (C), GPR-1/2 level is evenly elevated around the cortex (Colombo et al., 2003; Gotta et al., 2003). The spindle is then under equal pulling forces from both poles, and it undergoes violent movements in the center of the embryo. In the rab-11; let-99(RNAi) or rab-11; gpb-1(RNAi) embryo (D), the GPR-1/2 activity may be further up-regulated due to the disruption of these antagonizing regulators of G (Tsou et al., 2003), which results in even stronger pulling forces. Furthermore, because the embryo is still polarized, unlike in the rab-11; par-3(RNAi) embryo, the pulling forces may be unbalanced (not shown in the diagram) and thus lead to more violent and unpredictable spindle movements. The pulling force from the cortex is suppressed in the rab-11; par-2(RNAi) embryo (E) due to reduced GPR-1/2 activity (Colombo et al., 2003; Gotta et al., 2003). In the rab-11; gpr-1/2(RNAi) embryo (F), both GPR-1/2 and dynein-dynactin activities are reduced (Grill et al., 2003), accounting for the absence of pulling force from the posterior cortex. Inactivating the motor dynein-dynactin in rab-11; dnc-2(RNAi) embryo (G) also suppresses the pulling force from the cortex even though the GPR-1/2 activity may be normal.

RAB-11 Can Colocalize with and Contribute to the Organization of the ER
Rab11 localizes mainly to the peri-centrosomal region in interphase Chinese hamster ovary (CHO) cells with a lower concentration of puncta distributed throughout the cell (Ullrich et al., 1996), whereas in polarized MDCK cells during mitosis, Rab11 forms diffuse puncta in the cytosol during prophase and then becomes clustered near the spindle poles after metaphase; this spindle pole accumulation increases throughout telophase (Hobdy-Henderson et al., 2003). However, we found that C. elegans RAB-11 overlaps with ER extensively and is required for normal ER morphology specifically during metaphase. Although the astral MTs were short during metaphase in rab-11(RNAi) embryos, the perturbation in ER morphology that we observed is probably not a secondary effect of the MT disruption, as seen in a number of organisms (Voeltz et al., 2002), because disrupting the MT cytoskeleton by nocodazole, tba-2(RNAi) (Poteryaev et al., 2005) or zyg-9(RNAi) (this work) does not affect the ER morphological changes in C. elegans early embryos.

Why Are Astral MTs Shorter in rab-11(RNAi) Embryos than in WT at Metaphase?
One possible explanation is that RAB-11 (possibly in conjunction with its RE binding partner FIP3; see Supplementary Data) is required for delivery of proteins that can modulate MT lengths (such as MT-associated proteins [MAPs]) to astral MTs via the REs. When we examined the localizations of several known MAPs in C. elegans early embryos, such as EBP-2, ZYG-8 and -9, and dynactin (DNC-2), none were affected by RAB-11 depletion (Figure 4, H and J, and data not shown); however, some other MAPs may be involved. It is possible that changes in the cortical cytoskeleton contribute to the reduction of the observed MT length. However, we did not observe any significant perturbation in the distribution of cortical myosin before cytokinesis (Supplemental Data).

It may be significant that both astral MT length and ER morphology were affected in rab-11(RNAi) embryos predominantly during metaphase. This observation, together with the extensive colocalization between RAB-11 and ER that was seen, suggests that RAB-11 could mediate the cell-cycle–specific regulation of MT length through its effect on the ER. One possibility is that proteins regulating metaphase MT length could be processed and transported via the ER, whose normal morphology requires functional RAB-11 (this study). Our failure to demonstrate any mislocalization of MAPs in RAB-11 depleted embryos (see above) makes this possibility somewhat less likely.

It has been shown that ER-regulated calcium levels can alter MT dynamics (Facanha et al., 2002). Thus, as the ER cycles from its reticulate organization during metaphase to dispersed structures during anaphase (Poteryaev et al., 2005), it could provide cell-cycle–specific regulation of free calcium levels, which would affect MT growth or stability. In this scheme, RAB-11 could act directly in calcium regulation (such as interacting with a Ca²⁺ channel; van de Graaf et al., 2006) or indirectly (by affecting ER morphology, thereby regulating calcium levels in microdomains) to stabilize metaphase astral MTs.

When the ER disperses at the metaphase-to-anaphase transition, MTs become subject to different forms of cell-cycle–dependent regulation, such as by ZYG-8. Interestingly, we found that the ER also forms large aggregates during metaphase in zyg-8(b235) embryos. However, unlike in rab-11(RNAi) embryos, these aggregates persisted through anaphase. If the ER is regulating MT length, this may explain why zyg-8 mutant embryos exhibit defects in anaphase MT assembly (Gonczy et al., 2001), as well as a slight reduction in the MT growth during metaphase (Srayko et al., 2005). Possibly the doublecortin ZYG-8 may change MT dynamics in correspondence to the ER cycle. Although it is not known whether ZYG-8 is associated with the ER, it has been found that the chicken ortholog of ZYG-8 is associated with membrane structures (Capes-Davis et al., 2005). In genes that affect MT length independently of the cell cycle, such as zyg-9 (Matthews et al., 1998), the ER morphology was normal when these genes were depleted (Figure 8, G and H).

Our observations indicate that the length of astral MTs during metaphase is at least partially determined by RAB-11. The stage-specific perturbation of ER structure seen upon RAB-11 depletion suggests that the ER may in some way determine the length of astral MTs at metaphase. However, the depletion of RAB-11 must perturb the ER in a specific way because depletion of other proteins, such as CAR-1, can produce superficially similar disruptions of ER morphology without causing the shortened astral MTs or spindle alignment defects observed with RAB-11 depletion (Squirrell et al., 2006). Furthermore, depletion of the ER proteins OOC-3 and -5 as well as BFA treatment can cause perturbations of ER structure and spindle alignment defects without any concomitant shortening of MTs during metaphase. We should also mention that a normal ER organization alone is not sufficient to permit proper spindle alignment. For genes required for spindle alignment that regulate the interactions between the astral MTs and cortex, such as let-99 (Tsou et al., 2002; Tsou et al., 2003) and the trimeric G proteins (Labbe et al., 2003), the ER morphology was normal when these genes were depleted. However, given that nocodazole-mediated shortening of MTs during metaphase is sufficient to phenocopy the spindle alignment phenotype of RAB-11 depletion, we speculate that RAB-11 acts permissively (possibly via the ER) to specify an appropriate length for astral MTs at metaphase to allow the cortical interactions that mediate the alignment of the mitotic spindle.

	ACKNOWLEDGMENTS

 
We thank Koen Verbrugghe and Sebastian Bednarek for discussion and comments on the manuscript. We are grateful to Drs. Yuji Kohara and Dmitry Poteryaev and to Anne Spang, Chris Malone, Susan Strome, Geraldine Seydoux, Ken Kemphues, Tony Hyman, Pierre Gönczy, Iva Greenwald, Yun Chi, David Ron, and Judith Kimble, as well as the Caenorhabditis Genetics Center and Developmental Studies Hybridoma Bank for reagents and use of equipment. We also thank Bharti Solanki for constructing the gfp::nmy-2 strain used for the studies described in the supplement. This work was supported by the National Institutes of Health Grant GM052454-09 J.G.W.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-09-0862) on April 2, 2008.

Address correspondence to: John G. White (jwhite1{at}wisc.edu).

Abbreviations used: A-P, anterior-posterior; RNAi, RNA interference; G, subunit of the trimeric G protein; Gβ, β and subunit of the trimeric G protein; MT, microtubule; WT, wild-type; , deletion.

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