Depletion of RAB-5 results in an ER morphology defect similar to YOP-1/RET-1 depletion

A visual assay for formation of a tubular ER network from salt-washed ER-enriched membranes isolated from X. laevis oocytes revealed that tubule assembly requires GTP, in addition to Rtn4a and DP1/NogoA (Dreier and Rapoport, 2000; Voeltz et al., 2006). To investigate the origin of this GTP requirement, we compared the ability of X. laevis ER-enriched membranes to form tubules after incubation with either Rab or Rho GDI, which inhibit Rab or Rho family GTPases, respectively. Although Rho GDI failed to have any effect, addition of Rab GDI potently blocked ER tubule formation in vitro (Fig. 2 A). This result suggested that, in addition to YOP-1 and RET-1, ER structure is controlled by a Rab family GTPase.

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Figure 2.

Depletion of RAB-5 results in an ER morphology defect similar to depletion of YOP-1/RET-1. (A) Salt-washed membrane vesicles isolated from X. laevis eggs were incubated in the absence (top left) or presence of GTP (top right) or in the presence of GTP and 30 μM Rho GDI (bottom left) or 10 μM Rab GDI (bottom right). Data are representative of four independent experiments for each condition. Bar, 5 μm. (B) A rooted phylogenetic tree based on a sequence comparison of selected Rab-type GTPases from S. cerevisiae, C. elegans, and Homo sapiens. Asterisks denote redundancy between RAB-8 and RAB-10 or RAB-6.1 and RAB-6.2, which must be codepleted to observe a phenotype. Lethality of the embryos produced by the dsRNA-injected mother (Emb) and/or the failure of the injected mother to produce a normal number of embryos (Ste) is indicated. (C) Spinning-disk confocal optics were used to image RAB-5–depleted embryos (rab-5[RNAi]; n = 21) expressing the ER marker GFP:SP-12, as in Fig. 1 B. Bar, 10 μm. Higher magnification (2×) views of a portion of the adjacent cortical sections are shown to the right. Bar, 5 μm. (D) Metaphase control (left) and rab-5(RNAi) (right) embryos were fixed and stained with antibodies to the endosome marker RAB-5 (top) and the Golgi marker SQV-8 (bottom). Images are projections of deconvolved 3D datasets. Bar, 10 μm. (E) The number of mitotic ER clusters (>0.5 μm in diameter) measured in a cortical section of embryos expressing GFP:SP-12 collected 10 s before anaphase onset is plotted for at least 16 embryos for each condition. (F) Representative cortical sections used for the quantification in E showing GFP:SP-12 in control (left), RAB-5–depleted (middle), and YOP-1– and RET-1–depleted (right) embryos 10 s before anaphase onset. Bar, 10 μm.

To investigate whether a Rab GTPase contributes to ER structural dynamics in vivo, we systematically depleted each of the 29 C. elegans Rabs (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200701139/DC1). This analysis identified six Rab activities necessary for embryo production and/or early embryogenesis (Fig. 2 B). Of these, only reduction of RAB-5 levels resulted in an effect on ER structure similar to depletion of YOP-1/RET-1 (compare Fig. 2 C with Fig. 1, B and C; Video 1). Depletion of RAB-5, which resulted in 100% embryonic lethality, inhibited both the formation of a reticular network of thick tubules and the appearance of mitotic ER clusters (Fig. 2, C, E, and F; and Videos 1 and 2). In contrast to the dramatic effect on ER structure, staining of control and RAB-5–depleted fixed embryos with antibodies against a Golgi marker, the glucuronyltransferase SQV-8, revealed that Golgi size and distribution were not altered by RAB-5 depletion (Fig. 2 D).

The role of RAB-5 in ER structure is independent of its requirement during endocytosis

Rab5 has an established role in endocytosis and early endosome fusion (Somsel Rodman and Wandinger-Ness, 2000; D'Hondt et al., 2000; Pfeffer, 2001; Zerial and McBride, 2001). Previous work in C. elegans has shown that these functions are mediated by two RAB-5 guanine nucleotide exchange factors (GEFs), RME-6 and RABX-5, which have overlapping functions (Sato et al., 2005). Examination of the ER in embryos mutant for rabx-5 in which RME-6 was depleted by RNAi revealed a structural defect essentially identical to that in rab-5(RNAi) embryos (Fig. 3, A and E), indicating that the role of RAB-5 in ER structure is also mediated by RME-6 and RABX-5.

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Figure 3.

RAB-5 structures the ER independently of known effectors. (A) Spinning-disk confocal optics were used to image GFP:SP-12–expressing rabx-5(tm1512) mutant embryos depleted of RME-6 by RNAi (rabx-5[tm1512], rme-6[RNAi]; n = 7), as in Fig. 1 B. Bar, 10 μm. Higher magnification (2×) views of a portion of the adjacent cortical sections are shown to the right. Bar, 5 μm. (B) Differential interference contrast and spinning-disk confocal optics were used to image GFP:SP-12–expressing control (n = 18), yop-1, ret-1(RNAi) (n = 13), rab-5(RNAi) (n = 21), and chc-1(RNAi) (n = 15) embryos. Representative differential interference contrast (left) and GFP:SP-12 fluorescence images (right) of a single central section are shown. Bar, 10 μm. (C) Mitotic control (top) and chc-1(RNAi) (bottom) embryos were fixed and stained with antibodies to RAB-5. Images are projections of deconvolved 3D datasets. Bar, 10 μm. (D) A table listing the two known C. elegans RAB-5 GEFs (pink; Sato et al., 2005) and the C. elegans homologues of RAB-5 effectors (purple; Deneka et al., 2003) and their human homologues. In cases where a mutant allele was available, time-lapse sequences of living embryos from the mutant strain acquired using differential interference contrast microscopy (n = 6 for each condition) were analyzed for the appearance of four nuclei after the first embryonic cytokinesis, a phenotype indicative of a defect in ER structure (for details, see Fig. 5). The four-nuclei phenotype was observed when both RAB-5 GEFs were simultaneously inhibited but not in mutants for any RAB-5 effector. (E) The number of mitotic ER clusters (>0.5 μm in diameter) measured in a cortical section of embryos expressing GFP:SP-12 collected 10 s before anaphase onset is plotted for at least six embryos for each condition.

To determine whether the effect of RAB-5 depletion on ER structure is an indirect consequence of its effect on endocytosis, we inhibited endocytosis by depleting other proteins that function in the early endocytic pathway, including clathrin heavy chain (CHC-1; Fig. 3 B), dynamin, and α-adaptin (not depicted). Depletion of CHC-1 redistributed clathrin light chain into the cytoplasm (not depicted) and resulted in a dramatic decline in cytoplasmic yolk granule density (Fig. 3 B), a hallmark of a pronounced defect in endocytosis during oocyte development (Grant and Hirsh, 1999). However, despite a reduction in yolk density similar to that in RAB-5–depleted embryos, ER morphology was not perturbed by CHC-1 depletion (Fig. 3 B). RAB-5 still localized to punctate endosomal structures in CHC-1–depleted embryos (Fig. 3 C), although the RAB-5 structures were enlarged and relatively more concentrated in the embryo anterior compared with controls. Examination of ER structure in embryos individually depleted of candidate RAB-5 effector proteins, including the C. elegans homologues of EEA-1, Rabenosyn-5, Rabaptin-5, and the catalytic subunit of a type 3 PI 3-kinase, hVps34 (Deneka et al., 2003), also failed to reveal any detectable alteration in ER morphology (Fig. 3, D and E).

Depletion of RAB-5 also did not substantially alter the appearance of the microtubule cytoskeleton (Fig. S1 C), which has been shown to play a role in the distribution of the ER within cells (Du et al., 2004; Vedrenne and Hauri, 2006). In addition, mitotic ER tubules and clusters still formed in nocodazole-treated embryos that lacked detectable microtubule polymer (Fig. S1 D). As previous work suggested that the actomyosin cytoskeleton also plays a role in ER structure (Poteryaev et al., 2005), we examined the distributions of GFP fusions with myosin II (GFP:NMY-2; Nance et al., 2003) and the actin binding domain of moesin (GFP:Moe), an established probe for filamentous actin in the C. elegans embryo (Motegi et al., 2006). In the period leading up to metaphase, the actin cytoskeleton appeared similar in control and depleted embryos (Fig. S1 E). A comparison of the distribution of GFP:NMY-2 in control, rab-5(RNAi), and chc-1(RNAi) embryos revealed that during the establishment of polarity, the organization of cortical myosin II was also similar under all three conditions (Fig. S1 F). During mitosis, the organization of GFP:NMY-2 in rab-5(RNAi) embryos, although distinct from that in controls, was similar to that in the chc-1(RNAi) embryos (Fig. S1 G). These results suggest that although compromised endocytosis, which is common to both rab-5(RNAi) and chc-1(RNAi) embryos, does alter the organization of cortical NMY-2, this mild perturbation is not responsible for the impact of RAB-5 depletion on ER structure.

Cumulatively, these findings indicate that the defect in ER structure in RAB-5–depleted embryos is not a consequence of a general perturbation of membrane trafficking, a disruption of microtubule–ER interactions, or a defect in the organization of the actomyosin cytoskeleton, and instead support the conclusion that RAB-5 has a specific and previously unappreciated role in ER morphology.

Activated RAB-5 potentiates the formation of mitotic ER clusters

Small GTPases cycle between an active GTP-bound form and an inactive GDP-bound form. To determine whether ER structure is sensitive to activation as well as inhibition of RAB-5, we constructed transgenic strains that stably expressed RFP^mCherry (McNally et al., 2006) fused to either wild-type RAB-5 or a mutant form of RAB-5 that dramatically slows GTP hydrolysis (Q78L). Expression of the RFP fusion with wild-type RAB-5 did not affect either endosome distribution or ER morphology (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200701139/DC1). Expression of the RFP fusion with RAB-5^Q78L, at ~25% of the level of endogenous RAB-5 (Fig. 4 A), reduced the normal anterior bias of endosomes in the polarized one-cell embryo but did not dramatically alter endosome morphology as assessed by examination of the localization of endogenous RAB-5 (Fig. 4 B), or a GFP fusion with the endosomal marker EEA-1 (Fig. S2 F). However, expression of RFP:RAB-5^Q78L had a striking effect on ER structure, monitored by coexpression with GFP:SP-12. More mitotic clusters that were also considerably larger than those in control embryos were observed (Fig. 4 C and Video 3). Additionally, the clusters failed to fully disperse after anaphase and often persisted into the following cell division (Video 3). Depletion of YOP-1 and RET-1 in embryos expressing RFP:RAB-5^Q78L revealed that YOP-1 and RET-1 are required for the dramatic changes in ER structure caused by expression of the dominant-active RAB-5 mutant (Fig. 4 C). These results demonstrate that ER structure is sensitive to either depletion or activation of RAB-5, exhibiting opposing responses to the two perturbations.

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Figure 4.

Activated RAB-5 potentiates the formation of mitotic ER clusters. (A) An anti–RAB-5 immunoblot of a serially diluted extract prepared from embryos stably expressing RAB-5^Q78L fused to RFP^mCherry. (B) Metaphase control (left) and RFP:RAB-5^Q78L–expressing (right) embryos were fixed and stained with antibodies to RAB-5. Projections of deconvolved 3D datasets are shown. Bar, 10 μm. (C) Embryos expressing GFP:SP-12 alone (n = 18), GFP:SP-12 and RFP:RAB-5^Q78L (n = 15), or GFP:SP-12 and RFP:RAB-5^Q78L that were also depleted of YOP-1 and RET-1 by RNAi (n = 11) were imaged using spinning-disk confocal optics. Representative images of a central plane are shown. Bar, 10 μm. (D) Embryos expressing GFP:SP-12 that were depleted of YOP-1 and RET-1 (n = 13); RAB-5 (n = 21); or YOP-1, RET-1, and RAB-5 (n = 10) were imaged by spinning-disc confocal microscopy. Representative images of a single central section are shown. Bar, 10 μm. (E) Higher magnification (2×) views of a portion of the images in D. Arrowheads highlight aberrant ER loops present during interphase and mitosis in the triple-depleted embryos. Bar, 5 μm.

Expression of mammalian Rab5A^Q79L also potentiated the formation of mitotic ER clusters in human HeLa cells. Compared with early C. elegans embryos, control HeLa cells exhibited relatively few mitotic ER clusters, as did cells expressing Rab5A^S34N, a dominant-negative version of Rab5A. In contrast, expression of wild-type Rab5A resulted in a small increase, and expression of Rab5A^Q79L resulted in a dramatic and consistent increase in the number of mitotic ER clusters >0.5 μm in diameter (Fig. S3, A and B). Cumulatively, these results suggest a conserved role for RAB-5 in controlling ER structure.

Simultaneous inhibition of RAB-5 and YOP-1/RET-1 exacerbates ER structure defects

Depletion of RAB-5 or YOP-1/RET-1 has a similar effect on ER structure. In addition, YOP-1/RET-1 are required for RFP:RAB-5^Q78L to exert a dominant effect. These results raised the possibility that YOP-1 and RET-1 are downstream effectors of RAB-5. To determine whether YOP-1 and RET-1 are likely to be direct effectors of RAB-5, we tested whether recombinant FLAG-tagged YOP-1 or RET-1 could bind to GST:RAB-5 in vitro. However, no substantial binding of either protein to GST:RAB-5 prebound to either GDP or GTPγS was observed (Fig. S3 C). A role for YOP-1/RET-1 as downstream effectors of RAB-5 would also predict that simultaneous depletion of YOP-1/RET-1 would not enhance the effect of RAB-5 depletion on ER morphology. However, simultaneous depletion of YOP- 1/RET-1 with RAB-5 resulted in a dramatic synergistic defect evident throughout the cell cycle (Fig. 4, D and E). In the triple-depleted embryos, ER morphology was aberrant in interphase, when GFP:SP-12 was present in numerous loop-shaped structures. These ER loops accumulated around the spindle poles as the triple-depleted embryos entered mitosis, leaving the embryo periphery nearly devoid of ER (Fig. 4 D). In addition, as in the individual depletions, the ER failed to coalesce into a network of thick tubules and clusters (Fig. 4 D and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200701139/DC1). Although it is possible that RAB-5 and YOP-1/RET-1 are on a single pathway and the synergistic defect in ER structure is due to a greater effectiveness in inhibiting this pathway when two of its constituents are depleted by RNAi, we favor the idea that the strong synergistic phenotype indicates that RAB-5 and YOP-1/RET-1 make distinct essential contributions to ER structure. It is worth pointing out, however, that the idea that RAB-5 and YOP-1/RET-1 each retain some function when the other is absent does not rule out the possibility that RAB-5 exerts its effect in part by altering the function of YOP-1/RET-1.

Both RAB-5 and YOP-1/RET-1 promote nuclear envelope disassembly

The ER is a single contiguous compartment that includes the nuclear envelope. We were therefore interested in whether, in addition to the peripheral ER, inhibition of RAB-5 or YOP-1/RET-1 altered nuclear envelope dynamics. One clue that this might be the case came from the observation that depletion of either RAB-5 or YOP-1/RET-1 resulted in a characteristic “four-eyes” phenotype at the end of the first division, in which two nuclei instead of one re-formed in each daughter cell. In the C. elegans zygote, after the two pronuclei meet, the nuclear envelopes are permeabilized and cleared from the region near the centrosomes, allowing spindle microtubules to interact with and align chromosomes before their segregation (Fig. 5 A; Oegema and Hyman, 2005). After anaphase chromosome segregation, a single nuclear envelope reforms around each of the separated chromatin masses, thereby mixing the haploid sperm and oocyte genomes. In embryos in which either RAB-5 or YOP-1/RET-1 was depleted, separate nuclear envelopes formed around the sperm- and oocyte-derived chromosomes after their segregation, resulting in two nuclei in each daughter cell (Fig. 5 B).

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Figure 5.

RAB-5 and YOP-1/RET-1 promote nuclear envelope disassembly during mitosis. (A) Schematics highlighting the dynamics of the oocyte- and sperm-derived pronuclear envelopes (green) during the first division of the C. elegans embryo. (B) Spinning-disk confocal optics were used to image control (n = 8), rab-5(RNAi) (n = 16), and yop-1, ret-1(RNAi) (n = 12) embryos expressing GFP:SP-12. A representative central section immediately after the first embryonic cytokinesis is shown. Bar, 10 μm. (C–F) Spinning-disk confocal optics were used to image control (n = 9), rab-5(RNAi) (n = 11), yop-1, ret-1(RNAi) (n = 10), and npp-12(RNAi) (n = 10) embryos coexpressing RFP:histone and a GFP fusion with the resident INM protein LEM-2. Representative central sections are shown. Times are in seconds relative to anaphase onset. Schematics summarize nuclear envelope dynamics under each condition. Bars, 5 μm. (G) Representative central sections of npp-12(RNAi) embryos expressing GFP:SP-12 during metaphase and telophase. Bar, 10 μm. (H) A representative cortical section of an npp-12(RNAi) embryo expressing GFP:SP-12 10 s before anaphase onset (left). Bar, 10 μm. Mitotic ER clusters were quantified as in Fig. 3 E (right).

To understand the basis for this phenotype, we filmed embryos coexpressing a GFP fusion with the INM protein LEM-2 and an RFP^mCherry fusion with histone H2B. This analysis revealed that ~35 s before anaphase onset, the juxtaposed oocyte and sperm pronuclear envelopes in control embryos undergo a scission event in close proximity to the aligned chromosomes and are cleared from the region between the chromosomes, allowing the chromosomes from the two pronuclei to mix and form a single nucleus after segregation (Fig. 5 C and Video 5, left, available at http://www.jcb.org/cgi/content/full/jcb.200701139/DC1). In embryos in which either RAB-5 or YOP-1/RET-1 were depleted, this scission event never occurred, and the oocyte- and sperm-derived chromosomes remained separate during their segregation on the spindle (Fig. 5, D and E; and Video 5, right). Consequently, two nuclei were formed in each daughter cell, and the mixing of the genomes derived from the two gametes failed.

Depletion of NPP-12 phenocopies the nuclear envelope disassembly defect resulting from RAB-5 or YOP-1/RET-1 depletion without disrupting peripheral ER structure

The four-eyes phenotype observed in YOP-1/RET-1– or RAB-5– depleted embryos is highly unusual. Functional genomic analysis of cell division in C. elegans identified only one other protein, the nuclear pore component NPP-12, whose depletion reproducibly results in this phenotype (Sönnichsen et al., 2005). NPP-12 is the C. elegans homologue of gp210, one of two integral membrane proteins associated with the nuclear pore (Holaska et al., 2002; Hetzer et al., 2005). Imaging of embryos coexpressing GFP:LEM-2 and RFP:histone confirmed that depletion of NPP-12 results in a defect in nuclear envelope disassembly similar to that resulting from depletion of RAB-5 or YOP-1/RET-1 (Fig. 5 F). However, analysis of GFP:SP-12 indicated that, in contrast to depletion of YOP-1/RET-1 or RAB-5, depletion of NPP-12 does not noticeably perturb peripheral ER structure (Fig. 5, G and H). These results indicate that the defect in nuclear envelope disassembly in the YOP-1/RET-1– and RAB-5–depleted embryos is likely to be a consequence rather than a cause of the defect in ER structure. They also point to an interesting functional connection between the role of peripheral ER structure in nuclear envelope disassembly and events occurring at nuclear pores.

Control of ER structure by trans-acting small GTPases—an emerging theme?

RAB-5 was the only Rab GTPase whose depletion phenocopied the effect of YOP-1/RET-1 depletion on ER structure. This finding was surprising because RAB-5 localizes to endosomes and has well-studied functions in endocytosis and early endosome fusion (for reviews see D'Hondt et al., 2000; Somsel Rodman and Wandinger-Ness, 2000; Pfeffer, 2001; Zerial and McBride, 2001). Inhibition of endocytosis by other means, such as CHC-1 depletion, does not affect ER structure but also does not abolish localization of RAB-5 (Fig. 3 C), indicating that endosomes, although abnormal, are still present. In contrast to CHC-1 depletion, simultaneous inhibition of the two characterized RAB-5 GEFs, RME-6 and RABX-5, redistributes RAB-5 to the cytoplasm (Sato et al., 2005) and phenocopies the effect of RAB-5 depletion (Fig. 3, A and E). These results suggest that endosomal RAB-5 acts in trans to control ER structure. Consistent with this idea, ER tubule assembly in vitro does not require ongoing endocytosis but does require the function of a Rab-type GTPase.

We propose that active RAB-5 on endosomes transiently interacts with effectors on ER membranes to promote their homotypic fusion (Fig. 8 A). Consistent with the idea that interactions with endosomes might alter ER structure, the ER has also been shown to contact several other organelles, including the Golgi, plasma membrane, lysosomes, and late endosomes (Voeltz et al., 2002). In the one-cell embryo, the asymmetric clustering of the ER also mirrors the asymmetric distribution of endosomes (Fig. S2 A), consistent with a trans-acting mechanism. The hypothesis that RAB-5 acts in trans to control ER structure is similar to the proposal that the small GTPase Ran, together with chromatin, promotes the homotypic fusion of ER membranes to form the nuclear envelope (Hetzer et al., 2000). In this case, a chromatin-activated small GTPase signals in trans to promote ER membrane fusion. Depletion of RAB-5 does not prevent nuclear envelope assembly, and depletion of RAN-1 does not disrupt ER structure (unpublished data), indicating that these two reactions, which both require fusion of ER membranes, depend on signaling from distinct small GTPases. Another instance in which ER structural changes might be triggered by signals presented in trans is during autophagy, when a double-membrane layer that may be derived from ER membranes forms around a portion of the cytoplasm to create an autophagosome (Dunn, 1990; Klionsky and Emr, 2000). Interestingly, recent work suggests a role for the Rab24 GTPase during starvation-induced autophagy, although its precise function in this process remains unknown (Munafo and Colombo, 2002). Why would ER structure be controlled by trans-acting GTPases? One possibility is that trans-action restricts homotypic ER fusion to specific times and places within the cell to facilitate the formation of a network or envelope as opposed to large membrane aggregates.

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Figure 8.

A model for the role of RAB-5 in ER structure. (A) In this model, RAB-5 on endosomes interacts with effectors on ER membranes in trans to promote their homotypic fusion. Tubule formation also requires YOP-1 and RET-1, integral ER membrane proteins that serve a structural role. (B) A model for the role of peripheral ER morphology in nuclear envelope breakdown. During nuclear envelope breakdown, the peripheral ER promotes nuclear envelope disassembly by acting as a sink for the diffusion of components of the INM (red) after their release from chromatin and the nuclear lamina. When ER morphology is disrupted by depletion of RAB-5 or YOP-1/ RET-1, the diffusion to the peripheral ER is inhibited and the nuclear envelope fails to fully disassemble.

RNA-mediated interference and antibody production

Double-stranded RNA (dsRNA) was prepared as described previously (Oegema et al., 2001) from templates prepared by using the primers listed in Table S3 (available at http://www.jcb.org/cgi/content/full/jcb.200701139/DC1) to amplify N2 genomic DNA. For complete and partial depletions, L4 hermaphrodites were injected with dsRNA and incubated at 20°C for 45 or 22–24 h, respectively, before analysis. Antibodies against RAB-5 and RET-1 were generated by cloning nucleotides 124–612 of F26H9.6 (RAB-5) and the entire coding sequence of W06A7.3b (RET-1), amplified from a cDNA library, into pGEX6P-1 (GE Healthcare). Purified GST fusion proteins were outsourced for injection into rabbits (Covance). Both antibodies were affinity purified from serum as described previously (Desai et al., 2003) by binding to columns of the same antigen after removal of the GST tag by cleavage with PreScission protease. Antibodies directed against SQV-8 and GFP have been described previously (Audhya et al., 2005; Sato et al., 2006).

Microscopy and condensation analysis

For analysis of fixed embryos, images were acquired using a 100×, 1.35 NA U-Planapo oil-objective lens (Olympus) mounted on a DeltaVision microscope system (Applied Precision) equipped with an Olympus IX70 base and a charge-coupled device camera (CoolSnap; Roper Scientific). Immunofluorescence of fixed embryos was performed as described previously (Desai et al., 2003), using the following rabbit antibodies at a concentration of 1 μg/ml: α-RAB-5 (Cy3 labeled), the mouse monoclonal antibody DM1α (Oregon green 488 labeled; Sigma-Aldrich), α-SQV-8 (Cy-5 labeled), and the goat polyclonal GFP antibody (Oregon green 488 labeled). For live analysis, embryos were mounted as described previously (Oegema et al., 2001) and imaged at 20°C on a spinning-disc confocal microscope (Eclipse TE2000-E; Nikon) equipped with a Nikon 60×, 1.4 NA Planapo oil-objective lens and a charge-coupled device camera (Orca-ER; Hamamatsu). To depolymerize microtubules, meiotic embryos were dissected directly into meiosis media (25 mM Hepes, pH 7.4, 60% Leibowitz L-15 Media, 20% fetal bovine serum, and 500 μg/ml inulin) containing 10 μg/ml nocodazole and imaged in a depression slide sealed with petroleum jelly. Quantification of ER cluster formation and lamin and LacI fluorescence intensity measurements were performed using MetaMorph software. Analysis of chromosome condensation was performed as described previously (Maddox et al., 2006). The mean value of the condensation parameter was calculated after aligning the image sequences with respect to nuclear envelope permeabilization. To simplify presentation, the plots of the condensation parameters are displayed aligned with respect to the onset of condensation.

In vitro ER tubule assembly

A light membrane fraction from X. laevis egg extracts was prepared as described previously (Voeltz et al., 2006), stained with octadecyl rhodamine, and mounted on glass slides to visualize tubule formation. To test the effect of GDI addition, membranes were preincubated for 10 min with either 10 μM Rab GDI or 30 μM Rho GDI, followed by the addition of 1 mM ATP and 0.5 mM GTP. Reactions were allowed to proceed for 60 min before staining and pipette transfer onto slides. Purified Rho GDI was provided by G. Bokoch (The Scripps Research Institute, La Jolla, CA), and the construct to express Rab GDI was a gift from W. Balch (The Scripps Research Institute).

We thank Gary Bokoch, William Balch, Marino Zerial, Jennifer Lippincott-Schwartz, Barth Grant, and Peter Askjaer for strains and reagents. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources (NCRR).

A. Audhya is a fellow of the Helen Hay Whitney Foundation; K. Oegema is a Pew Scholar in the Biomedical Sciences; A. Desai is the Connie and Bob Lurie Scholar of the Damon Runyon Cancer Research Foundation (DRS 38-04). K. Oegema and A. Desai receive salary and additional support from the Ludwig Institute for Cancer Research.