The Golgi posttranslationally modifies proteins and sorts them

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1 Mitotic Golgi disassembly is required for bipolar spindle formation and mitotic progression Gianni Guizzunti a and Joachim Seemann a,1 a Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX Edited by Peter J. Novick, University of California, San Diego, La Jolla, CA, and approved September 2, 2016 (received for review July 2, 2016) During mitosis, the mammalian Golgi vesiculates and, upon partitioning, reassembles in each daughter cell; however, it is not clear whether the disassembly process per se is important for partitioning or is merely an outcome of mitotic entry. Here, we show that Golgi vesiculation is required for progression to metaphase. To prevent Golgi disassembly, we expressed HRP linked to a Golgi-resident protein and acutely triggered the polymerization of 3,3 -diaminobenzidine (DAB) in the Golgi lumen. The DAB polymer does not affect interphase cell viability, but inhibits Golgi fragmentation by nocodazole and brefeldin A and also halts cells in early mitosis. The arrest is Golgi specific and does not occur when DAB is polymerized in the endosomes. Cells with a DAB polymer in the Golgi enter mitosis normally but arrest with an intact Golgi clustered at a monopolar spindle and an active spindle assembly checkpoint (SAC). Mitotic progression is restored upon centrosome depletion by the Polo-like kinase 4 inhibitor, centrinone, indicating that the link between the Golgi and the centrosomes must be dissolved to reach metaphase. These results demonstrate that Golgi disassembly is required for mitotic progression because failure to vesiculate the Golgi activates the canonical SAC. This requirement suggests that cells actively monitor Golgi integrity in mitosis. Golgi disassembly mitosis spindle formation The Golgi posttranslationally modifies proteins and sorts them to their correct destinations. This basic secretory function is mediated by stacks of flattened cisternal membranes. Although the arrangement of cisternae into stacks is a common feature found in most eukaryotes (1), the morphological organization of stacks differs between species. Most organisms have numerous copies of stacks, present as individual units that are scattered throughout the cytoplasm (2). The exception are vertebrates, where the stacks are laterally connected to form a contiguous ribbon-like structure that is positioned in close proximity to the centrosomes in the perinuclear region of the cell (3). On one hand, the interconnection of cisternae facilitates the accommodation and processing of large cargos such as collagen (4, 5). On the other hand, the close positioning of the Golgi ribbon to the centrosomes guides membrane traffic for polarized secretion and thus plays a pivotal role in cell polarization (6 8). Such more complex organization, however, poses a challenge for cell division that requires a more elaborate mechanism to partition the continuous Golgi ribbon. To this end, the Golgi vesiculates in early mitosis and segregates into the daughter cells where it reassembles into a functional ribbon. Golgi disassembly is initiated in late G2 phase, when the ribbon is unlinked through severing of the lateral connections between the stacks (9). Interfering with this unlinking process, either by microinjection of antibodies against the Golgi stacking protein GRASP65 or by inhibiting the membrane fission protein CtBP1/ BARS, affects transition from G2 into M phase (10 12). After the cells progress into mitosis, the cisternae unstack and disassemble into a collection of vesicles and tubular membranes (13). Partitioning of these disassembled Golgi membranes is facilitated by the mitotic spindle, which carries essential Golgi proteins that are required to reorganize an intact ribbon structure in the daughter cells (14). Moreover, in early mitosis the Golgi matrix protein GM130 activates the spindle assembly factor TPX2 to locally promote microtubule (MT) nucleation and spindle formation, thereby linking Golgi membranes and the nascent spindle (15). This highly regulated sequence of ribbon unlinking, vesiculation, partitioning, and reassembly is unique to mammals, where the Golgi stacks are interconnected. In lower organisms, where the stacks are not laterally stitched together, the stacks remain intact throughout mitosis and the spindle has seemingly no role in Golgi partitioning (2). Together, these observations suggest that vesiculation of the mammalian Golgi and partitioning by the spindle are tightly controlled and functionally linked. To address whether Golgi vesiculation is a prerequisite for its partitioning and/or mitotic progression, we devised an approach to prevent Golgi remodeling by acutely filling the lumen of the cisternae with a 3,3 -diaminobenzidine (DAB) polymer and then monitored progression into mitosis. We found that the DAB polymer did not prevent transition into M phase, but inhibited the mitotic vesiculation of the Golgi. Strikingly, the cells became stalled in early mitosis with an active spindle assembly checkpoint (SAC) and monopolar spindles. The intact Golgi blocked centrosome separation and bipolar spindle formation. Depletion of centrosomes with the Polo-Like Kinase 4 (PLK4)-inhibitor, centrinone, restored bipolar spindle assembly and cells divided despite DAB polymers in the Golgi. Our results reveal that Golgi vesiculation in early mitosis is tightly coupled to spindle assembly and monitored to control mitotic progression. Results DAB Polymerizes in the Lumen of the Golgi in Living Cells Expressing Sialyltransferase HRP. To investigate the influence of Golgi disassembly on mitotic progression, we assembled an inorganic polymer in the lumen of the Golgi cisternae in live cells. This approach is based on horseradish peroxidase (HRP), which in the presence of H 2 O 2 converts the small molecule DAB into large insoluble polymers that can be visualized by light microscopy and EM (16). Significance During mammalian cell division, the contiguous Golgi ribbon is first vesiculated and then partitioned into the daughter cells with the aid of the mitotic spindle. Whether Golgi vesiculation is required for cell division is unclear. Here, we show that inhibition of Golgi disassembly by the acute formation of a polymer in the Golgi lumen prior to mitotic entry blocks bipolar spindle formation and arrests cells in early mitosis with an active spindle assembly checkpoint (SAC). Importantly, mitotic progression is fully restored upon depletion of centrosomes. Our work thus reveals that mitotic Golgi disassembly is tightly monitored and acts as an upstream surveillance point for the canonical SAC to ensure the fidelity of Golgi inheritance and cell division. Author contributions: G.G. and J.S. designed research, performed research, analyzed data, and wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. joachim.seemann@utsouthwestern. edu. E6590 E6599 PNAS Published online October 10,

2 Fig. 1. DAB polymerizes in the lumen of the Golgi in live cells expressing ST HRP. (A) SV589 fibroblasts stably expressing ST HRP were treated with doxycycline for 24 h to induce expression. Doxycycline was then removed for 12 h and the cells were immunostained for HRP, the Golgi marker GM130 (green), and labeled for DNA (blue). (Scale bar, 20 μm.) (B) Diagram of the experimental approach to assemble a DAB polymer in the lumen of the Golgi cisternae. (C) DAB polymerizes in the Golgi. ST HRP-expressing SV589 cells were treated for 4 min with DAB/H 2 O 2 or DAB alone as control. Upon fixation, cells were labeled for GM130 (green) and DNA (blue). Note that the dark DAB polymer, visible by transmitted light, is located in the perinuclear region of the cell and colocalizes with GM130. (Scale bar, 20 μm.) (D) Electron micrographs show the restriction of the DAB polymer to the lumen of the Golgi cisternae. Live cells expressing ST HRP were incubated for 4 min with DAB/H 2 O 2 to polymerize DAB and then fixed and processed for EM. The low power image at Top is magnified at Bottom. Note that polymerized DAB is present in Golgi cisternae (arrowheads), whereas the ER is not stained (arrows). (Scale bars, 0.5 μm.) Monomeric DAB can freely cross the membrane, but becomes trapped within the organelle where HRP activity is localized (17). To target HRP to the Golgi, we expressed HRP linked to the luminal side of the Golgi resident protein sialyltransferase (ST) (ST HRP) (18, 19). We noticed that cells constitutively expressing the construct lost HRP activity within a few passages. To overcome this limitation, we stably transduced human fibroblast SV589 cells to express the tet-repressor and ST HRP under the control of a doxycycline-inducible promoter. Transcription was turned on with a 24-h pulse of doxycycline, followed by a 12-h chase to suppress further expression and to allow ST HRP to exit the early secretory pathway and reach the Golgi. Immunofluorescence (IF) analysis with antibodies against HRP showed that ST HRP was restricted to the perinuclear region of the cells and colocalized with the Golgi marker GM130 (Fig. 1A), confirming that the fusion protein was targeted to the Golgi. To trigger polymer formation, SV589 cells expressing ST HRP were treated with DAB and low concentrations of H 2 O 2 for 4 min at room temperature (Fig. 1B). The short pulse was sufficient to polymerize DAB in the Golgi lumen, which appeared by transmitted light as a dark polymer in the perinuclear region and by IF colocalized with GM130 (Fig. 1C). We then confirmed by EM that DAB polymerized in the Golgi lumen (Fig. 1D). Live cells expressing ST HRP were first incubated with DAB/H 2 O 2 and then processed for EM. In line with reports where the DAB reaction was performed after fixation (18, 19), DAB polymers localized to the cisternae of the Golgi. Staining was not detected in the endoplasmic reticulum (ER). These results established that the DAB polymer is confined within the lumen of Golgi cisternae in living cells. DAB Polymers Prevent Brefeldin A- and Nocodazole-Induced Dispersal of the Golgi Ribbon. We then tested whether the DAB polymer renders the Golgi resistant to pharmacological agents that disrupt the Golgi structure. Brefeldin A (BFA) redistributes Golgi enzymes into the ER (20), leaving behind vesicular remnants containing the Golgi matrix protein GM130 that are scattered throughout the cytoplasm (21, 22). Because these BFA-induced morphological changes have been reported to phenocopy mitotic Golgi disassembly (23), we determined the extent to which the DAB polymer prevents BFA-triggered Golgi fragmentation. BFA dismantled the ribbon into punctate elements that redistributed across a significantly increased cellular area (Fig. 2A). However, the DAB polymer blocked the effects of BFA and the Golgi maintained its integrity as a single entity. We next challenged the Golgi ribbon with nocodazole (Noc), which depolymerizes MTs and unlinks the ribbon into individual ministacks that are dispersed throughout the cytoplasm. Control cells showed a perinuclear Golgi ribbon and an intact MT network (Fig. 2B). Nocodazole depolymerized MTs, indicated by a diffused staining of α-tubulin, and dispersed the Golgi into ministacks. By contrast, in cells containing the DAB polymer, the Golgi remained in its perinuclear position despite the absence of an intact MT network. We then investigated whether dispersed Golgi ministacks filled with the DAB polymer can reassociate into a perinuclear Golgi. Cells were first treated with nocodazole to disjoint the ribbon and to disperse the stacks. Upon incubation with DAB/H 2 O 2, the polymer colocalized with GM130 labeled structures (Fig. 2C). The number of nocodazole-induced Golgi ministacks was not altered compared with cells without the polymer (Fig. 2B). When nocodazole was washed out, a compact Golgi harboring a visible DAB polymer reformed in the perinuclear region that was indistinguishable from those in control cells. This result indicates that only the lumen, but not the cytoplasmic face of the Golgi is affected by the polymer, and that the machinery required for linking the Golgi to the MT cytoskeleton and for assembling a perinuclear Golgi structure is unaffected. Inhibition of Mitotic Golgi Disassembly Prevents Bipolar Division. We next examined the effect of the DAB polymer on cells entering mitosis. SV589 ST HRP cells released from a thymidine block were treated with DAB or DAB/H 2 O 2 and followed by live cell imaging. Control cells that rounded up first increased and then after 4 h gradually decreased, indicating a mitotic peak of the synchronized cell population (Fig. 3 A and B). Individual control cells rounded up, progressed through mitosis, and flattened out within 60 min (Fig. 3 C and D, arrowhead). By contrast, cells with a Golgi-localized DAB polymer also rounded up, but continuously accumulated (Fig. 3 A and B). These rounded cells did not divide (Fig. 3 C and D, arrows) and started to die after more than 3 h arrest. To assess whether the phenotype is specific to M phase, we polymerized DAB in thymidine-arrested cells and then kept them in thymidine during time-lapse microscopy to maintain the cells in interphase. Cells with a DAB polymer remained flat throughout the duration of the experiment and behaved as control-treated cells, indicating that the polymer by itself does not affect nondividing cells (Fig. 3 E and F). The DAB Polymer Arrests Cells in Mitosis with an Intact Golgi. To determine whether the rounded cells containing the DAB polymer were in fact arrested in mitosis, we labeled the cells with the MPM2 antibody, which recognizes mitosis-specific phosphoproteins, including centrosomal proteins (24). We identified mitotic control cells by their round appearance and condensed chromosomes CELL BIOLOGY PNAS PLUS Guizzunti and Seemann PNAS Published online October 10, 2016 E6591

3 aligned along the equatorial plane. These mitotic cells displayed a disassembled Golgi and were labeled by MPM2 (Fig. 4A). Similarly, MPM2 also marked cells that became arrested due to the DAB polymer, confirming that the cells were indeed in mitosis. Strikingly, MPM2 labeled two dot-like structures resembling a pair of nonseparated centrosomes that were surrounded by condensed chromosomes, which in the majority of cells were arranged in a circle. Moreover, in contrast to control cells, the mitotic Golgi membranes clustered at the nonseparated centrosomes (Fig. 4A, +DAB). The circular arrangement of chromosomes is reminiscent of cells treated with inhibitors of the mitotic kinesin, Eg5, which prevent centrosome separation and arrest cells in M phase with monopolar spindles (14, 25). Cells arrested in mitosis by the Eg5 inhibitor, S-trityl-L-cysteine (STLC), were positive for MPM2 (Fig. 4A) and showed a monopolar spindle organization that was comparable to cells arrested by the DAB polymer (Fig. 4B). STLC-treated cells progress through the initial phases of mitosis before becoming stalled in prometaphase (26). To analyze whether DAB polymer-containing cells entered mitosis normally, we examined the state of the nuclear envelope, which in metazoans breaks down during prophase. We labeled the cells with the nuclear pore complex antibody mab414, which has been widely used to determine mitotic changes of the nuclear envelope (27). Mitotic control cells and STLC-arrested cells with a disassembled Golgi and condensed chromosomes exhibited a loss of Ab414 staining compared with neighboring interphase cells (Fig. 4C). Similarly, in mitotic cells with DAB polymers the Golgi also contained condensed chromosomes and a disassembled nuclear envelope (Fig. 4C). This result demonstrates that despite the presence of an intact Golgi, cells progressed normally through the early phases of mitosis before becoming arrested. Because the mitotic disassembly of the Golgi was inhibited by the DAB polymer when observed by IF, it was important to analyze the Golgi structure by EM. When DAB is polymerized in cells with a disassembled Golgi after they transitioned into mitosis, the DAB reaction product marks Golgi-derived mitotic vesicles (19). By marked contrast, in cells that entered mitosis with a polymer and became arrested, as identified by condensed chromosomes and a disassembled nuclear envelope, the DAB polymer was found in stacks of intact cisternae and not detectable in the ER (Fig. 4D). Together, our results demonstrate that DAB polymers in the lumen of the Golgi block its mitotic disassembly. These cells still progress into M phase, but become arrested with monopolar spindles in prometaphase at a stage that is similar to Eg5 inhibitor-treated cells. Fig. 2. The DAB polymer prevents BFA- and nocodazole-induced Golgi disassembly. (A) The DAB polymer blocks BFA-induced Golgi disassembly. After induction of ST HRP expression for 24 h, doxycycline was removed for 12 h, and DAB/H 2 O 2 was added for 4 min to polymerize DAB. The cells were then treated for 30 min with BFA to fragment the Golgi before staining for GM130 and DNA. Polymerized DAB was visualized by bright field microscopy (Bottom). Quantitations presented in the bar diagrams show that BFA treatment significantly increased the cellular area over which the GM130 signal spread out (Golgi area) and the number of Golgi elements positive for GM130 (Golgi elements/cell). n = 3. (B) The DAB polymer blocks nocodazoleinduced Golgi disassembly. ST HRP-expressing cells were incubated with nocodazole to depolymerize MTs and to disperse the Golgi ribbon into ministacks in the absence or presence of the DAB polymer. Cells were stained for GM130, α-tubulin, and DNA. The effect of nocodazole was quantified as in A. n = 3. (C) The DAB polymer does not interfere with Golgi assembly in the perinuclear region. ST HRP cells were first treated with nocodazole to disperse the Golgi. After DAB was polymerized, nocodazole was washed out to allow MT assembly and reformation of a perinuclear Golgi ribbon. The cells were then labeled for GM130, α-tubulin, and DNA. The quantifications DAB Polymerization in Endosomes Does Not Affect Cell Division. In mitosis, the Golgi disassembles into vesicles that are then partitioned into the daughter cells (28, 29). If Golgi disassembly is important for mitotic progression, then DAB polymerized in a compartment that is not vesiculated in M phase, such as endosomes (30), should not interfere with cell division. To test this hypothesis, as well as to exclude that the mitotic arrest was due to secondary effects, we polymerized DAB in endosomes loaded with HRP by fluid-phase uptake for 10 min (Fig. 5A). The dark polymer colocalized with the early endosome (EE) marker EEA1 (Fig. 5B). We adjusted the HRP concentration to load endosomes with a comparable HRP activity as in the Golgi of ST HRP cells, which was confirmed by Western blotting (Fig. 5C) and measurement of HRP enzymatic activity in cell lysates (Fig. 5D). We then followed mitosis of cells with endosomal DAB polymers by time-lapse microscopy (Fig. 5E). Cells with shown in graphs indicate that the dispersed mini-golgi stacks reassembled into a compact single Golgi upon nocodazole removal. n = 3. (Scale bars in A C, 20μm.) Error bars represent mean ± SEM. E Guizzunti and Seemann

4 Fig. 3. DAB polymers in the Golgi block cell division. (A) SV589 cells expressing ST HRP were synchronized with thymidine for 16 h, released into fresh medium for 8 h, and treated with DAB. Polymerized DAB in the Golgi was detected by bright field microscopy, and the cells were followed by phase-contrast time-lapse microscopy. (B) The percentage of rounded-up control cells peaked at 4 h and gradually diminished, whereas rounded DAB-treated cells continuously increased over time. (C) Cells were treated as in A. Individual control cells rounded up and divided (arrowheads), whereas cells containing a DAB polymer remained rounded (arrows). (D) About 91% of control cells divided within 120 min after rounding up compared with 12% of DAB-treated cells. (E) DAB polymerization in the Golgi does not affect cell viability in interphase. SV589 cells expressing ST HRP were arrested in G1/S phase with thymidine and treated with DAB/H 2 O 2 before live cell imaging in the continued presence of thymidine. (F) Quantification of the number of rounded-up cells in E over time. (Scale bars in A, C,andE,50μm.) Error bars represent mean ± SEM, n = 3. DAB polymers in endosomes entered M phase and divided within 60 min, whereas ST HRP cells with DAB polymers in the Golgi mitotically arrested (Fig. 5F). This result shows that the mitotic block is specific for the DAB polymer in the Golgi. Inhibition of Golgi Disassembly Arrests Cells with Monopolar Spindles and Activates the SAC. The circular arrangement of chromosomes around monopolar spindles in DAB polymer-arrested cells is reminiscent of the Eg5 inhibition phenotype (25). We therefore labeled cells for the centrosomal marker γ-tubulin to examine whether the centrosomes failed to separate. Control cells in metaphase displayed well-separated γ-tubulin foci, a disassembled Golgi, and condensed chromosomes aligned at the equatorial plane (Fig. 6A). By contrast, in cells harboring a DAB polymer, an intact Golgi resided around nonseparated centrosomes, whereas in STLC-arrested cells the centrosomes also failed to separate, but the Golgi was more disassembled. To quantify these observations, we determined the distance between the two γ-tubulin foci as a measure of centrosome separation. Cells arrested with a DAB polymer and an intact Golgi showed a significantly reduced distance between the centrosomes, from 12.9 ± 0.3 μm incontrol metaphase cells to 2.3 ± 0.15 μm, which is comparable to STLCtreated cells with centrosomes separated by 2.2 ± 0.1 μm(fig.6b). The failure to form a bipolar spindle suggested that the spindle assembly checkpoint (SAC) could not be satisfied, which prevents progression into anaphase. We therefore examined the localization of the SAC kinase BubR1, which inhibits transition into anaphase when it is recruited to kinetochores (31). In mitotic control cells, BubR1 localized to kinetochores before metaphase, but it was absent in late mitosis after the SAC was satisfied (Fig. 6C). In cells arrested with the DAB polymer or STLC, BubR1 was present on kinetochores, indicating that the SAC was indeed active (Fig. 6C). Together, these results established that failure to disassemble the Golgi arrests cells in mitosis with monopolar spindles and an active SAC. Centrosome Depletion Bypasses the DAB-Induced Mitotic Arrest. Because an intact Golgi blocked transition into metaphase by inhibiting centrosome separation, we reasoned that depletion of centrosomes should bypass the arrest and restore mitotic progression. To test this reasoning, we removed centrosomes using centrinone, a recently developed inhibitor of Plk4 (32). Plk4 activity is required for centriole biogenesis (33, 34) and cells proliferating in the presence of centrinone progressively lose their centrosomes. Centrinone has been best characterized in HeLa cells, in which centrosomes are removed from >80% of cells within 3 d (32). We first confirmed that centrosomes were efficiently depleted from HeLa cells incubated with centrinone for 3 d CELL BIOLOGY PNAS PLUS Guizzunti and Seemann PNAS Published online October 10, 2016 E6593

5 Fig. 4. Preventing Golgi disassembly arrests cells in mitosis. ST HRP-expressing SV589 cells synchronized with thymidine were treated with DMSO (control), DAB/H 2 O 2, or STLC. (A) Cells were stained for GM130, DNA, and the mitotic marker MPM2. Mitotic control cells positive for MPM2 showed a dispersed Golgi and condensed chromosomes. MPM2 labeled mitotic cells with a DAB polymer (bright field) showed a more compact Golgi and a circular chromosome arrangement comparable to cells arrested in M phase with the Eg5 kinesin inhibitor STLC. (B) Cells were stained for GM130, DNA, and α-tubulin to label spindles. Mitotic control cells showed a bipolar spindle, whereas cells with a DAB polymer and STLC-arrested cells presented condensed chromosomes arranged around a monopolar spindle. (C) Cells were stained for GM130, DNA, and nucleoporins (Ab414) to mark the nuclear envelope. Mitotic control cells, cells that entered mitosis with a DAB polymer, and STLCtreated cells all showed a disassembled nuclear envelope and condensed chromosomes. (Scale bars in A C, 20 μm.) (D) Electron micrographs of cells that entered mitosis in the presence of a DAB polymer. Low power image showing a mitotic cell with condensed chromosomes. The boxed region is magnified in the Inset, revealing stacked Golgi cisternae filled by the DAB polymer (arrowhead). The ER is not labeled by the DAB polymer (arrow). (Scale bar, 5 μm.) (Scale bar in Inset, 0.2 μm.) before ST HRP induction for another 24 h (Fig. 7A). Controltreated cells contained distinct centrosomal γ-tubulin foci, which were undetectable in 82.3 ± 0.3% of centrinone-treated cells. The Golgi morphology was not affected by the absence of centrosomes, as was previously described (32) (Fig. 7 B and C). We then polymerized DAB in the Golgi and monitored mitotic progression (Fig. 7 D and E). In contrast to control- and centrinone-treated cells that divided, HeLa cells with a DAB polymer became mitotically arrested in a comparable manner to SV589 cells. Intriguingly, when cells were pretreated with centrinone to remove centrosomes, they divided despite the DAB polymer in the Golgi. These data show that removal of centrosomes relieves the mitotic arrest induced by an intact Golgi, allowing cells to progress through metaphase and divide. In line with previous reports, we observed that the duration of mitosis increased in cells lacking centrosomes (32, 35). Whereas control-treated cells finished mitosis within 60 min, centrinone-treated cells, either with or without a DAB polymer, required about 120 min (Fig. 7D). Discussion At the beginning of mitosis, the Golgi ribbon of a mammalian cell first disassembles into vesicles and then partitions into the two daughter cells with the aid of the spindle (14, 28, 29). In this study, we investigated whether Golgi vesiculation is required for its inheritance and for mitotic progression. Our results provide direct evidence that mitotic Golgi vesiculation is required for bipolar spindle formation. Golgi disassembly thereby functions as a control point that is tightly monitored and acts as an upstream requirement with the SAC. Our experimental approach further provides a system to acutely block the dynamic remodeling of Golgi such as upon challenging the Golgi structure with nocodazole and BFA (Fig. 2) or during mitosis (Fig. 3). Our approach prevents mitotic Golgi vesiculation by assembling a DAB polymer in the lumen of the Golgi cisternae in late G2 phase before mitotic entry. DAB is a small molecule that permeates cellular membranes. Upon oxidation, it assembles into large polymers that remain trapped within the reaction compartment. DAB can be polymerized by peroxidases, including HRP and the ascorbate peroxidase APEX, and both enzymes can be targeted as fusion proteins to specific subcellular locations. One advantage of HRP over APEX is its significantly higher enzymatic activity, although it is only active when expressed in the lumen of the secretory pathway (36). Furthermore, the HRP/DAB reaction occurs within seconds and is highly efficient, as a single HRP E Guizzunti and Seemann

6 Fig. 5. DAB polymerization in endosomes does not affect cell division. (A) Endosomes of thymidine-synchronized SV589 cells were loaded with HRP by fluid-phase uptake for 10 min before DAB polymerization and time-lapse imaging. (B) DAB polymerizes in endosomes. SV589 cells loaded with HRP were treated for 4 min with DAB/H 2 O 2 and stained for the early endosome marker EEA1 and DNA. (C) ST HRP-expressing cells contain a similar amount of HRP as cells with endocytosed HRP. Cells with endocytosed HRP at 4 C or 37 C and ST HRP-expressing cells were subjected to Western blotting analysis with antibodies against HRP. (D) ST HRP cells and HRP-loaded cells contain comparable HRP activity. Cells with HRP in endosomes and ST HRP-expressing cells were lysed and the peroxidase activity was determined by colorimetry using o-dianisidine as HRP substrate. n = 3intriplicates.(E) DAB polymers in endosomes do not affect cell division. SV589 cells released from thymidine block were loaded for 10 min with soluble HRP, incubated for 4 min with DAB/H 2 O 2, and then followed by time-lapse imaging. Cells with DAB polymers in the Golgi became mitotically arrested (arrows), whereas cells with a DAB polymer assembled in endosomes finished mitosis and divided (arrowheads). (F) Quantification of the number of cells with a Golgi- or endosome-localized DAB polymer completing mitosis within 60 min or 120 min. n = 3. (Scale bars in B and E,20μm.) molecule can polymerize DAB to fill an entire compartment (17). Another key advantage is that the dark DAB reaction product can be readily visualized by bright field microscopy in live cells and by EM. Because of its efficiency and sensitivity, HRP/DAB chemistry has been widely used in immunohistochemistry and EM applications to label specific cellular structures. To specifically target HRP to the Golgi, we generated inducible cell lines stably expressing HRP linked to the transmembrane domain of the Golgi enzyme sialyltransferase (ST HRP) in the lumen of the cisternae. ST HRP expression was induced by a pulse of doxycycline followed by a chase to clear the protein out of the early secretory pathway and restrict its localization to the Golgi (Fig. 1). The DAB reaction is thus limited to the Golgi, which is important because a trace of HRP activity in the ER could generate DAB polymers that interfere with ER remodeling and nuclear envelope breakdown in mitosis. When DAB is polymerized in ST HRP-expressing cells after fixation, the polymer localizes to the Golgi and is absent from other membranes, including the ER and nuclear envelope (18, 19). We found the same restricted localization in live cells by both light microscopy and EM (Fig. 1). Importantly, polymerization of DAB in live cells, either in the Golgi (Fig. 3) or in cells with HRP internalized into endosomes (Fig. 5), did not affect cell viability during interphase. In agreement, cell viability was also not disturbed when the DAB reaction was triggered to study transport through endosomal compartments (37 39), or Golgi biogenesis during interphase (16). The DAB polymer blocked Golgi fragmentation induced by nocodazole and BFA (Fig. 2), which in control cells redistribute Golgi membranes throughout the cell. Such pharmacological fragmentation of the interphase Golgi has been suggested to mimic its disassembly during mitosis (23). Similar to interphase cells treated with BFA or nocodazole, Golgi fragmentation in mitosis is driven by vesiculation as well as extensive MT remodeling. Consistent with our results in interphase cells, the DAB polymer also blocked mitotic Golgi vesiculation (Fig. 4). These results corroborate that the DAB polymer-fortified Golgi has sufficient strength to counteract the forces exerted through membrane and cytoskeleton remodeling under both physiological and pharmacological conditions. Our results further show that the cytoplasmic surface of the Golgi is not affected by the DAB polymer in the lumen of the cisternae. The cytoplasmic face of the Golgi serves as an assembly platform for a diverse array of protein complexes, including coatomer, clathrin, and various tethering proteins (40). The interphase Golgi also functions as a MT-organizing center that recruits components to assemble and organize a noncentrosomal MT network (41 43). When DAB was first polymerized in nocodazole-induced ministacks, the stacks reassembled back into a compact Golgi in the perinuclear region upon nocodazole washout (Fig. 3C), demonstrating that the interface between the Golgi membrane and the MT cytoskeleton is preserved. During mitosis, a variety of kinases including MEK1, Cdk1, and Plk1 target Golgin and GRASP proteins on the cytoplasmic side of the Golgi to drive Golgi disassembly (44 47). The inability to form a bipolar spindle and to progress through M phase caused by an intact Golgi was entirely restored upon centrosome depletion (Fig. 7E), indicating that the mitotic block was indeed triggered by active signaling instead of by an unspecific, adverseeffectofthetreatments. CELL BIOLOGY PNAS PLUS Guizzunti and Seemann PNAS Published online October 10, 2016 E6595

7 Fig. 6. Inhibition of mitotic Golgi disassembly arrests cells with monopolar spindles and an active spindle assembly checkpoint. ST HRP-expressing cells synchronized with thymidine were treated with DMSO (control), DAB/H 2 O 2, or STLC. Cells were then stained for GM130, DNA, and γ-tubulin to label centrosomes (A and B)orBubR1(C). (A) Control cells in metaphase (Top) contained separated centrosomes with condensed chromosomes aligned at the equatorial plane and a disassembled Golgi. In cells that entered mitosis with a DAB polymer in the Golgi (Middle) orstlc-arrestedcells(bottom), condensed chromosomes were arranged around unseparated centrosomes. (B) The distance between two γ-tubulin foci in A was measured as the spindle length. The distance was significantly decreased from 12.9 ± 0.3 μm in control metaphase cells to 2.3 ± 0.15 μm in DAB-treated cells and to 2.2 ± 0.1 μm in STLC-arrested cells (22 38 spindles per condition, n = 3). (C) The mitotic SAC is active in cells arrested by a DAB polymer. In control-treated cells, the SAC kinase BubR1 was localized to kinetochores in early mitosis and became dissociatedoncethe cells had progressed toanaphase (controlcells,bottom Left).In DAB-arrested cells (TopRight) and STLC-treated cells (Bottom Right), BubR1 was present at kinetochores, indicating that the SAC was indeed active. (Scale bars in A and C, 10μm.) Similarly, endosomal DAB polymers neither prevented mitotic progression (Fig. 5) nor affects the recruitment of clathrin and its adaptors to endosomes (48). These findings strongly argue that the cytoplasmic surface of the Golgi, as well as the cytoplasmic machinery that interacts with the Golgi, are not perturbed by the DAB polymer in the lumen of the organelle. The lateral tubular connections between stacks of the Golgi ribbon are severed in late G2 (9). Interfering with this step delays progression into M phase, which has been referred to as a checkpoint that monitors the integrity of the ribbon (10, 12). Because the DAB polymer inhibited Golgi dispersal in interphase (Fig. 2), as well as mitotic Golgi vesiculation (Fig. 4), it should also slow down the G2/M transition. Indeed, ST HRP-expressing cells with a DAB polymer released from a thymidine block rounded up later than control cells (Fig. 3B). In line with previous reports (44, 46), exit from interphase and M-phase entry was only delayed, not blocked, indicating that this G2/M control point is transient in nature and weaker compared with the canonical SAC that blocks mitotic progression. However, we cannot rule out the possibility that the DAB polymer-containing Golgi ribbon was partially unlinked in late G2, which could be sufficient to allow entry into M phase. Irrespective of the precise contribution of lateral Golgi unlinking in G2/M transition, our results establish that once the cells are committed to mitosis, Golgi vesiculation becomes essential to satisfy the canonical SAC and thereby for transition into metaphase. Cells with a DAB polymer failed to establish a bipolar spindle (Fig. 4) and arrested with a monopolar spindle and an active SAC, as marked by BubR1 on kinetochores (Fig. 6). The metaphase anaphase transition was thereby blocked and the cells died after prolonged arrest (Fig. 3). Under these conditions, the Golgi stacks remained intact (Fig. 4) and clustered around the nonseparated centrosomes (Fig. 6). These findings support the conclusion that mitotic Golgi vesiculation is tightly controlled and acts as an upstream requirement for the canonical SAC. Furthermore, Golgi vesiculation itself might have a mitotic checkpoint mechanisms that monitors its disassembly to ensure its subsequent partitioning by the mitotic spindle. The question then is how a Golgi that fails to be disassembled signals the downstream effectors to trigger mitotic arrest. When entering mitosis, cells with a DAB polymer in the Golgi became arrested with monopolar spindles, which phenocopies Eg5 kinesin inhibitors that block centrosome separation (Figs. 4 and 6). This result suggests that an intact Golgi physically limits centrosome separation and thus forfeits bipolar spindle formation. In support of this possibility, Golgi and centrosomes are linked in interphase through MTs and protein protein interactions (49). The mechanism by which the Golgi is anchored to centrosomes is not entirely clear, but may involve several proteins that simultaneously associate with both structures, including AKAP450, FTCD, GMAP210, and CDK5RAP2 (43, 50 52). Furthermore, the Golgi and centrosomes are functionally coupled and need to reorient as a unit during directional cell migration, as preventing remodeling and movement of the Golgi toward the leading edge blocks centrosome orientation (6). In a similar sense, it is likely that the release of the physical link between the Golgi and centrosomes in mitosis is required to segregate centrosomes to set up spindle bipolarity. One possibility to signal this event is to release proteins from the Golgi during its disassembly such that it could be further transduced downstream to uncouple centrosomes from the Golgi. In accordance with this possibility, several Golgi proteins are released during mitotic disassembly into the cytoplasm where they regulate different aspects of mitosis (53). On the other hand, vesiculating the cisternae may unlink the membrane subdomains connected to centrosomes, thus allowing centrosomes to move E Guizzunti and Seemann

8 Fig. 7. Depletion of centrosomes restores mitotic division of cells containing DAB polymers in the Golgi. (A) HeLa cells stably expressing ST HRP were treated with centrinone for 72 h to deplete centrosomes before 24-h induction of ST HRP. The cells were then synchronized with thymidine, released, treated with DAB to assemble a polymer in the Golgi, and followed by time-lapse microscopy. (B) Centrinone depletes centrosomes in HeLa ST HRP cells without affecting Golgi morphology and positioning. HeLa cells were treated with centrinone for 72 h before inducing ST HRP expression with doxycycline for 24 h in the continuous presence of centrinone and then stained for γ-tubulin to label centrosomes, GM130, and DNA. (Scale bar, 10 μm.) (C) Quantification of B. Centrosomal γ-tubulin foci were undetectable in 82% of centrinone-treated cells. (D) HeLa ST HRP cells treated as in A were followed by time-lapse imaging. Cells with a DAB polymer in the Golgi became mitotically arrested (arrows), whereas centrinone-treated cells with a DAB polymer finished mitosis and divided (arrowheads). (Scale bar, 25 μm.) (E) Quantification of D as the percentage of cells completing mitosis within 120 min. n = 3. and establish opposite poles of the spindle. In either case, releasing the connection between the Golgi and centrosomes is a prerequisite for progression into metaphase and is directly coupled to SAC signaling. In support of this notion, we showed that the DAB-induced mitotic block was fully rescued by centrosome depletion with centrinone (Fig. 7). Spindle assembly, although largely driven by centrosomes in animal cells, can be initiated by different pathways, including Randriven chromosome-derived, augmin-mediated MT-derived and GM130-triggered Golgi-derived spindle assembly (54 56). Such robustness is best exemplified in centrosome-depleted cells, which form bipolar spindles and continue to divide (32) (Fig. 7). In analogy, mitotic Golgi disassembly is also driven by several distinct cellular processes, including cytoskeletal rearrangements, lateral unlinking and unstacking of cisternae, persistent membrane fission, and inhibition of vesicle tethering and fusion. The requirement for multiple processes reflects the modular organization of the Golgi (3), where different parts of the Golgi use distinct mechanisms to ensure robust disassembly within a very short period of time. Interference with only one aspect of Golgi disassembly impacts, but does not block mitotic progression or Golgi partitioning, for example, by preventing GRASP protein phosphorylation to inhibit unlinking of the ribbon and unstacking of cisternae (46), COPI vesicle budding (57), BARS-mediated membrane fission (12), or p97/p47-mediated homotypic membrane fusion (58). Therefore, we used a global approach to prevent Golgi disassembly by filling the cisternae with a DAB polymer, which indeed blocked Golgi disassembly and progression into metaphase. Based on our data, we conclude that a bulky, unbreakable Golgi physically prevents the separation of the centrosomes so that the force generated by Eg5 kinesin is not sufficient to drive the centrosomes apart to form a bipolar spindle. The resultant failure in mitotic progression can be fully restored by unshackling the centrosomes from the Golgi, further unraveling a novel checkpoint that is in place and communicates with the canonical SAC to ensure the fidelity of cell division. Materials and Methods Plasmids and Cell Lines. SV589 (immortalized human fibroblasts) (59) and HeLa cell lines were maintained at 37 C and 5% CO 2 in medium A [DMEM (Mediatech), 10% (vol/vol) cosmic calf serum (CCS) (HyClone), 100 units/ml penicillin, and 100 μg/ml streptomycin (PenStrep)]. SV589 TetR cells stably expressing the tet-repressor were generated with plenti CMV TetR Blast (Addgene). Individual clones were selected with 10 μg/ml blasticidin S (Corning) and screened by IF with an anti-tetr antibody. The tet-inducible plasmid pqcxin/to was generated by replacing the promoter of pqcxin (Clontech) with the CMV promoter containing two tet-operator sites from pt- Rex DEST30 (Invitrogen). To allow selection with G418, the internal ribosome entry site of pqcxin/to between the multiple cloning sites and the neomycin resistance gene was replaced with the SV40 promoter from pcdna3.1( ) (Invitrogen) to yield pqcxin/to/sv. The insert ST HRP [transmembrane domain and cytoplasmic tail of sialyltransferase (residues 1 45) fused to HRP], was amplified from psr ST HRP (18) and either cloned into pqcxin/to/sv40 to generate pqcxin/to/sv ST HRP or into plvx TetOne Puro (Clontech) to obtain plvx ST HRP. Stable SV589 cells expressing ST HRP were produced by transduction of SV589 TetR cells with pqcxin/to/sv ST HRP and selection with 1 mg/ml G418 (Assay Designs) to obtain SV589 ST HRP cells. HeLa ST HRP cells were made with lentivirus generated with plvx ST HRP followed by selection with 5 μg/ml puromycin (RPI). CELL BIOLOGY PNAS PLUS Guizzunti and Seemann PNAS Published online October 10, 2016 E6597

9 Drug Treatments. ST HRP expression was induced with 1 μg/ml doxycycline (Sigma) in medium A for 24 h. Twelve hours before the experiment, doxycycline was removed. Cells were arrested in S phase with 2 mm thymidine (EMD Millipore) for 16 h and released into medium A to allow cell cycle progression. MTs were depolymerized with 5 μg/ml nocodazole (EMD Millipore) for 2 h at 37 C. BFA (LC Laboratories) was used at 5 μg/ml for 60 min at 37 C. STLC (Acros Organics) was used at 20 μm to arrest cells with monoasters. Centrosomes were depleted from HeLa ST HRP cells with 125 nm centrinone (LCR-263) for at least 4 d. Upon ST HRP induction with 1 μg/ml doxycycline for 24 h in the presence of centrinone, doxycycline was removed and the cells were incubated with 2 mm thymidine for 16 h. Thymidine and centrinone were then washed out before DAB polymerization. Antibodies. The following primary antibodies were used: rabbit polyclonal against HRP (Jackson ImmunoResearch), GM130 (60), EEA1 (Santa Cruz), mouse monoclonal against α-tubulin (TAT1) (61), γ-tubulin (Sigma), GM130 (BD), nuclear pore complex proteins (Ab414) (27), BubR1 (Millipore), MPM-2 (Millipore), and Tet-repressor (MoBiTec). Secondary antibodies were as follows: Alexa Fluor 488- or Alexa Fluor 594 goat anti-mouse or goat anti-rabbit (Invitrogen) and HRP-conjugated goat anti-rabbit (Jackson ImmunoResearch). DAB Polymerization Reaction. DAB was freshly dissolved in DMSO at 100 mg/ml and used within 30 min. The cells were washed with medium A and DAB was polymerized for 4 min at room temperature after changing to medium B [DMEM, 25 mm Hepes-KOH ph 7.2, 10% (vol/vol) CCS, PenStrep] containing 0.1 mg/ml DAB and 0.003% H 2 O 2. The cells were then washed with medium A. HRP Uptake and Western Blotting. Cells were incubated with 1.5 mg/ml HRP (Worthington) in medium C (medium B without serum) for 10 min at 37 C and washed with medium A before DAB polymerization. To measure HRP activity, the cells were incubated for 20 min in 50 mm Na 2 HPO 4 /NaH 2 PO 4 ph 5.0, 0.3% Triton X-100, mmo-dianisidine dihydrochloride and 0.003% H 2 O 2. The reaction was stopped by adding NaN 3 to 10 mm and the OD was measured at 460 nm. For Western blotting, cells were solubilized with SDS sample buffer, cleared lysates were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore), and probed with anti-hrp antibodies followed by HRP-conjugated secondary antibodies. IF, Live Cell Imaging, and EM. Cells grown on glass coverslips were fixed and permeabilized in methanol at 20 C for 15 min and incubated at 37 C for 30 min with primary antibodies followed by secondary antibodies. DNA was stained with 1 μg/ml Hoechst (Invitrogen) before mounting cells in Mowiol (EMD) solution (62). Images were captured using a Axiovert 200M microscope (Zeiss) with a Plan-Neofluar 40 /1.3 differential interference contrast objective (Zeiss), a Orca-285 camera (Hamamatsu), and the software Openlab (Improvision). For phase-contrast time-lapse microscopy, cells were switched to medium D [CO 2 -independent medium (Invitrogen), 2 mm GlutaMAX (Invitrogen), 10% (vol/vol) CCS, PenStrep]. The temperature was maintained at 37 C using a XL-3 incubator (Zeiss). Phase-contrast images were captured every 10 min on a Axiovert 200 microscope with a Plan- Neofluar 10 /0.3 Ph1 objective (Zeiss), Retiga 2000R camera (Qimaging), and MetaMorph For EM, the cells were fixed for 30 min with 2.5% (wt/vol) glutaraldehyde in 0.1 M sodium cacodylate ph 7.4, treated with 1% osmium tetroxide and 1.5% (wt/vol) potassium cyanoferrate in 0.1 M cacodylate ph 7.4 for 30 min, and embedded in Epon 812 (Electron Microscope Sciences). The coverslips were removed by hydrofluoric acid. Thin sections were stained with 2% (wt/vol) uranyl acetate and lead citrate and were imaged on a Tecnai G2 Spirit EM (FEI) using a USC1000 camera (Gatan). Image Analysis and Quantitation. Image analysis was performed with ImageJ. Data from three or more independent experiments are shown as mean. Error bars represent the SEM. The statistical significance was assessed by Student s t tests. The Golgi area (Fig. 2), defined as the cellular area over which the GM130 signal is distributed, was encircled using the Freehand tool and the area was calculated using the Measure function (ImageJ). The average numbers of cells from three independent experiments per condition per experiment are presented: (Fig. 2A) control: 30, BFA: 30, DAB -> BFA: 30; (Fig. 2B) control: 24, Noc: 10, DAB -> Noc: 34; and (Fig. 2C) control: 22, Noc -> DAB: 23, Noc -> DAB -> Noc washout: 22. For analyzing the number of Golgi elements per cell, GM130-stained structures were identified by applying a fixed threshold and objects were counted using the Analyze Particles function or manually for the BFA experiments in Fig. 2A. Cells from three independent experiments per condition per experiment were analyzed: (Fig. 2A) control: 30, BFA: 30, DAB -> BFA: 30; (Fig. 2B) control: 24, Noc: 10, DAB -> Noc: 34; and (Fig. 2C) control: 22, Noc -> DAB: 23, Noc -> DAB -> Noc washout: 22. The percentage of rounded-up cells in Fig. 3 was analyzed from the average number of cells per duplicate, condition, and from three independent experiments: (Fig. 3B) control: 278+DAB: 263 and (Fig. 3F) control: 327, +DAB: 305. The percentage of cell division was determined from the average number of cells per duplicate, condition, and from three independent experiments: (Fig. 3D) control: 100 cells, +DAB: 100 cells; (Fig. 5F) DAB in the Golgi: 100 cells, DAB in endosomes: 100 cells; and (Fig. 7E) control: 40, CN: 40, DAB: 18, +CN +DAB: 15. The duration of mitosis was determined as the time elapsed from rounding up of the cell to telophase/g1. The spindle length (Fig. 6B) was measured as the shortest distance between two γ-tubulin foci using the Measure function. Mitotic cells (control: 22, +DAB: 38, +STLC: 22) of randomly selected fields from three independent experiments were analyzed. Theeffectofcentrinonetreatmentonγ-tubulin foci in Fig. 7C was determined from 50 cells per condition and from three independent experiments. ACKNOWLEDGMENTS. 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