Mitosis involves considerable membrane remodelling and vesicular trafficking to generate two independent cells. Consequently, endocytosis and endocytic proteins are required for efficient mitotic progression and completion. Several endocytic proteins also participate in mitosis in an endocytosis-independent manner. Here, we report that the sorting nexin 9 (SNX9) subfamily members – SNX9, SNX18 and SNX33 – are required for progression and completion of mitosis. Depletion of any one of these proteins using siRNA induces multinucleation, an indicator of cytokinesis failure, as well as an accumulation of cytokinetic cells. Time-lapse microscopy on siRNA-treated cells revealed a role for SNX9 subfamily members in progression through the ingression and abscission stages of cytokinesis. Depletion of these three proteins disrupted MRLCS19 localization during ingression and recruitment of Rab11-positive recycling endosomes to the intracellular bridge between nascent daughter cells. SNX9 depletion also disrupted the localization of Golgi during cytokinesis. Endocytosis of transferrin was blocked during cytokinesis by depletion of the SNX9 subfamily members, suggesting that these proteins participate in cytokinesis in an endocytosis-dependent manner. In contrast, depletion of SNX9 did not block transferrin uptake during metaphase but did delay chromosome alignment and segregation, suggesting that SNX9 plays an additional non-endocytic role at early mitotic stages. We conclude that SNX9 subfamily members are required for mitosis through both endocytosis-dependent and -independent processes.
Endocytosis is thought to shut down during mitosis then resume during the final stage, cytokinesis (Schweitzer et al., 2005). In support of this idea, several endocytic proteins including disabled-2 (Dab2), dynamin II (dynII), epsin, eps15, α-adaptin and amphiphysin II, are inactivated by Cdk1-mediated phosphorylation during mitosis (Chen et al., 1999; Chircop et al., 2010; Dephoure et al., 2008; He et al., 2003; Kariya et al., 2000). Furthermore, clathrin, epsin and cyclin-G-associated kinase (GAK) have been shown to play non-endocytic roles in chromosome alignment and segregation during mitosis (Liu and Zheng, 2009; Royle et al., 2005; Shimizu et al., 2009).
Cytokinesis results in the generation of two independent daughter cells and can be divided into two stages: (1) membrane ingression to generate a cleavage furrow between segregated chromosomes followed by (2) membrane abscission at a site along the intracellular bridge (ICB) connecting the two nascent daughter cells (Glotzer, 2005). Vesicles derived from internal organelles, including the Golgi, lysosomes, early endosomes and recycling endosomes, traffic from both daughter cells to the ICB. These vesicles accumulate asymmetrically on one side of the centrally located midbody ring (MR) and are required for abscission (Goss and Toomre, 2008). However, the role of these vesicles in abscission is unknown.
During cytokinesis, endocytosis contributes to the generation of recycling endosomes that are delivered to the ICB. Depletion or functional inhibition of dynII and Arf6 results in multinucleate cells, an indicator of cytokinesis failure (Chircop et al., 2011a; Fielding et al., 2005; Joshi et al., 2010). Arf6 depletion blocks trafficking of Rab11-positive recycling endosomes to the ICB (Fielding et al., 2005). In contrast, dynII and clathrin localise to the midbody (Chircop et al., 2010; Gromley et al., 2005; Low et al., 2003; Warner et al., 2006), suggesting that they participate more directly in the abscission process. The identification of all endocytic proteins required for mitosis and their roles during this process are yet to be revealed.
Sorting nexin 9 (SNX9) is one of the major binding partners for dynII in endocytosis (Lundmark and Carlsson, 2004). SNX9 belongs to the sorting nexin (SNX) superfamily of proteins that consists of 33 proteins in mammals (Seet and Hong, 2006). All members contain a SNX–Phox–homology (PX) domain and associate with phosphotidylinositol-3-monophosphate-enriched elements of the early endocytic network via their PX domain (Yarar et al., 2008). The SNXs function in diverse processes, including endocytosis, endosomal sorting and endosomal signalling (Carlton et al., 2005; Seet and Hong, 2006). Together with SNX18 and SNX33, SNX9 belongs to the SNX9 subfamily due to the presence of a Bin–Amphiphysin–Rvs (BAR) domain that allows dimerization and contributes to modulation and shaping of membrane curvature (Carlton et al., 2004), as well as a Src-homology 3 (SH3) domain that allows interaction with a wide range of proline-rich PXXP motif containing proteins (Alto et al., 2007; Mayer, 2001). Of the three SNX9 subfamily members, SNX9 is the best characterized. It is a major binding partner of a number of proteins involved in clathrin-mediated endocytosis (CME), such as clathrin, cargo adaptor protein-2 (AP-2), dynamin, synaptojanin, actin-related protein-2/3 (ARP2/3) complex activator, Wiskott-Aldrich syndrome protein (WASp) and Cdc42-associated kinase (ACK) (Badour et al., 2007; Lundmark and Carlsson, 2002; Lundmark and Carlsson, 2004; Shin et al., 2008; Soulet et al., 2005; Yarar et al., 2007; Yeow-Fong et al., 2005). SNX9 is also essential for dorsal-ruffle formation and clathrin-independent, actin-dependent fluid phase endocytosis (Yarar et al., 2007). Although the three SNX9 subfamily members share highly similar structures, they appear to have non-overlapping functions and it remains unclear if they can form heteromeric complexes (Dislich et al., 2011; Håberg et al., 2008). SNX18 functions in clathrin-independent endosomal trafficking that is dependent on AP1 and the retrograde trafficking protein PACS1 (Håberg et al., 2008). SNX18 also plays a redundant role to SNX9 in CME (Park et al., 2010). SNX33 has been implicated in dynamin-dependent endocytosis by modulating amyloid precursor protein (APP) endocytosis and α-secretase cleavage (Schöbel et al., 2008) and the formation of prion protein (Heiseke et al., 2008). Of the three SNX9 subfamily members, only SNX33 has been shown to play a role in mitosis, as its overexpression results in micronucleate cells (Zhang et al., 2009). Its role in mitosis is thought to be linked to its ability to regulate actin polymerization via its association with WASp (Zhang et al., 2009). Thus it is proposed to be a modulator of the actinomyosin II contractile ring for membrane ingression during cytokinesis.
In this study we aimed to determine the roles of all three SNX9 subfamily members in mitosis. We show that all are required for successful completion of both the ingression and abscission stages of cytokinesis and for the delivery of a subset of vesicles to the ICB during this process. We also show that SNX9 plays an additional role during metaphase that is not dependent on its endocytic function.
Depletion of SNX9, SNX18 or SNX33 causes multinucleation and delays mitotic exit
SNX33 is required for successful completion of mitosis (Zhang et al., 2009). Therefore, we aimed to determine whether SNX9 and SNX18 are also required for mitosis by assessing the mitotic phenotype of HeLa cells depleted of these proteins using siRNA. Two siRNAs were targeting each SNX9 subfamily member was assessed. At 72 h post-transfection, immunoblot analyses revealed satisfactory reduction in the expression level of SNX9, SNX18 and SNX33 by their specific siRNAs compared to untransfected and luciferase (Luc) siRNA controls (Fig. 1A–C). A similar reduction in the protein level of these proteins was also observed by immunofluorescence microscopy (supplementary material Fig. S1). The siRNAs were highly specific to each SNX protein as depletion of any one of the three SNX proteins by their specific siRNA did not affect expression of the other two SNX9 subfamily members (Fig. 1A–C). Depletion of any one of these proteins caused a significant increase in the percentage of multinucleate cells, an indicator of cytokinesis failure (Fig. 1D). Specifically, depletion of SNX9 resulted in a fourfold increase in multinucleation (untreated: 3.8±0.9%; Luc siRNA: 3.4±0.7%; SNX9-1 siRNA: 11.9±3.2%; SNX9-2 siRNA: 12.1±1%; Fig. 1D), whereas depletion of SNX18 or SNX33 caused a sevenfold increase in multinucleation (SNX18-1 siRNA: 20.5±1.7%; SNX18-2 siRNA: 30.4±0.5%; SNX33-1 siRNA: 17.6±2.3%; SNX33-2 siRNA: 30.5±3%; Fig. 1D). At 6 h post-mitotic entry, there was a significant increase in cells undergoing cytokinesis following depletion of SNX9 subfamily members (untreated: 2.0±0.52%; Luc siRNA: 2.8±0.4%; SNX9-1 siRNA: 8.2±3.2%; SNX9-2 siRNA: 10.9±2.8%; SNX18-1 siRNA: 10.9±0.9%; SNX18-2 siRNA: 13.6±1.5%; SNX33-1 siRNA: 15.4±1.5%; SNX33-2 siRNA: 19.3±1.5%; Fig. 1E), suggesting that depletion of any one of these proteins delays mitotic exit. Since both siRNAs assessed per protein induced similar phenotypes, SNX9-1, SNX18-1 and SNX33-1 siRNAs were used in all subsequent experiments. Depletion of all three SNX proteins produced similar phenotypes in U2OS human osteosarcoma cells (supplementary material Fig. S2). We next aimed to rescue the multinucleation phenotype induced by SNX9, SNX18 or SNX33 depletion by overexpressing GFP–SNX9 (Fig. 1F), HA–SNX18 (Fig. 1G) and GFP–SNX33 (Fig. 1H), respectively. Indeed, ectopic expression of these proteins completely rescued the multinucleation phenotype (Fig. 1F–H). We conclude that all three SNX9 subfamily members are required for successful completion of mitosis.
To further characterise the role of the SNX9 subfamily in mitosis we carried out time-lapse video microscopy to measure the time spent by individual cells in mitosis (Fig. 2A). Consistent with our fixed cell analysis (Fig. 1D,E), an increased number of SNX9-subfamily-depleted cells failed mitosis and either became binucleate or did not complete mitosis within the 20 h experimental period (Fig. 2B). Regardless of whether or not these depleted cells completed or failed mitosis they spent a longer period of time in mitosis than cells treated with Luc siRNA (Fig. 2C,D). Specifically, cells depleted of SNX9, SNX18 and SNX33 spent a median time of 210, 220 and 270 min in mitosis, respectively, compared to 120 min for Luc siRNA control cells (Fig. 2C,D). Thus, the function of these three proteins is associated with efficient mitotic progression and completion.
SNX9, 18 and 33 have multiple points of action throughout mitosis
To determine the point of action of the three SNX9 subfamily members during mitosis, we determined the time siRNA-treated cells took to progress through the following four mitotic stages: prophase to anaphase, and anaphase to completion of cytokinesis, either successfully or unsuccessfully generating a multinucleate cell (Fig. 2A). Neither SNX18 nor SNX33 appear to play a role at early mitotic stages from prophase to anaphase since cells depleted of these proteins completed this transition with similar kinetics to Luc siRNA control cells (Fig. 3A). In contrast, completion of this mitotic transition was delayed in SNX9-depleted cells, with a median time of 60 min vs 50 min for Luc siRNA control cells (Fig. 3A). Depletion of all three proteins caused a significant delay at the anaphase to completion or multinucleation transition (Fig. 3B). These findings indicate that all three SNX9 subfamily members are required for efficient progression through cytokinesis and that SNX9 may have an additional role in ensuring efficient completion of chromosome alignment and segregation in early mitosis.
We next examined the subcellular localizations of SNX9 subfamily members during mitosis. In interphase, all three proteins were observed in the cytoplasm in punctate structures (Fig. 4A–C), consistent with previous observations (Håberg et al., 2008). We also observed SNX18 and SNX33 located in the nucleus. Upon mitotic entry (prometaphase), SNX9, but not SNX18 and SNX33, accumulated at the spindle poles (Promet; Fig. 4A). SNX9 dissociated from the spindle poles upon chromosome alignment (Met; Fig. 4A) and segregation (Ana; Fig. 4A), then accumulated at the ingressing furrow during early stages of cytokinesis (Tel; Fig. 4A). In contrast, SNX18 and 33 were localized to puncta in the cytoplasm throughout mitosis (Fig. 4B,C). Towards the end of the abscission stage, a small amount of SNX9 and SNX33, but not SNX18, located to the midbody region within the ICB (Cyto; Fig. 4A,C). The ICB contains a three ring structure: the centrally located MR and twin flanking midbody rings (FMRs) that reside on either side of the MR (Chircop et al., 2010; Julian et al., 1993). Co-immunofluorescence microscopy revealed that SNX9 and SNX33 localize to a region that overlaps all three rings as indicated by their colocalization with γ-tubulin (a MR marker; Fig. 4D) and phospho-dynII (a FMR marker; Fig. 4E). SNX18 did not localize to any of the ICB rings (Fig. 4D,E). The specificity of these cell cycle specific localizations were confirmed as they were not observed in cells depleted of the relevant SNX protein (supplementary material Fig. S1). The distinct mitotic localizations of SNX9, SNX18 and SNX33 suggest that they have different mitotic roles.
Effects of the depletion of SNX9 subfamily members on endocytosis during interphase and mitosis
We next sought to determine if the mitotic role(s) of SNX9, SNX18 and SNX33 are dependent on their endocytic functions. We used the cellular uptake of Transferrin (Tfn) conjugated to Texas Red as a marker for endocytic function. We found that Tfn uptake was significantly reduced during metaphase compared to during interphase and this resumed during cytokinesis (Fig. 5), as previously reported (Schweitzer et al., 2005). Depletion of SNX9 blocked endocytosis of Tfn by >77% in interphase cells (Fig. 5), as previously reported (Lundmark and Carlsson, 2003; Soulet et al., 2005). An analogous reduction in Tfn uptake in interphase cells were also observed in SNX18 and SNX33 depleted cells (Fig. 5). This is presumably an indirect effect, due to a block in endosomal trafficking (Park et al., 2010; Schöbel et al., 2008). Tfn uptake was also blocked by the depletion of these proteins in cytokinetic cells (Fig. 5). In contrast, although Tfn uptake was already low in control metaphase cells, there was no further reduction in uptake in SNX9-subfamily-depleted metaphase cells (Fig. 5). This finding is consistent with previous reports illustrating that endocytic proteins are inactivated during mitosis. We conclude that all SNX9 subfamily proteins are required for efficient endocytosis during interphase and cytokinesis but not during metaphase.
SNX9 participates in chromosome alignment and segregation
Mitotic stages from prophase to anaphase involve chromosome condensation and alignment at the metaphase plate followed by their equal segregation. Mitotic progression through these earlier mitotic stages is slower in SNX9-depleted cells (Fig. 3A). To gain insight into the specific role of SNX9 during the earlier stages of mitosis, we again examined the progression of SNX9-depleted cells from prophase to anaphase by time-lapse video microscopy but in this case HeLa cells stably expressing GFP–H2B were analysed to clearly define each mitotic stage. Moreover, cells were observed at 2 min intervals (instead of 10 min) to more accurately assess the point of mitosis that is delayed. Similar to the results shown in Fig. 3A, HeLa-GFP–H2B cells depleted of SNX9 also spent a significantly longer period of time in the prophase to anaphase transition with a median time of 75 min (SNX9-1 siRNA) and 71 min (SNX9-2 siRNA) vs 38 min in untreated cells (Fig. 6A). We next separated this mitotic transition into distinct events: chromosome alignment (prophase–metaphase) and chromosome segregation (metaphase–anaphase). The results indicated that SNX9 plays a role in both mitotic events as HeLa-GFP–H2B cells depleted of SNX9 were delayed in their mitotic progression through both transitions [median time of 34 min (SNX9-1 siRNA) and 38 min (SNX9-2 siRNA) vs 20 min for untreated cells during prophase–metaphase (Fig. 6B); median time of 31 min (SNX9-1 siRNA) and 34 min (SNX9-2 siRNA) vs 22 min for untreated cells during metaphase–anaphase; Fig. 6C]. Again, consistent with our findings in HeLa cells (Fig. 3), depletion of SNX18 or SNX33 did not affect mitotic progression of HeLa-GFP–H2B cells through these mitotic phases (Fig. 6A–C).
We next investigated the requirement of SNX9 in regulating chromosome dynamics. The width of the centre of the metaphase plate was significantly wider in metaphase HeLa-GFP–H2B cells that were depleted of SNX9 compared to untreated cells (Fig. 6D,G). Consistent with the time-lapse data indicating that SNX18 and SNX33 are not required for progression from prophase to anaphase, the width of the metaphase plate was not affected in HeLa-GFP–H2B cells – depleted of these proteins (Fig. 6E–G). These findings indicate that the metaphase function of SNX9 contributes to chromosome congression and alignment.
Role of SNX9, SNX18 and SNX33 during the ingression and abscission phases of cytokinesis
Cleavage furrow formation during the ingression phase of cytokinesis is driven by the activity of the actinomyosin II contractile ring, which assembles at the cell equator between segregated chromosomes. Myosin regulatory light chain (MRLC) is a component of the myosin II filaments, and its phosphorylation on T18 and S19 drives the assembly of the actinomyosin II contractile ring (Ikebe et al., 1988) and activity of the myosin II motor (Komatsu et al., 2000; Yamakita et al., 1994). Phosphorylation of the major site, S19, allows myosin II to interact with actin to assemble an actin–myosin II complex and initiate contraction (Scholey et al., 1980). Given that all three SNX9 subfamily members are required for cytokinesis (Fig. 3B), we assessed the activity of the myosin II motor using an antibody that specifically recognises MRLC phosphorylated at S19. MRLCS19 localization was disrupted in SNX9-subfamily-depleted cells during anaphase and telophase (Fig. 7A). In contrast to control cells where MRLCS19 was concentrated at the ingressing furrow during anaphase and telophase, it was diffusely distributed in the cytoplasm in cells depleted of any one of the SNX9 subfamily proteins (Fig. 7A). The ratio of the fluorescence intensity of MRLCS19 staining at the ingressing furrow or cleavage furrow:the fluorescence intensity of MRLCS19 at the polar region was used to quantitate this effect (Fig. 7A, right panel). A significant reduction of >40% in MRLCS19 fluorescence signal at the ingressing furrow and cleavage furrow was detected (Fig. 7A–C). This was not due to an overall loss of protein as MRLCS19 levels were not significantly different in depleted cells compared to untreated controls cells (supplementary material Fig. S3). These findings indicate that all SNX9 subfamily members contribute to the efficient accumulation of active myosin II at the cleavage furrow during ingression.
All three SNX9 members are required for endocytosis (Fig. 5) and vesicle trafficking is required for the abscission phase of cytokinesis. Therefore, we next assessed the role of these proteins in regulating vesicle trafficking from the Golgi, recycling endosomes (RE), lysosomes and endoplasmic reticulum (ER) to the ICB. In untreated cells, GM130 (a Golgi marker) was observed as a network of punctate structures distributed throughout the cytoplasm in metaphase (Fig. 8A, Met), consistent with the early stages of Golgi reformation (Sütterlin and Colanzi, 2010). During cytokinesis, GM130 accumulated in two pools in each of the nascent daughter cells (Fig. 8A, Cyto), as previously reported (Gaietta et al., 2006). Depletion of SNX9 resulted in an accumulation of GM130 at the inner pool, as indicated by an increase in the ratio of the fluorescence intensity of the inner pool:fluorescence intensity of the outer pool in each nascent daughter cell (Fig. 8A, bottom panel; Fig. 8B). The overall protein level of GM130 was unaffected by the depletion of SNX9 during metaphase (supplementary material Fig. S4A) or cytokinesis (supplementary material Fig. S4B), suggesting that SNX9 is involved in vesicle trafficking from the Golgi to the ICB. Depletion of SNX18 and SNX33 had no effect on the distribution and the level of GM130 during metaphase and cytokinesis (Fig. 8A,B; supplementary material Fig. S4).
We next examined the recycling endosomes (RE). The RE marker, Rab11, localized at the spindle poles and the cleavage furrow during telophase and along the ICB during cytokinesis in untreated cells (Fig. 8C), as previously observed (Wilson et al., 2005). Depletion of any one of the SNX9 members disrupted Rab11 localization at both mitotic stages (Fig. 8C–E) but did not affect the overall level of Rab11 in telophase (supplementary material Fig. S5A) or cytokinesis (supplementary material Fig. S5B). In contrast, the level and localization of PDI (an ER marker) and Lamp1 (a lysosome marker) were unaffected by the depletion of these proteins (supplementary material Figs S6, S7). We conclude that all three SNX9 members are required for localization of the recycling endosomes to the cleavage furrow and ICB during cytokinesis. SNX9 plays an additional role in the accumulation of Golgi at the ICB during abscission.
In this study, we confirmed the requirement of SNX33 for mitotic progression and completion. In addition, our findings show that its two closest-related SNX members, SNX9 and SNX18, also play important roles in mitosis. Collectively, these three SNX9 subfamily members are essential for efficient progression and completion of cytokinesis. These proteins are also required for endocytosis during these mitotic phases, suggesting that they contribute to vesicle trafficking during cytokinesis. Indeed, all three proteins are required for the correct localization of recycling endosomes during cytokinesis. SNX9 also contributes to the localization of Golgi to the ICB. Our additional major finding indicates that SNX9 is also required for chromosome alignment and segregation during metaphase and anaphase. SNX9 does not participate in endocytosis during metaphase, suggesting that its role in this process is independent of its endocytic function. These findings demonstrate that SNX9 subfamily proteins participate in multiple stages throughout mitosis in both endocytic-dependent and -independent manners.
Only SNX9 was shown to have a role during metaphase. The increased width of the metaphase plate observed in SNX9-depleted cells supports a role for SNX9 in chromosome alignment. Consistent with this role, it is found to be enriched at the spindle poles during prometaphase. Clathrin is a major binding partner for SNX9 in endocytosis (Lundmark and Carlsson, 2003) and is also required for chromosome alignment and segregation (Royle et al., 2005). Like SNX9, its role in this process is independent of its role in endocytosis (Royle et al., 2005). During metaphase, clathrin localizes to the mitotic spindle and spindle poles (Royle et al., 2005). Depletion or mutation of clathrin leads to destabilization of the kinetochore fibres, which results in a defect in chromosome alignment and delay in chromosome segregation (Royle et al., 2005). This results in persistent activation of the spindle assembly checkpoint. It will be interesting to determine if SNX9 is also involved in stabilizing kinetochore fibres and if it co-operates with clathrin at the mitotic spindle for chromosome alignment and segregation, thus providing further mechanistic insight into the non-endocytic mitotic function(s) of these proteins.
All SNX9 subfamily members participate in cytokinesis. Our findings suggest that these proteins function in an endocytic-dependent manner during this mitotic phase. However, they may also participate in cytokinesis in an endocytic-independent manner. Consistent with this idea, depletion of any of the three SNX9 members prevented accumulation of active myosin II at the cleavage furrow and delayed progression through the ingression phase. SNX33 has previously been shown to be required for cytokinesis and its role in this process was hypothesized to be due to its ability to regulate actin polymerization via interaction with WASp (Badour et al., 2007; Lundmark and Carlsson, 2003; Zhang et al., 2009). SNX9 and SNX18 also bind WASp and regulate actin dynamics (Badour et al., 2007). During clathrin-mediated endocytosis, SNX9 is thought to recruit N-WASP to the plasma membrane, which in turns stimulates actin recruitment and polymerization (Cullen, 2008). Thus, it is possible that SNX9 and its subfamily members may contribute to the recruitment of WASp and actin polymerization for efficient formation and activity of the actinomyosin II contractile ring at the ingressing cleavage furrow. In line with this idea, depletion of any one of the three SNX9 members resulted in disruption of MRLCS19 cleavage furrow localization, which is predicted to disrupt contractile ring function.
The role of SNX9 members during cytokinesis appears to be dependent on their endocytic activity. Supporting this idea, we have shown that depletion of any one of these proteins delays or blocks cytokinesis and also blocks Tfn uptake during this process. Endocytosis is thought to contribute to the generation of recycling endosomes that are delivered to the ICB for abscission. We show that Rab11-positive recycling endosomes do not accumulate at the cleavage furrow and ICB in cytokinetic cells depleted of the SNX9 subfamily members. This could be due to a block in (i) the generation of recycling endosomes, (ii) endocytic activity at internalized endosomes or (iii) recruitment of vesicles derived from recycling endosomes to the cleavage furrow and ICB.
SNX9 and SNX33 may also participate in abscission more directly. We show that these proteins localise to the FMRs and MR within the ICB. We have recently shown that dynII locates to the FMRs (Chircop et al., 2010) and functions here in an endocytic-independent manner for abscission (Chircop et al., 2011b). Numerous proteins that locate to the MR are involved in protein and vesicular recruitment. Some of these include the endocytic proteins, Arf6, and components of the ESCRT machinery. Thus, it is possible that SNX9 and SNX33 are involved in recruiting key proteins and/or vesicles to the abscission site. Not only did the depletion of all three SNX9 members prevent recruitment of Rab11-positive recycling endosomes to the ICB, but depletion of SNX9 also caused an accumulation of Golgi at the edge of the ICB. We hypothesize that SNX9 is required for the recruitment of Golgi-derived vesicles to the ICB. This was highly specific as the localization of vesicles derived from lysosomes or the ER was unaffected. The exact role of vesicles at the ICB is unclear, however they are proposed to (i) provide extra total cell surface area, an increase of at least 25% is required to complete division (Boucrot and Kirchhausen, 2007), (ii) deliver critical cytokinetic proteins to the abscission site (Low et al., 2003), and/or (iii) be directly involved in compound fusion, whereby numerous vesicles fuse with the plasma membrane during abscission to separate the daughter cells (Goss and Toomre, 2008; Gromley et al., 2005; Low et al., 2003; Prekeris and Gould, 2008). The role of SNX9 subfamily members in this process is critical to understand as cytokinesis failure leads to aneuploidy.
Overall, our findings revealed that the three SNX9 subfamily proteins are involved in ensuring efficient progression and completion of mitosis at several distinct stages. We have shown that all three SNX9 subfamily members most likely function during cytokinesis in an endocytic-dependent manner. We have further revealed a non-endocytic function for SNX9, specifically in chromosome alignment.
Materials and Methods
Cell culture and transfection
HeLa human cervical carcinoma cells and HeLa cells stably expressing GFP–H2B were maintained in RPMI 1640 medium. U2OS human osteosarcoma cells were maintained in DMEM medium. All medium were supplemented with 10% foetal bovine serum (FBS) and cells were grown at 37°C in a humidified 5% CO2 atmosphere. Cells were seeded at 50–60% confluence (1×105 cells per 10 cm dish, 0.5×105 cells per well of a 6-well plate; 0.2×105 cells per well of a 12-well plate). For siRNA analyses, cells were transfected with 1000 pmol of siRNA (per 10 cm dish for immunoblotting), 200 pmol of siRNA (per well of a 6-well plate for immunofluorescence and time-lapse microscopy experiments) or 100 pmol (per well of a 12-well plate for immunofluorescence and time-lapse microscopy experiments). For DNA transfections, 1.5 µg of the indicated plasmid DNA was used in each well of a 6-well plate. In both cases, cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Cell synchronization and treatment
HeLa, HeLa-GFP-H2B and U2OS cells grown on glass coverslips were synchronized at the G2–M border by treatment with the selective Cdk1 small-molecule inhibitor RO-3306 (9 µM) for at least 18 h. Cells were allowed to progress through mitosis upon RO-3306 wash out. Following RO-3306 wash out, cells were incubated at 37°C/5% CO2 for 80 min (metaphase), 2.5 h (cytokinesis) or 6 h (multinucleation and cytokinesis accumulation scoring).
GFP-SNX9 was a gift from Sandra Schmid (Soulet et al., 2005). HA-SNX18 and GFP-SNX33 constructs were kindly provided by Stefan F. Lichtenthaler (Dislich et al., 2011). The siRNA target sequences in the sense orientation for the following proteins are: SNX9-1: 5′-AACCUACUAACACUAAUCGAU-3′ (Lundmark and Carlsson, 2004); SNX9-2: 5′-AACAGUCGUGCUAGUUCCUCA-3′ (Shin et al., 2008); SNX18-1: 5′-CGUCAUGGACCUAUUAGCGCUGUAU-3′; SNX18-2: 5′-CACCGACGAGAAAGCCUGGAAUU-3′ (Qiagen); SNX33-1: 5′-CAAGAUCGCUGAGACAUACUCCA-3′; SNX33-2: 5′-CACACGGGCCGUACCUAUUGAA-3′; Luciferase: 5′-CGUACGCGGAAUACUUCGA-3′.
HeLa cells were transfected with siRNA targeted the indicated SNX9 subfamily member using Neon Transfection System as per manufacturer's instruction (Invitrogen). On the next day, the indicated plasmid construct (GFP-SNX9, HA-SNX18 and GFP-SNX33) were transfected using the Neon Transfection System (Invitrogen) to overexpress the relevant SNX9 member in the appropriate siRNA-depleted cells. For western blot analyses, cells were collected 24 h post-transfection. For multinucleation analyses, cells were synchronized with RO-3306 for 18 h followed by RO-3306 wash out for 6 h.
Time-lapse microscopy analyses
Immediately following release into the cell cycle G2–M synchronized HeLa or HeLa-GFP–H2B cells were viewed with an Olympus IX81 inverted microscope and a time-lapse series was acquired using a fully motorized stage, 10× objective, and Metamorph software using the Time-lapse modules as previously described (Chircop et al., 2010; Joshi et al., 2010). Temperature control was achieved using the Incubator XL, providing a humidified atmosphere with 5% CO2. Imaging was performed for 20 h with a lapse time of 2 min or 10 min as indicated.
Cells were fixed in ice-cold 100% methanol for 3 min at −20°C and blocked in 3% bovine serum albumin (BSA)/PBS for 45 min prior to incubation with the primary antibody. For membrane trafficking studies, cells were fixed in ice-cold 4% PFA in PBS for 20 min and then permeabilised with 0.2% Triton X-100 in PBS for 15 min before blocking in 3% BSA/PBS for 45 min prior to primary antibody incubation. The following antibodies were used: anti-SH3PX1 (anti-SNX9, NB100-2813, Novus Biological), anti-SNX18 (GTX106319, GeneTex), anti-SH3PX3 (anti-SNX33, H00257364-D01P, Abnova), anti-Lamp1 (ab25630, Abcam), anti-PDI (ab2792, Abcam), anti-Rab11 (610657, BD Transduction), anti-phospho-myosin light chain S19 (3675S, Cell Signaling), anti-GM130 (610822, BD Transduction), anti-dynI phospho-S778 which detects dynII phospho-S764 (Anggono et al., 2006), anti-α-tubulin (clone DM1A; Sigma) and anti-γ-tubulin (GTU88; Sigma). Fluorescein- or Texas-Red-conjugated AffiniPure secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) were then applied. Cell nuclei were counterstained with DAPI (4′6′-diamidino-2-phenylindole; Sigma). Cells were washed three times with PBS between each step except for after blocking. Cells were viewed and scored with a fluorescence microscope (Olympus IX80). Fluorescence images were captured under an Olympus IX81 inverted microscope using 60× or 100× oil immersion lenses and deconvolved using AutoDeblur v9.3 (AutoQuant Imaging, Watervliet, NY). Integrated fluorescence intensity was measured using Metamorph software (Version 18.104.22.168.).
Cellular extracts were prepared as described previously (Fabbro et al., 2004). In brief, cells were collected by centrifugation, washed with PBS, then resuspended in ice-cold lysis buffer for sonication [25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1% Triton X-100, and EDTA-free Complete protease inhibitor cocktail (Roche)] followed by incubation on ice for 30 min. The supernatant was collected following centrifugation at 13,000 rpm for 30 min at 4°C. Cell lysates (200 µg) were fractionated by SDS-PAGE for immunoblot analysis with the following antibodies: anti-SNX9 (Santa Cruz), anti-SNX18 (GeneTex), anti-SNX33 [a gift from Stefan F. Lichtenthaler (Dislich et al., 2011)], anti-GFP (Clonetech), anti-HA.11 (Covance) and anti-γ-tubulin (Sigma). Antibody bound to the indicated protein was detected by incubation with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.). Blotted proteins were visualized using the ECL detection system (Pierce).
Quantitative high-throughput receptor-mediated endocytosis (RME) assays were performed as previously described (Hill et al., 2009; Odell et al., 2010) using Transferrin (Tfn) conjugated to Texas Red in untreated and siRNA-transfected HeLa cells for 10 min. Cells were then washed with ice-cold PBS and acid washed (0.2 M acetic acid/0.5 M NaCl, pH 2.8) for 15 min on ice. Cells were then fixed with ice-cold 4% PFA in PBS for 15 min and processed for immunofluorescence microscopy analysis as described above. Cells were imaged on an ImageXpressmicro platereader (Molecular Devices) and Tfn uptake was analysed using MetaXpress software (V22.214.171.124, Molecular Devices) by calculating the cell:vesicle integrated intensity.
We thank Sandra Schmid for providing the GFP-SNX9 constructs and Stefan F. Lichtenthaler for providing the anti-SNX33 antibody, as well as HA-SNX18 and GFP-SNX33 constructs. Scott L. Page is thanked for his technical assistance. We also thank Phillip J. Robinson, Rose Boutros and Scott L. Page for critic reading of the manuscript.
This work was supported by grants from the National Health and Medical Research Council of Australia [grant number 477102 to M.C.] and the National Health and Medical Research Council Biomedical Career Development Award Fellowship [grant number 477104 to M.C.].
Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.105981/-/DC1
- Accepted May 24, 2012.
- © 2012. Published by The Company of Biologists Ltd