Abstract
Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin), increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through γ-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.
Introduction
As the primary microtubule-organizing centers, centrosomes are vital for the maintenance of genomic integrity in animal cells (Nigg and Stearns 2011). The centrosome consists of two barrel-shaped centrioles surrounded by a network of proteins termed pericentriolar materials (PCM). To maintain the fidelity of cell division, each cell must duplicate a pair of centrioles precisely once per cell cycle, one daughter per mother centriole. Mishaps in centrosome assembly result in chromosome missegregation and other cell cycle defects. Thus, stringent regulation of centrosome assembly is imperative for proper cell division and survival.
Studies in C. elegans have discovered five evolutionarily conserved proteins (ZYG-1, SPD-2, SAS-4, SAS-5 and SAS-6) that are required for centrosome assembly (Delattre et al., 2006; Habedanck et al., 2005; Pelletier et al., 2006; Shimanovskaya et al., 2014). Many other factors, including protein phosphatase 2A, also regulate the production, activity, or turnover of core regulators, and are equally important in regulating centrosome assembly (Kitagawa et al., 2011; Song et al., 2011). Like other biological processes, centrosome assembly is regulated by a combined action among negative and positive regulators (Kemp et al., 2007). While the kinase ZYG-1 promotes centriole duplication in C. elegans, szy-20 acts as a genetic suppressor of zyg-1 (O’Connell et al., 2001; Song et al., 2008). The szy-20 gene encodes a centrosome-associated RNA-binding protein that negatively regulates centrosome assembly by opposing ZYG-1. Centrosomes in szy-20 mutants exhibit elevated levels of centrosomal proteins, resulting in defective microtubule (MT) behavior and embryonic lethality. SZY-20 contains putative RNA-binding domains (SUZ, SUZ-C). Mutating these domains has been shown to perturb in vitro RNA-binding of SZY-20 and its capacity to regulate centrosome size in vivo (Song et al., 2008). Other studies have shown that a number of RNAs and RNA-binding proteins are associated with centrosomes and MTs, and influence proper mitotic spindles and other aspects of cell division. In mammalian cells, several RNA-binding proteins (e.g., RBM8A, Hu antigen R, and the Ewing sarcoma protein) associate with centrosomes and play a role in regulating centrosome assembly during cell division (Castro et al., 2000; Filippova et al., 2012; Ishigaki et al., 2014; Leemann-Zakaryan et al., 2009; Ugrinova et al., 2007). In yeast, spindle pole body duplication is linked to translational control via the action of RNA-binding proteins (Sezen et al., 2009). In Xenopus, MT-guided localization of transcripts followed by spatially enriched translation is important for proper MT behavior and cell division, suggesting the importance of local translational control (Blower et al., 2005; Blower et al., 2007; Groisman et al., 2000).
Despite the finding that SZY-20 negatively regulates ZYG-1, no direct interaction between the two proteins has been found. Thus, identifying additional factors that function between SZY-20 and ZYG-1 should provide further insights into the molecular mechanism by which the putative RNA-binding protein, SZY-20, influences centrosome assembly. Toward this end, we report here our identification of an RNA-binding protein ATX-2 that physically associates with SZY-20. ATX-2 is the C. elegans ortholog of human Ataxin-2 that is implicated in human neurodegenerative disease (Lastres-Becker et al., 2008). Specifically, human spinocerebellar ataxia type 2 is shown to be associated with an extended poly-glutamine (Q) tract in Ataxin-2 (Elden et al., 2010; Huynh et al., 1999; Lastres-Becker et al., 2008). Ataxin-2 is an evolutionarily conserved protein that contains an RNA-binding motif (LSm: Sm-like domain) and a PAM domain for binding the poly-(A) binding protein (PABP1) (Achsel et al., 2001; Jimenez-Lopez and Guzman 2014; Satterfield and Pallanck, 2006). It has been shown that Ataxin-2 binds directly to the 3’UTR of mRNAs and stabilize target transcripts, and that poly-Q expansion blocks the RNA-binding by Ataxin-2 in vitro (Yokoshi et al., 2014). Ataxin-2 homologs have been implicated in a wide range of RNA metabolism-dependent processes including translational control of circadian rhythm (Zhang et al., 2013; Lim and Allada, 2013). While ATX-2 in C. elegans is known to be responsible for embryonic development and translational control in germline development (Ciosk et al., 2004; Kiehl et al., 2000; Maine et al., 2004; Skop et al., 2004), the action of this RNA-binding protein in centrosome assembly and cell division has not been fully explored.
In this study, we investigate the role of C. elegans ATX-2 in early cell cycles and how ATX-2 acts together with SZY-20 and ZYG-1 in controlling centrosome size and MT behavior. We show that ATX-2 negatively regulates the key centriole factor ZYG-1. In the centrosome assembly pathway, SZY-20 acts upstream of ATX-2 and positively regulates embryonic levels of ATX-2; proper levels of ATX-2 contributes in turn to normal centrosome size and subsequent MT dynamics.
Results
ATX-2 physically interacts with SZY-20 in vivo independently of RNA
To further elucidate the role of SZY-20 in regulating centrosome assembly, we looked for additional factors interacting with SZY-20. By immunoprecipitating (IP) endogenous SZY-20 from worm protein lysates with anti-SZY-20, followed by mass spectrometry, we generated a list of proteins associated with SZY-20 in vivo. As for a putative RNA-binding protein SZY-20, we found many known RNA-binding proteins co-precipitated with SZY-20, including ATX-2 (11 peptides, 19% coverage = 181/959aa) and PAB-1 (15 peptides, 32% coverage = 206/646aa) (S1A Fig). In C. elegans, ATX-2 is shown to form a cytoplasmic complex with PAB-1 (Ciosk et al., 2004), and the ATX-2-PAB-1 interaction is conserved from yeast to mammals (Ciosk et al., 2004; Nonhoff et al., 2007; Satterfield and Pallanck, 2006; Yokoshi et al., 2014). pab-1 encodes Poly-A-binding protein, a C. elegans homolog of a human PABP1 that plays a role in RNA stability and protein translation (Ciosk et al, 2004; Kahvejian et al., 2005).
To confirm the physical interaction between SZY-20 and ATX-2, we used anti-SZY-20 to pull down SZY-20 and its associated proteins from embryonic lysates and examined co-precipitates by western blot (Fig 1A). Consistent with our mass spectrometry data, we detected ATX-2 and SZY-20 in the SZY-20-immunoprecipitates from wild-type embryonic extracts. Given that this protein complex consists of RNA-binding proteins, we asked if the physical association is mediated through RNA. To test RNA dependence, we repeated IP to pull down SZY-20 interacting proteins in the presence of RNaseA or RNase inhibitor and found that they co-precipitated in either condition (Fig 1A, S1B Fig), suggesting ATX-2 and SZY-20 physically interact in an RNA-independent manner although we cannot exclude the possibility that RNA bound by the ATX-2-SZY-20 complex could have been protected from RNase treatment.
The szy-20(bs52) mutation results in a truncated protein, deleting the C-terminal 197 aa residues including the SUZ-C domain, one of the putative RNA-binding domains in SZY-20 (Song et al., 2008). We utilized szy-20(bs52) embryos for IP analysis to determine if the C-terminal truncation of SZY-20 affected physical association of SZY-20 with ATX-2 (S1B Fig).
Whereas ATX-2 co-precipitated with SZY-20 in wild-type extracts, ATX-2 was undetectable in co-precipitates from szy-20(bs52) extracts, suggesting that the C-terminus of SZY-20 influences physical interaction with ATX-2. IP assay using embryos expressing SZY-20-GFP-3xFLAG yielded a similar result: ATX-2 is undetectable in co-precipitates of SZY-20 tagged with GFP-3xFLAG at the C-terminus (S1E Fig), supporting that proper folding of the C-terminal domain is critical for SZY-20 to interact with ATX-2. Further, the C-terminal deletion in either ATX-2 or SZY-20 appears to alleviate its interaction with SZY-20 (S1C and S1D Fig). By additional IP assays with anti-GFP using embryos expressing various GFP-tagged proteins, we further confirmed that both ATX-2 and PAB-1 physically interact with SZY-20 or with each other (S1E Fig). Together, our data suggest SZY-20 forms a complex with known RNA-binding proteins ATX-2 and PAB-1 in vivo, via direct or indirect interaction.
ATX-2 is required for proper cell division during embryogenesis
We next asked if these SZY-20 interacting proteins play a similar role to SZY-20 during early cell division. In C. elegans, ATX-2 is required for translational control in gonadogenesis and for normal cytokinesis during embryogenesis (Ciosk et al., 2004; Kiehl et al., 2000; Maine et al., 2004; Skop et al., 2004). To date, PAB-1 is only known to be required for germline development (Ciosk et al., 2004; Ko et al., 2013). Consistent with prior studies, we observed strong embryonic lethality in both atx-2(RNAi) and atx-2(ne4297) but a very low embryonic lethality in pab-1(RNAi), and sterility by loss of pab-1 or atx-2 (Fig 1B).
Embryonic lethality by loss of atx-2 might result from defective cell division. To examine what role ATX-2 plays in cell division, we immunostained embryos for microtubules (MTs), centrosomes and DNA (Fig 1D-1H). Confocal microscopy of immunofluorescence (IF) revealed that knocking down atx-2 by RNAi results in multiple cell division defects including polar body extrusion failure (22%; S3 Movie), abnormal spindle positioning (3%), chromosome missegregation (10%) and cytokinesis failure (36%; n=114). We observed similar cell division phenotypes, but with higher penetrance in temperature sensitive (ts) atx-2(ne4297) mutants. By 4D time-lapse confocal microscopy, we observed that incomplete cytokinesis following successful centrosome duplication results in tetrapolar spindles in one-cell embryo (S1 and S2 Movie). In these embryos, the cytokinetic furrow initiates but cytokinesis fails to complete, resulting in a multi-nucleated cell with four centrosomes after the second mitosis. All of these cell division phenotypes resemble cell cycle defects observed previously in szy-20(bs52) embryos (Song et al, 2008), suggesting that ATX-2 functions closely with SZY-20 in cell division. In contrast, pab-1(RNAi) produced only minor cell cycle defects such as atypical spindle positioning (S2A Fig).
Since ATX-2 appears to play a similar role to SZY-20, we then asked if atx-2 genetically interacts with szy-20. Genetic analysis by combining atx-2(RNAi) or pab-1(RNAi) with a hypomorphic mutant allele szy-20(bs52) suggest a positive genetic interaction among these factors (Fig 1B, Table 1). At 24°C, the semi-restrictive temperature, szy-20(bs52) animals fed with atx-2(RNAi) produced higher embryonic lethality than single knockdown of either szy-20(bs52) or atx-2(RNAi). Similarly, pab-1(RNAi) in szy-20(bs52) led to a synergistic increase in embryonic lethality compared to szy-20(bs52) or pab-1(RNAi) alone.
Cytological analyses further confirmed the functional interaction among these factors. Double-knockdown significantly enhanced the penetrance of cell division phenotypes, compared to mock-treated szy-20(bs52) or RNAi alone: 55% (n=128) of szy-20(bs52); atx-2(RNAi) embryos exhibit a failure of polar body extrusion, compared to 22% (n=114) of atx-2(RNAi) and 18% (n=145) of szy-20(bs52). This synergistic effect is even stronger for cytokinesis: 36% of atx-2(RNAi) and 6% of szy-20(bs52), but 68% of szy-20(bs52); atx-2(RNAi) embryos failed to complete cytokinesis at first division (Fig 1C, 1H). Consistent with embryonic lethality, pab-1(RNAi); szy-20(bs52) produced highly penetrant cytokinesis defect, in contrast to the weak phenotype by pab-1(RNAi) alone (Fig 1C). Combining pab-1(RNAi) and szy-20(bs52) mutation produced 82% (n=68) of cytokinesis failure and 24% (n=68) of polar body extrusion failure, while pab-1(RNAi) (n=68) alone showed a minor cell cycle defect (S2A Fig). In addition, atx-2(RNAi) or pab-1(RNAi) enhances sterility in szy-20(bs52) animals, consistent with the sterility associated with all three genes. Together, our data suggest that RNA-binding proteins ATX-2 and PAB-1 function in close association with SZY-20 during embryogenesis.
ATX-2 negatively regulates centrosome assembly
As szy-20 is known as a genetic suppressor of zyg-1 (Song et al., 2008), we asked if ATX-2/PAB-1 functions in centrosome assembly as well. To address this, we first examined centrosome duplication in zyg-1(it25) embryos fed with atx-2 or pab-1(RNAi) at 24°C (Fig 2). When grown at 24°C the restrictive temperature, zyg-1(it25) embryos fail to duplicate centrosomes during the first cell cycle, producing monopolar spindles at the second cell division (O’Connell et al., 2001). By recording live imaging of zyg-1(it25) embryos expressing GFP-α-Tubulin, mCherry-γ-Tubulin and mCherry-Histone, we scored centrosome duplication at the second mitosis (Fig 2A-2C). Over 60% of atx-2 or pab-1(RNAi) treated zyg-1(it25) embryos produced bipolar spindles, indicating successful centrosome duplication during the first cell cycle, while only 3% of mock treated zyg-1(it25) embryos formed bipolar spindles. We noticed, however, that a great majority of atx-2(RNAi) treated zyg-1(it25) embryos exhibit four centrosomes in one-cell embryo after the second cell cycle, suggestive of cytokinesis failure due to loss of atx-2 (Fig 2C, S1 and S2 Movie). Consistent with RNAi-mediated knockdown, we made a similar observation in zyg-1(it25); atx-2(ne4297) double mutants (S3A and S3B Fig).
Given the positive genetic interactions among szy-20, atx-2 and pab-1, we further asked if co-depleting these factors could enhance the suppression of zyg-1 (Fig 2D, 2E). At 24°C, co-depleting either atx-2 or pab-1 with szy-20 restored nearly 100% of centrosome duplication to zyg-1(it25) embryos, while single depletion produced ~60% duplication in zyg-1(it25). Despite the restoration in centrosome duplication at 24°C, none of these embryos hatched, owing to other cell cycle defects such as cytokinesis failure described above. However, we were able to show partial restoration of embryonic viability in zyg-1(it25) by co-depleting either atx-2 or pab-1 with szy-20 at semi-restrictive temperature 23°C, whereas single depletion of atx-2 or pab-1 showed no effect on embryonic viability of zyg-1(it25). Our data indicate that like szy-20, atx-2 and pab-1 act as genetic suppressors of zyg-1. Thus, these RNA-binding proteins in a complex function together to negatively regulate centrosome assembly.
Finally, we asked if any RNA-binding protein could suppress zyg-1 through global translational control. We chose the RNA-binding protein CAR-1 that is required for cytokinesis (Audhya et al., 2005; Squirrell et al., 2006) to test if car-1 suppresses zyg-1 (S3C Fig). We found no sign of centrosome duplication in car-1(RNAi); zyg-1(it25) embryos, while atx-2(RNAi) restored centrosome duplication to zyg-1(it25) in a parallel experiment. This result suggests that RNA-binding proteins are not general suppressors of zyg-1, but that actions of RNA-binding proteins (SZY-20/ATX-2/PAB-1) are specific to the ZYG-1 dependent centrosome assembly pathway.
SZY-20 promotes ATX-2 abundance in early embryos
To understand how these RNA-binding proteins coordinate their actions within the cell, we examined the subcellular localization in early embryos. Immunostaining embryos with α-ATX-2 shows a diffuse pattern throughout the cytoplasm with occasional small foci; as expected, this signal is significantly diminished in atx-2(RNAi) or atx-2(ne4297) embryos (Fig 3A, 3B). We also generated a transgenic strain expressing atx-2-gfp-3x flag (S1 Table) and observed the dynamics of ATX-2-GFP using time-lapse movies, which shows consistent patterns to endogenous ATX-2 (S4 Movie). C. elegans PAB-1 is also found in the cytoplasm and is often associated with P-bodies or stress granules (Gallo et al., 2008) and SZY-20 localizes to the nuclei, centrosome and cytoplasm in early embryos (Song et al., 2008). Co-staining embryos show that ATX-2 partially coincides with cytoplasmic SZY-20 (Fig 3C, S2B Fig) or PAB-1 (Fig 3D, S2C Fig). A similar co-localization is also observed for GFP-PAB-1 and SZY-20 (S2D Fig). Thus, these RNA-binding proteins appear to function together in the cytoplasm to regulate early cell division.
To gain insights into the hierarchical relationship among these factors, we used mutant strains to determine how the embryonic expression is affected by the other factors (Fig 3E-3I). Quantitative immunofluorescence revealed that ATX-2 levels in the cytoplasm are significantly reduced (~50%) in the hypomorphic szy-20(bs52) embryo relative to wild-type control (Fig 3E, 3H), while the zyg-1(it25) mutation had no significant effect on the cytoplasmic ATX-2 (Fig 3E, 3I). SZY-20 localization was, however, unaffected by either atx-2 or pab-1 depletion (S2D Fig). Consistently, prior study showed that szy-20 acts upstream of zyg-1 (Song et al., 2008). Thus, it seems likely that szy-20 function upstream of atx-2, with both acting upstream of zyg-1. Quantitative western blots further support this hierarchical relationship (Fig 3J-3L). szy-20(bs52) embryos exhibit significantly reduced ATX-2 levels (~40%; Fig 3K) relative to wild-type control, while atx-2(ne4297) embryos show normal levels of SZY-20 (~98%; Fig 3L) and zyg-1 mutants contain normal levels of both ATX-2 and SZY-20 (Fig 3J-3L). However, no significant changes are found in the level of a centriole factor, SAS-6 in either mutant embryo (Fig 3J). Taken together, our data suggest that SZY-20 acts upstream of ATX-2, and positively regulates ATX-2 abundance in early embryos.
Loss of atx-2 leads to increased levels of centrosomal ZYG-1
Because atx-2 acts as a genetic suppressor of zyg-1, we reasoned that inhibiting ATX-2 might enhance ZYG-1 activity, thereby restoring centrosome duplication and embryonic viability to zyg-1(it25) embryos. By staining embryos for ZYG-1 and microtubules (Fig 4A), we quantified the fluorescence intensity of ZYG-1 at first metaphase centrosomes, finding that atx-2 mutant centrosomes possess twice as much ZYG-1 levels as those in control embryos (p <0.001, Fig 4B). Using the CRISPR-Cas9 method (Dickinson et al., 2015; Paix et al., 2015), we also generated a strain expressing HA-tagged ZYG-1 at endogenous levels from the native genomic locus (S4A-S4C Fig, S2 Table). By labeling endogenous ZYG-1 with anti-HA (S4A and S4B Fig), we observed a similar pattern of ZYG-1 localization at centrosomes to that seen by anti-ZYG-1 (S4E Fig). The levels of centrosomal HA-ZYG-1 are also increased by two-fold in atx-2(RNAi) embryos (S4C Fig).
Given that ZYG-1 localizes to centrosomes in a cell cycle-dependent manner, the observed increase in centrosomal ZYG-1 at first metaphase in atx-2 mutants could result from a shift in the cell cycle due to loss of atx-2. To examine cell cycle dependence of ZYG-1 localization to centrosomes, we utilized a strain expressing GFP-ZYG-1-C-term that contains a C-terminal portion (217-706 aa) lacking most of the kinase domain, but including the Cryptic Polo Box (CPB) that is sufficient for centrosomal targeting (Peters et al., 2010; Shimanovskaya et al., 2014). To observe dynamics of centrosome-associated ZYG-1 over time, we acquired 4D time-lapse movies of early embryos starting from pronuclear meeting up to separation of the centriole pair at first anaphase (Fig 4C, 4D, S5 and S6 Movie). Using these recordings, we first quantified the fluorescence intensity of the intensely labeled sub-centrosomal GFP signal, which presumably reflects the centriolar structure. Then, we measured pericentriolar GFP signal that likely represents PCM. Throughout the cell cycle, we observe a nearly two-fold increase in both centriolar and PCM-associated GFP signal in atx-2(RNAi) embryos compared to controls. It could be that the elevated levels of centrosomal ZYG-1 in atx-2 mutants reflect a global increase in ZYG-1 throughout the cells. To test this possibility we compared overall ZYG-1 levels by measuring cytoplasmic GFP signals, but found no increase in the cytoplasmic levels of atx-2(RNAi) embryos. In fact, we noticed a small decrease (p=0.2) in the cytoplasmic GFP signal in atx-2(RNAi) embryos compared to controls, suggesting that increased centrosomal ZYG-1 levels are unlikely due to an increase in overall ZYG-1 expression. Similar results were also observed in atx-2(ne4297) mutants (S6 Movie). Together, our data show that inhibiting ATX-2 results in elevated levels of ZYG-1 at centrosomes without affecting overall ZYG-1 levels. Thus, we speculate that elevated levels of centrosomal ZYG-1 in atx-2 depleted embryos might partially compensate for the reduced activity of mutant ZYG-1 (P442L) in zyg-1(it25) (Kemp et al., 2007), restoring centrosome duplication in zyg-1 mutants.
ATX-2 negatively regulates centrosome size
Prior work has shown that SZY-20 negatively regulates centrosome size in a ZYG-1 dependent manner (Song et al., 2008). Here, our data indicate that ATX-2, acting downstream of SZY-20, affects centrosomal ZYG-1 levels. We thus tested if ATX-2 influences centrosome size as well. Quantitative IF using α-SPD-5 revealed that at first metaphase, atx-2 mutant centrosomes possess twice as much SPD-5 as wild-type centrosomes (Fig 5A, 5B). Consistent with the results in szy-20 mutants (Song et al., 2008), centrosomal SPD-2 levels are also significantly increased without affecting overall levels in atx-2 mutants (S5A and S5B Fig). The centrosome factors SPD-5 and SPD-2, acting as a scaffold, are required for localization of other PCM factors including γ-Tubulin (Hamill et al., 2002; Kemp et al., 2004; Woodruff et al., 2015).
γ-Tubulin is a PCM factor that plays a key role in MT nucleation and anchoring (Hannak et al., 2002; O’Toole et al., 2012; Srayko et al., 2005). By 4D-time lapse confocal microscopy of embryos expressing mCherry-γ-Tubulin (Toya et al., 2010), we measured the fluorescent intensity of centrosomal mCherry signal at first metaphase (Fig 5C, 5D). Consistent with SPD-5 levels, atx-2(RNAi) embryos display a nearly two-fold increase in centrosomal γ-Tubulin, equivalent to the increase seen in szy-20(bs52) embryos (Song et al., 2008). A similar observation is made in a strain overexpressing GFP-γ-Tubulin (S5C Fig). Note that szy-20(bs52) embryos possess ~50% of normal ATX-2 levels, which is comparable with the 50% average knockdown of ATX-2 by feeding atx-2(RNAi) (Fig 3E, 3K).
Remarkably, atx-2(ne4297) mutant centrosomes show even greater increase (4-fold) than szy-20(bs52) or atx-2(RNAi) embryos, suggesting a dose-dependent correlation between the amount of ATX-2 and centrosomal γ-Tubulin. Compared to single knockdown by szy-20(bs52) or atx-2(RNAi), co-depletion by szy-20(bs52); atx-2(RNAi) further exacerbated centrosomal enlargement, in a manner nearly equivalent to those in the strong loss-of-function mutation atx-2(ne4297) embryo. Thus, centrosomal γ-Tubulin levels correlate inversely with embryonic ATX-2 levels. Cytoplasmic mCherry-γ-Tubulin levels, however, are found to be similar in all embryos examined (Fig 5D). Furthermore, quantitative immunoblot confirms no significant effect on endogenous γ-Tubulin levels in atx-2(ne4297) compared to wild-type embryos (Fig 5E). Therefore, our data show that centrosomal PCM levels (SPD-2, SPD-5, γ-Tubulin) correlate inversely with ATX-2 abundance, suggesting that ATX-2 negatively regulates centrosome size in proportion to its abundance, while not affecting overall cellular levels of centrosome factors.
ATX-2 limits the number of microtubules emanating from the centrosome
The PCM factors, SPD-2, SPD-5 and γ-Tubulin, play a critical role in positively regulating the MT nucleating capacity of the centrosome (Hamill et al., 2002; Hannak et al., 2002; O’Toole et al., 2012; Srayko et al; 2005). As atx-2 mutant embryos exhibit enlarged centrosomes with increased centrosomal SPD-5 and γ-Tubulin, we examined if atx-2 mutant centrosomes affected MT nucleating capacity. To investigate MT nucleation, we used a strain expressing EBP-2-GFP to mark the plus-ends of growing MTs (Srayko et al., 2005) and acquired a series of 500 msec-interval snap shots at the center plane of first metaphase centrosomes (Fig 6, S7 Movie). First, we found that atx-2 mutant embryos exhibit a three-fold increase (p<0.0001) in centrosomal EBP-2-GFP signal compared to wild-type controls (Fig 6A, 6B). To assess the level of MT nucleation by the centrosome, we measured the fluorescent intensity of EBP-2-GFP in line regions proximal (25 pixels) to the centrosome, finding that atx-2 mutant centrosomes exhibit a two-fold increase (p<0.001) in MT nucleation (Fig 6C). Consistent with increased MT nucleation, kymograph analysis at the proximal region to the centrosome revealed a drastic increase (p=0.002) in the number of astral MTs emanating from atx-2 mutant centrosomes over a 5-sec period, compared to controls (Fig 6F). Further, line scans of the kymograph in a 355° arc around the centrosome show that mutant centrosomes emanate increased numbers of spindle fibers as well as astral MTs (Fig 6D). Thus, atx-2 mutant centrosomes possessing elevated levels of PCM factors nucleate the increased number of astral and spindle MTs.
Excessive MT nucleation leads to aberrant MT growth in atx-2 mutants
Next we examined how these MTs nucleated by mutant centrosomes continue to grow out toward the cortex (Fig 6E, 6G, 6H, S7 Movie). To measure the number of MTs reaching the cortex, we used kymograph analysis over a 5-sec period and counted the number of EBP-2-GFP dots crossing an arc drawn 1.5 μm (10 pixels) inside the cortex. In mutant embryos, fewer MTs grew out to reach the cortex than in controls (p=0.001; Fig 6H). Consistently, only a small portion of MTs nucleated by mutant centrosomes grew beyond the midpoint between the centrosome and cortex compared to controls (p=0.029; Fig 6G). In fact, astral MT growth rates are significantly slower in mutant embryos (0.70 μm/sec ± 0.007) than in controls (0.88μm/sec ± 0.013, p=0.0014) (S5E Fig). Thus, MT growth appears to be impeded in the mutant embryo, presumably due to an excess of MT nucleation.
In C. elegans embryos, it has been shown that fast MT growth is subject to the MT stabilizing complex (ZYG-9/TAC-1) and the amount of free tubulin (Srayko et al., 2005). atx-2 mutant embryos, however, exhibit a significant increase in both centrosomal and overall TAC-1 levels (S5F and S5G Fig), indicating that defective MT growth in atx-2 mutants is unlikely due to insufficient MT stabilization. Studies in other systems also showed that the amount of free tubulin influences the rate of MT polymerization in vitro (Belmont et al., 1990; Walker et al., 1988) and such mechanism appears to be pertinent in C. elegans embryos (Lacroix et al., 2016; Song et al., 2008; Srayko et al., 2005). We thus hypothesized that atx-2 mutant centrosomes possessing increased PCM nucleate more MTs, reducing the supply of free tubulin available for MT polymerization. Subsequently MT growth is interfered, resulting in shorter than normal MTs and cytokinesis failure. To test this, we generated atx-2 mutants overexpressing GFP-Tubulin to see if increasing Tubulin levels in the atx-2 mutant could partially rescue cytokinesis defects. As predicted, the incidence of cytokinesis failure is significantly (p<0.001) reduced in GFP-Tubulin overexpressing atx-2 embryos compared to control mutant embryos (Fig 7A). This partial rescue of cytokinesis failure further led to a significant decrease (p<0.001) in embryonic lethality (Fig 7B), suggesting that MT growth in atx-2 embryos is partly affected by limited supply of free tubulins, likely resulting from excessive MT nucleation. Next, we examined atx-2 mutant embryos overexpressing GFP-γ-Tubulin, a PCM factor, to see if further enhancing MT nucleation by increasing γ-Tubulin levels could exacerbate cytokinesis defects through worsened MT growth. Consistent with our hypothesis, cytokinesis defects in mutant embryos were significantly increased by GFP-γ-Tubulin overexpression compared to control mutants, leading to an increase in embryonic lethality (Fig 7A, 7B). Together, our data suggest that excessive MT nucleation and subsequent MT growth defects result in cytokinesis defects in atx-2 mutants.
Cytokinesis defects in atx-2 mutants result from excessive MT nucleation due to enlarged centrosomes
If defective cytokinesis results from elevated PCM levels at mutant centrosomes through excessive MT nucleation, reducing PCM levels should reconcile proper centrosomal PCM levels and MT nucleation, thereby restoring proper cytokinesis in atx-2 mutants. Whereas tbg-1(RNAi) produces cytokinesis failure in wild-type embryos, reducing γ-Tubulin in atx-2 embryos partially restores normal cytokinesis (p<0.001, Fig 7C), and embryonic viability (p<0.01, Fig 7D) to mutant embryos. Interestingly, not only did tbg-1(RNAi) rescue cytokinesis defects in atx-2 mutants, but atx-2 mutation also reduced cytokinesis failure in tbg-1(RNAi) embryos, suggesting a mutual suppression between atx-2 and tbg-1 (Fig 7C, 7D): At 20°C, the semi-permissive temperature for atx-2 mutants, single knockdown of atx-2(ne4297) and tbg-1(RNAi) produces 24% (n=91) and 66.5% (n=73), respectively, compared to 10.8% (n=57) of cytokinesis failure by double knockdown in atx-2(ne4297); tbg-1(RNAi) embryos. Consistently, atx-2(ne4297); tbg-1(RNAi) embryos exhibited centrosome-associated γ-Tubulin nearly to the normal level, although tbg-1(RNAi) almost abolished centrosomal γ-Tubulin in wild-type embryos (Fig 7E). It appears that ATX-2 influences MT nucleation and MT dynamics through γ-Tubulin at the centrosome, suggesting an epistatic relationship between atx-2 and tbg-1. A prior study reported a mutual suppression between szy-20 and zyg-1 (Song et al., 2008). Given the genetic and functional relationship between atx-2 and szy-20, we speculated that loss of zyg-1 might have a similar effect on atx-2. At semi-restrictive conditions for the atx-2 mutant, double homozygous mutants zyg-1(it25); atx-2(ne4297) produce a small but significant increase (p=0.02) in embryonic viability compared to the atx-2(ne4297) single mutant, accompanied by the restoration of normal cytokinesis (Table 1). Restoring normal cytokinesis appears to correlate with zyg-1(it25)-mediated restoration of centrosome size in atx-2 embryos (Fig 7F). In fact, zyg-1(it25) embryos exhibit decreased levels of centrosomal γ-Tubulin at the permissive temperature 20°C where centrosome duplication occurs normally. Moreover, inhibiting ZYG-1 reduces centrosomal γ-Tubulin levels in atx-2(RNAi) or mutant embryos, which likely leads to the partial restoration of normal cytokinesis and embryonic viability in ATX-2 depleted embryos (Fig 7F, 7G, Table 1).
Together, our results suggest a model in which loss of ATX-2 leads to elevated PCM levels at centrosomes and excessive MT nucleation, resulting in MT growth defects and subsequent cytokinesis failure (Fig 7H). Consistently, embryos depleted of ATX-2 exhibit a prominent cytokinesis failure phenotype, accompanied by enlarged centrosomes that cause excessive MT nucleation and aberrant MT growth, which results in cytokinesis failure and embryonic lethality. Thus, the proper level of centrosomal PCM factors is critical for normal MT nucleating activity to support normal MT dynamics. In this pathway, ATX-2 acts upstream to establish the proper centrosome size and MT nucleating activity.
Discussion
ATX-2 forms a complex with SZY-20 and acts as a negative regulator of centrosome assembly in C. elegans embryos
In this study, we identified that ATX-2, together with PAB-1, physically associates with SZY-20 in vivo. While RNA-binding proteins ATX-2 and SZY-20, each containing unique RNA-binding motifs (LSm, SUZ and SUZ-C, respectively), form a complex, these two proteins appear to physically interact independently of RNA. While it remains unknown what RNA molecules associate with these RNA-binding proteins, each RNA-binding protein in a ribonucleoprotein (RNP) complex might recruit a specific group of RNA through each own RNA-binding motif. In fact, Ataxin-2 has been shown to bind directly to mRNAs through its LSm domain and promote the stability of transcripts independently of its binding partner, poly (A)-binding protein (PABP) in flies and humans (Satterfield and Pallanck 2006; Yokoshi et al., 2014). Our prior study in C. elegans embryos showed that SUZ and SUZ-C RNA-binding motifs in SZY-20 exhibit RNA-binding capacity in vitro and that mutating these domains perturbs in vitro RNA-binding of SZY-20 and its capacity to regulate centrosome size in vivo (Song et al., 2008). However, there is no evidence that RNA-binding role of ATX-2 is directly involved in centrosome regulation, while C. elegans ATX-2 is shown to function in translational regulation during germline development (Ciosk et al., 2004), In a multi-protein complex, ATX-2 and SZY-20 function closely to regulate cell division and centrosome assembly. While knocking down ATX-2 phenocopies a loss of function szy-20(bs52) mutation, the different degree of phenotypic penetrance in atx-2(RNAi), a strong loss of function atx-2(ne4297) mutation, and a hypomorphic szy-20(bs52) mutation suggests a dose-dependent regulation of ATX-2. Double knockdown by combining atx-2(RNAi) and szy-20(bs52) mutation further enhances embryonic lethality, cytokinesis failure, the restoration of centrosome duplication to zyg-1(it25) embryos and the levels of centrosome-associated factors (ZYG-1, SPD-5, γ-Tubulin). Furthermore, the atx-2(ne4297) mutation produces a similar effect to that of atx-2(RNAi) combined with the szy-20(bs52) mutation. Our data thus suggest that atx-2 exhibits a positive genetic interaction with szy-20 in regulating cell cycle and centrosome assembly. In this pathway, SZY-20 acts upstream of ATX-2 to promote ATX-2 levels, with both acting upstream of zyg-1. We propose that SZY-20 influences centrosome size and MT dynamics indirectly through ATX-2 (Song et al., 2008). While it remains unclear whether C. elegans ATX-2 directly acts on ZYG-1, it has been shown that Plx4 (a Xenopus homolog of Plk4/ZYG-1) forms a complex with Atxn-2 (Xenopus homolog of ATX-2; Hatch et al., 2010). Although a direct role for Atxn-2 in centrosome assembly has not been demonstrated, a physical connection between Xenopus Atxn-2 and Plx4 suggests a possible role of Xenopus Atxn-2 in centrosome assembly, via a mechanism that seems likely to be conserved between nematodes and vertebrates.
ATX-2 negatively regulates centrosome size in a dose-dependent manner
Our data indicate that ATX-2 acts as a negative regulator of centrosome size. A priori, increased PCM levels at atx-2 mutant centrosomes could be achieved by several mechanisms. First, centrosome factors might be overexpressed in atx-2 mutant cytoplasm via translational control, leading to increased recruitment of these factors to the centrosome by equilibrium, as shown by Decker et al (2011). Second, atx-2 mutants might enhance the recruitment of factors to centrosomes post-translationally, without affecting overall levels of these factors. Third, loss of ATX-2 might promote local translation near centrosomes, leading to locally enriched centrosome factors. The first scenario is unlikely because our quantitative analyses reveal no significant changes in overall levels of centrosome factors (SPD-2, SAS-6, γ-Tubulin) or cytoplasmic levels of ZYG-1 by loss of ATX-2. Our current data do not differentiate between the second and third scenarios, but certain observations in C. elegans and other systems are consistent with the latter. It has been shown that Ataxin-2 assembles with polyribosomes and that ribosomes are associated with the MT cytoskeleton (Hamill et al., 1994; Satterfield and Pallanck 2006). Furthermore, neuronal RNA granules are shown to contain translational machinery, allowing local translation upon arrival of transcripts at the right location (Shiina et al., 2005). We also observed ribosomal protein S6 spatially associated with C. elegans centrosomes (S3D Fig), suggesting the possibility of local translation around the centrosome. Indeed mRNA localization linked to the cytoskeleton and the following local translation is shown to be an efficient means of concentrating proteins at the functional site (Groisman et al., 2000; Huttelmaier et al., 2005; Kindler et al., 2005). For example, spatial control of β-actin translation is executed by localizing its transcripts to actin-rich protrusions, which facilitates neuronal outgrowth (Huttelmaier et al., 2005). Also, RNA-binding protein (CPEB/maskin) mediated localization of cyclin B1 transcripts to the mitotic apparatus leads to locally enriched Cyclin B proteins in the vicinity of spindles and centrosomes, supporting cell cycle progression (Griosman et al., 2000). Together such mechanism has been demonstrated to provide a tight control for temporal and spatial translation. How then, might ATX-2 regulate translation? For negative translational control, it has been proposed that Ataxin-2 binding to PABP inhibits translation by blocking the interaction between PABP and translational machinery or by directly blocking translation of mRNA targets at the initiation stage (Besse and Ephrussi, 2008; Roy et al., 2004; Satterfield and Pallanck 2006; Yokoshi et al., 2014). Alternatively, ATX-2 may facilitate the interaction between a microRNA and its mRNA target, leading to translational inhibition (McCann et al., 2011). ATX-2 might also mediate translational control via its RNA-binding role as a component of the RNP complex comprising SZY-20/ATX-2/PAB-1. In fact, C. elegans PAB-1 is shown to associate with stress granules and processing bodies (P-bodies) that have been implicated in translational repression (Gallo et al., 2008). It has been observed that RNP complexes including P-bodies are involved in subcellular targeting of RNAs and precise timing of local translation at the final subcellular destination (Barbee et al., 2006; Besse and Ephrussi, 2008). Further identification of specific RNAs that bind ATX-2 and/or SZY-20 will help to understand how RNA-binding role of an ATX-2 associated RNP complex plays a role in defining proper centrosome size.
ATX-2 contributes to proper MT nucleating activity of centrosomes and MT growth through γ-Tubulin
We have shown here that ATX-2 plays an essential role in cell division. atx-2 mutant embryos with enlarged centrosomes exhibit multiple cell division defects including cytokinesis failure and aberrant spindle positioning. In animal cells, contact between astral MT and the cortex is critical for the initiation of the cleavage furrow that is required for proper cytokinesis (Green et al., 2012; Normand and King, 2010). Thus cell cycle defects in atx-2(ne4297) embryos might associate with aberrant MT behavior, likely due to enlarged centrosomes. Recent work showed an additional role of ZYG-1 in regulating centrosome size, independently of its role in centriole duplication (Song et al., 2008). PCM factors SPD-2, SPD-5 and γ-Tubulin are known to positively regulate the MT nucleating activity of the centrosome (Hamill et al., 2002; Hannak et al., 2002; O’Toole et al., 2012; Wueseke et al., 2015). atx-2 mutant centrosomes possessing increased (2-5 folds) levels of centrosome factors nucleate the drastically increased number of MTs. Such increase in MT nucleation by mutant centrosomes may lead to a substantial reduction in cytoplasmic free tubulins available for timely MT polymerization. Given the rapid cell divisions (~20 min/cell cycle) and high MT growth rate (0.88 µm/sec) in early C. elegans embryos, timely and sufficient supply of free tubulins in the cytoplasm must be immensely critical for proper cell cycle progression. In support of this, either overexpressing Tubulin or reducing positive regulators (ZYG-1 or γ-Tubulin) of MT nucleation partially restores normal cytokinesis, MT growth and embryonic viability to atx-2 mutants. Partial restoration of normal MT-dependent processes correlates with proper levels of centrosomal γ-Tubulin. Therefore, enlarged centrosomes are likely to be a primary cause of embryonic lethality in atx-2 mutants. Mutant centrosomes possess significantly increased levels of γ-Tubulin, and reducing its levels at mutant centrosomes partially reinstalls free tubulins available for MT growth, which in turn restores normal cell divisions and embryonic viability in atx-2 mutants. Our data suggest that ATX-2 contributes to MT nucleation and MT dynamics, in part, through γ-Tubulin at centrosomes, although it remains unknown how RNA-binding protein, ATX-2, regulates centrosomal levels of γ-Tubulin. Given our observation that overall levels of γ-Tubulin are not altered in atx-2 mutant embryos, it is curious how γ-Tubulin levels are locally enriched at the centrosome, perhaps via the regulation of recruitment to the centrosome or locally enriched translational control. Thus, it will be interesting to see if tbg-1 transcripts are elevated at the proximity of centrosomes by in situ hybridization. A recent study in Xenopus egg extracts reported that RNase treatment leads to defective spindle assembly due to hyperactive MT destabilizer MCAK (the ortholog of C. elegans KLP-7) by depleting RNA, suggesting a direct involvement of RNA in regulating MT organization (Jambhekar et al., 2014). Interestingly, we also found that atx-2 mutants show increased levels of KLP-7 at centrosomes and cytoplasm (S5H Fig), and that atx-2 and klp-7 exhibit a synergistic effect on MT dynamics and embryonic lethality (S6 Fig).
Another centriole factor, SAS-4, positively regulates centrosome size in C. elegans (Kirkham et al., 2003). Interestingly, while we identify ATX-2 as a negative regulator of centrosome size, depleting ATX-2 had no effect on centriolar SAS-4 levels (S4G Fig), suggesting that ATX-2 regulates centrosome size independently of SAS-4, perhaps through a separate pathway. Thus, a balance between positive (e.g., SAS-4) and negative (e.g., ATX-2) regulators may contribute to establish proper centrosome size and MT nucleating activity, which in turn influences MT dynamics during the cell cycle. Improper levels of ATX-2 disrupt this balance, resulting in deregulated MT dynamics and subsequently abnormal cell divisions.
In summary, our work uncovers a role for ATX-2, the C. elegans ortholog of human Ataxin-2 in regulating centrosome size and MT cytoskeleton. In this pathway, SZY-20 positively regulates levels of ATX-2, which contributes to defining the proper centrosome size, leading to proper levels of MT nucleation and subsequent MT growth. While human Ataxin-2 and its poly Q stretch have been implicated in spinocerebellar ataxia (Lastres-Becker et al., 2008), it remains largely unknown how this RNA-binding protein is linked to human diseases. Many RNA-binding proteins in neuronal RNA granules have been implicated in neuronal functions and growth via controlled local translation (Bette and Ephrussi, 2008; Shiina et al., 2005), suggesting a link between RNA-binding role and neuronal activity. In this regard, our work provides insights into a mechanistic link between the RNA-binding role of Ataxin-2 and the MT cytoskeleton.
Materials and Methods
Strains and Genetics
All C. elegans strains were grown on MYOB plates seeded with E. coli OP50, and were derived from the wild-type Bristol N2 strain (Church et al., 1995). All strains were maintained at 18 or 20°C unless otherwise indicated. Transgenic strains were generated by standard particle bombardment transformation (Praitis et al., 2001) and the CRISPR/Cas-9 method (S2 Table, Dickinson et al., 2015; Paix et al., 2015). The list of worm strains used in this study is shown in S1 Table. Standard genetics were used for strain construction and genetic manipulations (Brenner, 1974). RNAi feeding was performed as described and L4440 clone with the empty dsRNA-feeding vector served as a negative control (Kamath et al., 2003).
Cytological Analysis
For immunostaining, we used the following antibodies at a 1:400-3000 dilution: α-ATX-2 (Ciosk et al., 2004), DM1A (Sigma), α-GFP (Roche), α-SAS-4 (Song et al., 2008), α-SPD-2 (Kemp et al., 2004), -SPD-5 (Hamill et al., 2002), α-SZY-20 (α-S20N: Song et al., 2008), and secondary antibodies Alexa Fluor 488 and 568 (Invitrogen). Affinity-purified rabbit polyclonal antibodies were generated (YenZym) against the following peptides: for γ-Tubulin (S4D Fig, aa31-44): Ac-HGINERGQTTHEDD-amide, for ZYG-1 (S4E Fig, aa671-689): Ac-PAIRPDDQRFMRTDRVPDR-amide.
Immunofluorescence and confocal microscopy were performed as described (Song et al., 2008). For confocal microscopy, MetaMorph software (Molecular Devices) was used to acquire images from a Nikon Eclipse Ti-U microscope equipped with a Plan Apo 60 X 1.4 NA lens, a Spinning Disk Confocal (CSU X1) and a Photometrics Evolve 512 camera. Fluorescence intensity measurements were made using the MetaMorph software, and image processing with Photoshop CS6.
For quantification of centrosomal signals, the average intensity within a 25-pixel (pixel = .151 μm) diameter region was measured throughout a region centered on the centrosome. For each region, a focal plane with the highest average intensity was recorded. The same sized regions were drawn in the cytoplasm for cytoplasmic signal, and outside the embryo for background subtraction unless otherwise indicated. For centriolar signals, analysis was done the same way, but a 5-pixel diameter region was used.
For EBP-2-GFP movies, pre-mitotic embryos were selected and monitored under DIC until before metaphase I. Upon entrance into metaphase, 61 images were captured every 500 msec for a 30 sec period. For spindle MTs, a 355° circular region was drawn around each centrosome, and kymographs were generated for the first 5 seconds of recordings. Linescan measurements of the kymographs were taken to obtain average EBP-2 signal along every point of the circular region. Values for each linescan were averaged and plotted on a graph. To measure MT nucleation, the fluorescence intensity was quantified at a line region drawn in a semi-circle (25 pixel radius) around the centrosome. Linescan (5 pixel wide) measurements were taken in 5-sec time projections from recordings. Pixel intensities were generated for each point along the line and averaged for comparison. For background subtraction, cytoplasmic intensity in the same size region was used. To quantify the number of growing astral microtubules proximal to the centrosome, line regions were drawn in a semi-circle (25 pixel radius) around the centrosome. For midpoint measurements, line regions were drawn at a radius of 60 pixels from the centrosome. Kymographs were generated for the first 5 seconds of each movie. Individual pixel measurements from the kymographs were obtained, and pixels with GFP intensity over 40 were counted. Cortex measurements were obtained by generating 5 sec kymographs of a region drawn at 1.5 μm inside of the cell cortex. The number of EBP-2 signal was counted manually. For MT polymerization rate, 4.5 μm-long lines were drawn along the individual EBP-2-GFP tracks outward to the cortex. Kymographs were generated, and rate was calculated by distance over time (μm/sec).
Immunoprecipitation and Quantitative Western Analysis
20-25 μl of Dynabeads Sheep-anti-Rabbit, Protein A magnetic beads (Invitrogen) or mouse-anti-GFP magnetic beads (MBL) were used per IP reaction. Beads were washed 2x 15 min in 1 ml PBS with 0.1% Triton-X (PBST). For SZY-20 IP, beads were incubated overnight at 4°C in a ratio of 1mg/ml (α-SZY-20 or α-HA/beads volume) and 3 mg/ml (α-IgG/beads volume). Embryos were collected by bleaching worms grown in liquid culture, snap-frozen in liquid nitrogen, and stored at -80°C until use. Embryo pellets were ground up in microcentrifuge tubes in equal volumes of worm lysis buffer (50 mM HEPES; pH 7.4, 1 mM EDTA, 1 mM MgCl2, 200 mM KCl, 10% glycerol; Cheeseman et al., 2004) containing cOmplete protease inhibitor cocktail (Roche) and MG132 (Tocris). For RNase A or RNase Inhibitor treatment, RNase (10 μg/ml, Roche) or RNase Inhibitor (200U/ml, Roche) was added to lysis buffer prior to grinding. Embryos were ground for 5 min, and sonicated for 3 min. The lysate was then centrifuged at 4°C for 2x 20 min at 15K rpm in a desktop centrifuge, and the supernatant were collected. For enzyme reaction, lysates were incubated for 15 min at room temperature (RT). Protein concentration was determined and adjusted before IP. Beads were washed for 2x 15 min with PBST + 0.5% BSA at RT, followed by 1x 15 min wash with 1X worm lysis buffer. Beads were resuspended in 1X lysis buffer, mixed with embryonic lysates and incubated at 4°C for 1 hour. Following IP, beads were washed 2x 5 min in PBST. Samples were dissolved in 2X Laemmli Sample Buffer (Sigma) with 10% β-mercaptoethanol, fractionated on a 4-12% NuPAGE Bis-Tris Gel (Invitrogen) and blotted to nitrocellulose membrane using the iBlot Gel Transfer System (Invitrogen). α-ATX-2 (Ciosk et al., 2004), α-GFP (Roche), mouse α-HA (Sigma), α-SAS-6 (Song et al., 2011), α-SPD-2 (Song et al., 2008), α-SZY-20 (Song et al., 2008), DM1A (Sigma), α-TAC-1 (Hadwiger et al., 2010) and α-γ-Tubulin at 1:400-2500 dilution, and IRDye secondary antibodies (LI-COR Biosciences) were used at 1:10,000 dilution. Blots were imaged on a Licor Odyssey IR scanner, and analyzed using the Odyssey Infrared Imaging System (LI-COR Biosciences). Mass spectrometry analysis was performed as described (Song et al., 2011).
Acknowledgements
We are grateful for the assistance of Mass Spectrometry Facility at National Institutes of Health. We also thank Song lab members for experimental assistance, David Weisblat for helpful discussion, Craig Mello, Kevin O'Connell, Ahna Skop, Masaki Shirayama and Caenorhabditis Genetics Center for worm strains and RNAi stains.
Footnotes
Contact Information: msong2{at}oakland.edu 01-248-370-4494 (Phone) 01-248-370-4225 (Fax)