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Interphase Positioning of Centromeres Sets Up Spindle Assembly
Developmental Cell
Previews Interphase Positioning of Centromeres Sets Up Spindle Assembly Hironori Funabiki1,* 1Laboratory of Chromosome and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.11.014
It has been known for many years that centromeres cluster at the spindle pole body in fission yeast. In this issue of Developmental Cell, Ferna´ndez-A´lvarez et al. (2016) reveal that the functional significance of clustering is to promote spindle assembly by modulating nuclear envelope integrity at the onset of mitosis. ‘‘They are all clustered at the SPB!’’ In 1991, as a second-year PhD student in Mitsuhiro Yanagida’s lab at Kyoto University, I inherited a project by a graduating student, Satoru Uzawa, who developed a technique allowing simultaneous visualization of fluorescence in situ hybridization (FISH) signals and indirect immunofluorescence in the fission yeast Schizosaccharomyces pombe. Soon after, Iain Hagan, a post doc in the lab, found that a new antibody against Sad1, a protein required for spindle formation, beautifully stained the spindle pole body (SPB), the centrosome-like organelle in fungi. Combining the anti-Sad1 antibody and FISH, I observed that centromeres cluster together adjacent to the SPB in interphase nucleus (Funabiki et al., 1993). The result was striking, but it could have simply reflected the preservation of the ‘‘Rabl configuration,’’ in which centromeres pulled to the pole during anaphase remain there during the following cell cycle. To understand the functional significance of this result, I wished I could identify a mutant defective in centromere clustering, but Sad1 and g-tubulin were the only known fission yeast SPB proteins at the time. Thus, it is ironic that Ferna´ndez-A´lvarez et al. (2016), in this issue of Developmental Cell, solve this puzzle in part by using a separation-of-function mutant of Sad1, a protein we now know as the founding member of the SUNdomain family proteins and a subunit of the LINC complex, playing a number of important roles in nuclear integrity and movement. Retrospectively, I am glad I did not pursue the molecular mechanism underlying clustering as a graduate student, since detailed studies of meiosis were first needed to pave the road.
The existence of an active mechanism underlying the centromere-SPB association was suggested by an observation made in Yasushi Hiraoka’s lab, demonstrating that the SPB switches its association from centromeres to telomeres when cells enter meiosis (Chikashige et al., 1994). That is, whereas centromeres cluster at the SPB during mitosis, at the transition into the meiotic program, centromeres dissociate from the SPB and telomeres are then recruited, forming a ‘‘bouquet’’ of chromosomal configuration that is prevalent in meiotic prophase of many eukaryotes. This switch during meiosis from centromeres to telomeres thus provided a basis for isolating mutants defective in telomere clustering at the SPB, and their characterization led to a model in which telomere-binding protein Taz1 recruits telomeres to the SPB by interacting with Sad1 via Rap1, Bqt1, and Bqt2 (Chikashige et al., 2006; Tomita and Cooper, 2007). Surprisingly, disruption of telomere clustering at the SPB in Dtaz1 and Dbqt1 mutants causes defects in spindle formation and chromosome segregation in the subsequent meiosis I and meiosis II (Fennell et al., 2015; Tomita and Cooper, 2007), although bipolar spindles still form in about 25%–50% of cells. Further close inspection revealed that a prior stable contact between the SPB and the centromere during meiotic prophase correlated with subsequent functional spindle formation. Indeed, artificial anchoring of the centromere to the SPB rescued spindle formation, demonstrating that centromeres can potentiate the ability of the SPB to nucleate spindle microtubules (Fennell et al., 2015). But how? Another piece of the puzzle relates to the fact that in fission yeast, unlike in animals, chromosome segregation involves
endomitosis, which means that the nuclear envelope (NE) does not break down. However, the NE does exhibit a local rearrangement at the SPB during mitosis. The main body of the SPB sits on the cytoplasmic side of the NE in interphase, but at mitotic entry, a large fenestra forms in the NE to insert the SPB into the NE. The study by Cooper and colleagues (Ferna´ndez-A´lvarez et al., 2016) now suggests that centromere association with the SPB promotes this fenestration process. To study the functional significance of the centromere-SPB linkage, the team executed a systematic alanine-scanning mutation analysis on Sad1, a protein known to accumulate at the inner nuclear membrane beneath the SPB, and isolated a temperature-sensitive sad1.2 mutant that abrogates centromere clustering at the SPB during mitotic interphase without interfering with SPB integrity and reproduction. In this mutant, spindle formation in mitosis is severely inhibited. To monitor fenestration, the authors took advantage of a cut11 mutant. Cut11 is a homolog of the nuclear pore complex component Ndc1, which is recruited to the SPB. In wild-type cells, the fenestration process is so restricted that cytoplasmic leakage of nuclear GFP is undetectable. In contrast, in cut11.1 mutant, fenestra remained open during mitosis, causing nuclear GFP leaks into the cytoplasm. However, introducing the sad1.2 mutation suppressed the efflux, suggesting that nuclear fenestration was compromised by a defect in Sad1. Remarkably, artificial targeting of the centromere to the SPB rescued GFP efflux, spindle formation, and temperature-sensitive growth of sad1.2. Similarly, bqt1D cells defective in telomere clustering at the SPB during
Developmental Cell 39, December 5, 2016 ª 2016 Elsevier Inc. 527
Developmental Cell
Previews meiotic prophase also failed to insert the SPB into the NE in the subsequent meiotic spindle-formation stages. Altogether, these results indicate that centromereSPB and telomere-SPB associations promote spindle assembly through facilitating NE fenestration and SPB insertion. The study leaves a number of open questions. First, it remains unclear whether the centromere is the only locus that promotes NE fenestration. In meiosis, an integrated single stretch of telomere sequence that recruits Taz1 is sufficient to confer spindle formation (Tomita et al., 2013). It is possible that any stable linkage between chromatin and the SPB may suffice to support this function. Second, nothing is known about the mechanism by which the centromere promotes NE fenestration. Mitotic protein kinases Cdk1 and Polo-like kinase (Plo1) are localized to the SPB and are required for proper spindle formation, but it is unknown whether their activities can be regulated by the centromere. Third, it remains to be established whether SPB access to the nucleoplasm is the only essential process that the SPB-centromere interaction and fenestration stimulate. Metazoan centrosomes are also tethered to the cytoplasmic surface of inter-
phase nuclei by Sad1 homologs, and this interaction facilitates NE rupture and membrane removal from chromosomes via microtubule-dependent forces (Turgay et al., 2014). However, NE breakdown (NEBD) can proceed in the absence of microtubules, and microtubules are not required for NE fenestration in fission yeast. Because the NE interacts with chromatin at multiple points, it is tempting to speculate that such contacts play a role in NEBD in animal cells as well. The coincidence of the SPB/chromatin colocalization with the onset of mitosis provides a narrow spatiotemporal window in which the SPB can insert into the NE, restricting the free exchange of nuclear and cytoplasmic materials. The size of this window varies greatly among species (Drechsler and McAinsh, 2012). Even in open mitoses where the NE appears to completely disassemble, a membranous ‘‘spindle envelope’’ helps to promote molecular crowding and excludes cytoplasmic organelles (Schweizer et al., 2015). Thus, allowing chromosomal access to the centrosome/SPB without losing enrichment of spindle materials may be an evolutionarily conserved task. Characterizing mechanistic differences and similarities among diverse
528 Developmental Cell 39, December 5, 2016
mitoses may further our understanding of key principles regulating the assembly of two distinct chromatin-associated architectures, the NE and the spindle.
REFERENCES Chikashige, Y., Ding, D.Q., Funabiki, H., Haraguchi, T., Mashiko, S., Yanagida, M., and Hiraoka, Y. (1994). Science 264, 270–273. Chikashige, Y., Tsutsumi, C., Yamane, M., Okamasa, K., Haraguchi, T., and Hiraoka, Y. (2006). Cell 125, 59–69. Drechsler, H., and McAinsh, A.D. (2012). Open Biol. 2, 120140. Fennell, A., Ferna´ndez-A´lvarez, A., Tomita, K., and Cooper, J.P. (2015). J. Cell Biol. 208, 415–428. Ferna´ndez-A´lvarez, A., Bez, C., O’Toole, E., Morphew, M., and Cooper, J.P. (2016). Dev. Cell 39, this issue, 544–559. Funabiki, H., Hagan, I., Uzawa, S., and Yanagida, M. (1993). J. Cell Biol. 121, 961–976. Schweizer, N., Pawar, N., Weiss, M., and Maiato, H. (2015). J. Cell Biol. 210, 695–704. Tomita, K., and Cooper, J.P. (2007). Cell 130, 113–126. Tomita, K., Bez, C., Fennell, A., and Cooper, J.P. (2013). EMBO Rep. 14, 252–260. Turgay, Y., Champion, L., Balazs, C., Held, M., Toso, A., Gerlich, D.W., Meraldi, P., and Kutay, U. (2014). J. Cell Biol. 204, 1099–1109.
Previews Interphase Positioning of Centromeres Sets Up Spindle Assembly Hironori Funabiki1,* 1Laboratory of Chromosome and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.11.014
It has been known for many years that centromeres cluster at the spindle pole body in fission yeast. In this issue of Developmental Cell, Ferna´ndez-A´lvarez et al. (2016) reveal that the functional significance of clustering is to promote spindle assembly by modulating nuclear envelope integrity at the onset of mitosis. ‘‘They are all clustered at the SPB!’’ In 1991, as a second-year PhD student in Mitsuhiro Yanagida’s lab at Kyoto University, I inherited a project by a graduating student, Satoru Uzawa, who developed a technique allowing simultaneous visualization of fluorescence in situ hybridization (FISH) signals and indirect immunofluorescence in the fission yeast Schizosaccharomyces pombe. Soon after, Iain Hagan, a post doc in the lab, found that a new antibody against Sad1, a protein required for spindle formation, beautifully stained the spindle pole body (SPB), the centrosome-like organelle in fungi. Combining the anti-Sad1 antibody and FISH, I observed that centromeres cluster together adjacent to the SPB in interphase nucleus (Funabiki et al., 1993). The result was striking, but it could have simply reflected the preservation of the ‘‘Rabl configuration,’’ in which centromeres pulled to the pole during anaphase remain there during the following cell cycle. To understand the functional significance of this result, I wished I could identify a mutant defective in centromere clustering, but Sad1 and g-tubulin were the only known fission yeast SPB proteins at the time. Thus, it is ironic that Ferna´ndez-A´lvarez et al. (2016), in this issue of Developmental Cell, solve this puzzle in part by using a separation-of-function mutant of Sad1, a protein we now know as the founding member of the SUNdomain family proteins and a subunit of the LINC complex, playing a number of important roles in nuclear integrity and movement. Retrospectively, I am glad I did not pursue the molecular mechanism underlying clustering as a graduate student, since detailed studies of meiosis were first needed to pave the road.
The existence of an active mechanism underlying the centromere-SPB association was suggested by an observation made in Yasushi Hiraoka’s lab, demonstrating that the SPB switches its association from centromeres to telomeres when cells enter meiosis (Chikashige et al., 1994). That is, whereas centromeres cluster at the SPB during mitosis, at the transition into the meiotic program, centromeres dissociate from the SPB and telomeres are then recruited, forming a ‘‘bouquet’’ of chromosomal configuration that is prevalent in meiotic prophase of many eukaryotes. This switch during meiosis from centromeres to telomeres thus provided a basis for isolating mutants defective in telomere clustering at the SPB, and their characterization led to a model in which telomere-binding protein Taz1 recruits telomeres to the SPB by interacting with Sad1 via Rap1, Bqt1, and Bqt2 (Chikashige et al., 2006; Tomita and Cooper, 2007). Surprisingly, disruption of telomere clustering at the SPB in Dtaz1 and Dbqt1 mutants causes defects in spindle formation and chromosome segregation in the subsequent meiosis I and meiosis II (Fennell et al., 2015; Tomita and Cooper, 2007), although bipolar spindles still form in about 25%–50% of cells. Further close inspection revealed that a prior stable contact between the SPB and the centromere during meiotic prophase correlated with subsequent functional spindle formation. Indeed, artificial anchoring of the centromere to the SPB rescued spindle formation, demonstrating that centromeres can potentiate the ability of the SPB to nucleate spindle microtubules (Fennell et al., 2015). But how? Another piece of the puzzle relates to the fact that in fission yeast, unlike in animals, chromosome segregation involves
endomitosis, which means that the nuclear envelope (NE) does not break down. However, the NE does exhibit a local rearrangement at the SPB during mitosis. The main body of the SPB sits on the cytoplasmic side of the NE in interphase, but at mitotic entry, a large fenestra forms in the NE to insert the SPB into the NE. The study by Cooper and colleagues (Ferna´ndez-A´lvarez et al., 2016) now suggests that centromere association with the SPB promotes this fenestration process. To study the functional significance of the centromere-SPB linkage, the team executed a systematic alanine-scanning mutation analysis on Sad1, a protein known to accumulate at the inner nuclear membrane beneath the SPB, and isolated a temperature-sensitive sad1.2 mutant that abrogates centromere clustering at the SPB during mitotic interphase without interfering with SPB integrity and reproduction. In this mutant, spindle formation in mitosis is severely inhibited. To monitor fenestration, the authors took advantage of a cut11 mutant. Cut11 is a homolog of the nuclear pore complex component Ndc1, which is recruited to the SPB. In wild-type cells, the fenestration process is so restricted that cytoplasmic leakage of nuclear GFP is undetectable. In contrast, in cut11.1 mutant, fenestra remained open during mitosis, causing nuclear GFP leaks into the cytoplasm. However, introducing the sad1.2 mutation suppressed the efflux, suggesting that nuclear fenestration was compromised by a defect in Sad1. Remarkably, artificial targeting of the centromere to the SPB rescued GFP efflux, spindle formation, and temperature-sensitive growth of sad1.2. Similarly, bqt1D cells defective in telomere clustering at the SPB during
Developmental Cell 39, December 5, 2016 ª 2016 Elsevier Inc. 527
Developmental Cell
Previews meiotic prophase also failed to insert the SPB into the NE in the subsequent meiotic spindle-formation stages. Altogether, these results indicate that centromereSPB and telomere-SPB associations promote spindle assembly through facilitating NE fenestration and SPB insertion. The study leaves a number of open questions. First, it remains unclear whether the centromere is the only locus that promotes NE fenestration. In meiosis, an integrated single stretch of telomere sequence that recruits Taz1 is sufficient to confer spindle formation (Tomita et al., 2013). It is possible that any stable linkage between chromatin and the SPB may suffice to support this function. Second, nothing is known about the mechanism by which the centromere promotes NE fenestration. Mitotic protein kinases Cdk1 and Polo-like kinase (Plo1) are localized to the SPB and are required for proper spindle formation, but it is unknown whether their activities can be regulated by the centromere. Third, it remains to be established whether SPB access to the nucleoplasm is the only essential process that the SPB-centromere interaction and fenestration stimulate. Metazoan centrosomes are also tethered to the cytoplasmic surface of inter-
phase nuclei by Sad1 homologs, and this interaction facilitates NE rupture and membrane removal from chromosomes via microtubule-dependent forces (Turgay et al., 2014). However, NE breakdown (NEBD) can proceed in the absence of microtubules, and microtubules are not required for NE fenestration in fission yeast. Because the NE interacts with chromatin at multiple points, it is tempting to speculate that such contacts play a role in NEBD in animal cells as well. The coincidence of the SPB/chromatin colocalization with the onset of mitosis provides a narrow spatiotemporal window in which the SPB can insert into the NE, restricting the free exchange of nuclear and cytoplasmic materials. The size of this window varies greatly among species (Drechsler and McAinsh, 2012). Even in open mitoses where the NE appears to completely disassemble, a membranous ‘‘spindle envelope’’ helps to promote molecular crowding and excludes cytoplasmic organelles (Schweizer et al., 2015). Thus, allowing chromosomal access to the centrosome/SPB without losing enrichment of spindle materials may be an evolutionarily conserved task. Characterizing mechanistic differences and similarities among diverse
528 Developmental Cell 39, December 5, 2016
mitoses may further our understanding of key principles regulating the assembly of two distinct chromatin-associated architectures, the NE and the spindle.
REFERENCES Chikashige, Y., Ding, D.Q., Funabiki, H., Haraguchi, T., Mashiko, S., Yanagida, M., and Hiraoka, Y. (1994). Science 264, 270–273. Chikashige, Y., Tsutsumi, C., Yamane, M., Okamasa, K., Haraguchi, T., and Hiraoka, Y. (2006). Cell 125, 59–69. Drechsler, H., and McAinsh, A.D. (2012). Open Biol. 2, 120140. Fennell, A., Ferna´ndez-A´lvarez, A., Tomita, K., and Cooper, J.P. (2015). J. Cell Biol. 208, 415–428. Ferna´ndez-A´lvarez, A., Bez, C., O’Toole, E., Morphew, M., and Cooper, J.P. (2016). Dev. Cell 39, this issue, 544–559. Funabiki, H., Hagan, I., Uzawa, S., and Yanagida, M. (1993). J. Cell Biol. 121, 961–976. Schweizer, N., Pawar, N., Weiss, M., and Maiato, H. (2015). J. Cell Biol. 210, 695–704. Tomita, K., and Cooper, J.P. (2007). Cell 130, 113–126. Tomita, K., Bez, C., Fennell, A., and Cooper, J.P. (2013). EMBO Rep. 14, 252–260. Turgay, Y., Champion, L., Balazs, C., Held, M., Toso, A., Gerlich, D.W., Meraldi, P., and Kutay, U. (2014). J. Cell Biol. 204, 1099–1109.