- Email: [email protected]
Chromosome Segregation: Pulling from the Poles
Current Biology, Vol. 12, R651–R653, October 1, 2002, ©2002 Elsevier Science Ltd. All rights reserved.
Chromosome Segregation: Pulling from the Poles Duane A. Compton
Dual wavelength video microscopy has been used to evaluate how chromatids move poleward upon chromosome separation at anaphase. The data reveal that poleward microtubule flux provides the dominant force for separating chromatids in Drosophila embryos during anaphase A.
By one count in the middle of the 20th century there were as many as nine different mechanisms proposed to explain how chromatids segregate to spindle poles after chromosomes split in anaphase [1]. Over the past five decades, genetic, biochemical and cell biological experiments in different model systems have whittled that list down to just a handful, with merely two mechanisms implicated in vertebrate cells [2]. One mechanism involves kinetochore-associated motors that work in concert with microtubule plus-end disassembly to actively pull chromatids toward the pole (the so-called pac-man model). The other involves the poleward translocation, or poleward flux, of the entire spindle microtubule lattice, including attached chromatids, in conjunction with disassembly of microtubule minus ends at spindle poles (the so-called traction fiber model). Up until now, technical limitations have made it impossible to determine the extent to which poleward microtubule flux contributes to chromatid movement in experimental systems other than cultured vertebrate cells or egg extracts. With a new paper in this issue of Current Biology, Maddox and co-workers overcome these technical hurdles and provide stunning video images that not only reveal vigorous poleward microtubule flux in mitotic spindles in living Drosophila embryos, but also demonstrate that poleward microtubule flux is the dominant mechanism pulling chromatids poleward during anaphase A [3]. Poleward microtubule flux was first observed in cultured vertebrate cells using photoactivation of caged fluorescent tubulin [4]. Spindle microtubules saturated with caged tubulin were photoactivated with a focused beam of light to create a fluorescent bar across an otherwise dark spindle. Over time, the fluorescent bar translocated toward the spindle pole because microtubule assembly at plus ends was exactly matched by disassembly at minus ends at spindle poles. While photoactivation has proven powerful in observing flux in vertebrate cells and extracts, its application was limited to those experimental systems that had largesized spindles due to the need to photoactivate the caged tubulin. Department of Biochemistry, Dartmouth Medical School, 410 Remsen Building, Hanover, New Hampshire 03755, USA.
PII S0960-9822(02)01158-2
Dispatch
For these new studies in Drosophila embryos, Maddox and co-workers turned to fluorescent speckle microscopy (FSM), a relatively new technique to monitor microtubule dynamics in living cells [5]. This technique involves incorporation of sub-saturating quantities of fluorescently conjugated tubulin subunits into microtubules. At sub-saturating concentrations, microtubules containing the tagged tubulin subunits are non-uniformly labeled and appear to have fluorescent speckles. These speckles serve as reference marks to follow microtubule polymer dynamics in a similar way to the fluorescent bar created by the photoactivation technique. However, because FSM does not require photoactivation to create fluorescent marks on spindle microtubules, it is ideally suited to study microtubule behavior in cells with small spindles such as Drosophila embryos. Results from these new experiments generate two major conclusions. First, mitotic spindles in Drosophila embryos undergo robust poleward microtubule flux during both metaphase and anaphase A. Fluorescent speckles on microtubules were observed moving poleward during metaphase and anaphase A at uniform average velocities of 3.2 and 5.2 µm/min when measured at 18oC and 23–25oC, respectively. These velocities compare favorably with those measured in other experimental systems at metaphase such as cultured PtK1 cells (~0.5 µm/min) [4] and frog egg extracts (2.0 µm/min) [6]. These data provide definitive evidence of poleward microtubule flux in insects, an idea that was previously only supported by indirect evidence [7,8]. Furthermore, these data clearly establish that poleward microtubule flux is a general phenomenon that is not limited to spindles in vertebrate cells. Second, when chromosomes split at the onset of anaphase, the velocity of poleward chromatid movement very nearly matched the velocity of poleward microtubule flux. Using dual wavelength video microscopy the authors simultaneously tracked chromatid to pole movement and poleward microtubule flux in the same embryos. Whole chromatids were labeled with the DNA-binding dye DAPI or centromeres alone were labeled with green fluorescent protein (GFP)-tagged MeiS332, a Drosophila centromere protein. At 18oC, average poleward chromatid velocity was 3.6 µm/min, very close to the velocity of microtubule flux measured at that temperature (3.2 µm/min). At 23–25oC, chromatid movement was more variable with average velocities of 6.6 µm/min, slightly faster than the velocity of microtubule flux measured at that temperature (5.2 µm/min). These results indicate that the movement of the entire spindle lattice through poleward microtubule flux is the dominant mechanism for poleward chromatid movement as it contributes between 79% and 89% (depending on temperature) to the poleward movement of chromatids in anaphase A during early mitotic divisions in Drosophila embryos.
Dispatch R652
Metaphase
Anaphase
Current Biology
Figure 1. Poleward microtubule flux drives chromosome segregation. Fluorescent speckle microscopy reveals poleward microtubule flux in spindle microtubules as fluorescently tagged tubulin subunits non-uniformly incorporated into spindle microtubules (red) move progressively poleward (dashed line). In metaphase, net microtubule length remains constant as tubulin subunit assembly at plus ends matches disassembly at minus ends (small arrows). At anaphase, chromosomes separate and individual chromatids move poleward under the force of poleward microtubule flux (large arrows) as spindle microtubules primarily disassemble at minus ends at spindle poles.
At 23–25oC, the authors observed variable rates of poleward chromatid movement. Slow phases that closely matched the velocity of poleward microtubule flux were interspersed with fast phases that exceeded the velocity of poleward microtubule flux. During some of the fast phases of chromatid movement, kinetochores labeled with GFP–MeiS332 were observed to overtake and eliminate the speckles on spindle microtubules as the chromatid moved poleward. This indicates that a second mechanism utilizing kinetochore activity coupled to microtubule plus-end disassembly actively pulls chromatids poleward. This kinetochore activity most likely involves the minus-end-directed motor cytoplasmic dynein as previous studies showed that injection of Drosophila embryos with inhibitory dynein antibodies or the dominantly acting p50 subunit of dynactin suppressed only the rapid phase of poleward chromatid movement in anaphase [9]. Thus, as in vertebrate systems, both kinetochore activity and poleward microtubule flux act simultaneously to drive chromatid segregation in Drosophila embryos at 23–25oC. These two different mechanisms driving poleward chromatid movement most likely evolved to provide
redundancy to insure the fidelity of chromosome transmission at each cell division. However, the contribution that each mechanism makes to segregate chromatids differs among experimental systems. In cultured vertebrate cells, kinetochore activity dominates and poleward microtubule flux contributes only 25–30% to poleward chromatid movement [10]. On the other hand, in Drosophila embryos [3], as well as frog egg extracts [6], poleward microtubule flux dominates and kinetochore activity provides little contribution. How does poleward microtubule flux drive chromatid movement in anaphase A? Disassembly of microtubule minus ends at spindle poles appears to be a continuous process in mitosis (Figure 1). However, during metaphase, net microtubule length remains unchanged and no chromosome displacement occurs because the rate of microtubule disassembly at minus ends is exactly matched by the rate of microtubule assembly at plus ends. At anaphase onset, an abrupt change occurs when sister chromatids separate because the addition of tubulin subunits to kinetochore-associated microtubule plus ends ceases (in some cases, kinetochore-associated microtubule plus ends begin actively losing tubulin subunits). Under these conditions, microtubules shorten through subunit loss primarily at minus ends and the microtubules and their associated chromatids are dragged poleward. The molecular details of how poleward microtubule flux is generated are currently not known. Two phenomena lie at the heart of this issue. First is the continuous disassembly of microtubule minus ends at spindle poles and nothing is currently known about the molecules responsible for that process. Second is microtubule translocation poleward. Maddox and colleagues [3] show that all spindle microtubules, both kinetochore and non-kinetochore microtubules, undergo poleward flux. This has been observed in other systems as well and argues against mechanisms for poleward microtubule translocation predicated on the poleward sliding of one group of microtubules against another group of stationary microtubules. The fact that poleward microtubule flux in frog egg extracts is inhibited by non-hydrolyzable ATP analogs fueled speculation that microtubule motors power microtubule translocation [11]. However, other models such as one that combines minus end disassembly with biased microtubule diffusion (a thermal ratchet) cannot be excluded. The field can look forward to rapid progress in the hunt for the molecular mechanism driving poleward microtubule flux in the spindle now that the genetically tractable Drosophila system is available to attack the problem. References 1. Schrader, F. (1953). Mitosis: The movements of chromosomes in cell division, 2nd edn. (Columbia University Press, New York). 2. Gorbsky, G.J. (1992). Chromosome motion in mitosis. Bioessays 14, 73–80. 3. Maddox, P., Desai, A., Oegema, K., Mitchison, T.J. and Salmon, E.D. (2002). Poleward microtubule flux is a major component of spindle dynamics and anaphase A in mitotic Drosophila embryos. Curr. Biol. 12, this issue. 4. Mitchison, T.J. (1989). Poleward microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J. Cell Biol. 109, 637–652.
Current Biology R653
5. Waterman-Storer, C.M., Desai, A., Bulinski, J.C. and Salmon, E.D. (1998). Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr. Biol. 8, 1227–1230. 6. Desai, A., Maddox, P.S., Mitchison, T.J. and Salmon, E.D. (1998). Anaphase A chromosome movement and poleward spindle microtubule flux occur at similar rates in Xenopus extract spindles. J. Cell Biol. 141, 703–713. 7. Wilson, P.J., Forer, A. and Leggiadro, C. (1994). Evidence that kinetochore microtubules in crane-fly spermatocytes disassemble during anaphase primarily at the poleward end. J. Cell Sci. 107, 3015–3027. 8. LaFountain, J.R., Oldenbourg, R., Cole, R.W. and Rieder, C.L. (2001). Microtubule flux mediates poleward motion of acentric chromosome fragments during meiosis in insect spermatocytes. Mol. Biol. Cell 12, 4054–4065. 9. Sharp, D.J., Rogers, G.C. and Scholey, J.M. (2000). Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos. Nat. Cell Biol. 2, 922–930. 10. Mitchison, T.J. and Salmon, E.D. (1992). Poleward kinetochore fiber movement occurs during both metaphase and anaphase A in newt lung cell mitosis. J. Cell Biol. 119, 569–582. 11. Sawin, K.E. and Mitchison, T.J. (1991). Poleward microtubule flux in mitotic spindles assembled in vitro. J. Cell Biol. 112, 941–954.
Chromosome Segregation: Pulling from the Poles Duane A. Compton
Dual wavelength video microscopy has been used to evaluate how chromatids move poleward upon chromosome separation at anaphase. The data reveal that poleward microtubule flux provides the dominant force for separating chromatids in Drosophila embryos during anaphase A.
By one count in the middle of the 20th century there were as many as nine different mechanisms proposed to explain how chromatids segregate to spindle poles after chromosomes split in anaphase [1]. Over the past five decades, genetic, biochemical and cell biological experiments in different model systems have whittled that list down to just a handful, with merely two mechanisms implicated in vertebrate cells [2]. One mechanism involves kinetochore-associated motors that work in concert with microtubule plus-end disassembly to actively pull chromatids toward the pole (the so-called pac-man model). The other involves the poleward translocation, or poleward flux, of the entire spindle microtubule lattice, including attached chromatids, in conjunction with disassembly of microtubule minus ends at spindle poles (the so-called traction fiber model). Up until now, technical limitations have made it impossible to determine the extent to which poleward microtubule flux contributes to chromatid movement in experimental systems other than cultured vertebrate cells or egg extracts. With a new paper in this issue of Current Biology, Maddox and co-workers overcome these technical hurdles and provide stunning video images that not only reveal vigorous poleward microtubule flux in mitotic spindles in living Drosophila embryos, but also demonstrate that poleward microtubule flux is the dominant mechanism pulling chromatids poleward during anaphase A [3]. Poleward microtubule flux was first observed in cultured vertebrate cells using photoactivation of caged fluorescent tubulin [4]. Spindle microtubules saturated with caged tubulin were photoactivated with a focused beam of light to create a fluorescent bar across an otherwise dark spindle. Over time, the fluorescent bar translocated toward the spindle pole because microtubule assembly at plus ends was exactly matched by disassembly at minus ends at spindle poles. While photoactivation has proven powerful in observing flux in vertebrate cells and extracts, its application was limited to those experimental systems that had largesized spindles due to the need to photoactivate the caged tubulin. Department of Biochemistry, Dartmouth Medical School, 410 Remsen Building, Hanover, New Hampshire 03755, USA.
PII S0960-9822(02)01158-2
Dispatch
For these new studies in Drosophila embryos, Maddox and co-workers turned to fluorescent speckle microscopy (FSM), a relatively new technique to monitor microtubule dynamics in living cells [5]. This technique involves incorporation of sub-saturating quantities of fluorescently conjugated tubulin subunits into microtubules. At sub-saturating concentrations, microtubules containing the tagged tubulin subunits are non-uniformly labeled and appear to have fluorescent speckles. These speckles serve as reference marks to follow microtubule polymer dynamics in a similar way to the fluorescent bar created by the photoactivation technique. However, because FSM does not require photoactivation to create fluorescent marks on spindle microtubules, it is ideally suited to study microtubule behavior in cells with small spindles such as Drosophila embryos. Results from these new experiments generate two major conclusions. First, mitotic spindles in Drosophila embryos undergo robust poleward microtubule flux during both metaphase and anaphase A. Fluorescent speckles on microtubules were observed moving poleward during metaphase and anaphase A at uniform average velocities of 3.2 and 5.2 µm/min when measured at 18oC and 23–25oC, respectively. These velocities compare favorably with those measured in other experimental systems at metaphase such as cultured PtK1 cells (~0.5 µm/min) [4] and frog egg extracts (2.0 µm/min) [6]. These data provide definitive evidence of poleward microtubule flux in insects, an idea that was previously only supported by indirect evidence [7,8]. Furthermore, these data clearly establish that poleward microtubule flux is a general phenomenon that is not limited to spindles in vertebrate cells. Second, when chromosomes split at the onset of anaphase, the velocity of poleward chromatid movement very nearly matched the velocity of poleward microtubule flux. Using dual wavelength video microscopy the authors simultaneously tracked chromatid to pole movement and poleward microtubule flux in the same embryos. Whole chromatids were labeled with the DNA-binding dye DAPI or centromeres alone were labeled with green fluorescent protein (GFP)-tagged MeiS332, a Drosophila centromere protein. At 18oC, average poleward chromatid velocity was 3.6 µm/min, very close to the velocity of microtubule flux measured at that temperature (3.2 µm/min). At 23–25oC, chromatid movement was more variable with average velocities of 6.6 µm/min, slightly faster than the velocity of microtubule flux measured at that temperature (5.2 µm/min). These results indicate that the movement of the entire spindle lattice through poleward microtubule flux is the dominant mechanism for poleward chromatid movement as it contributes between 79% and 89% (depending on temperature) to the poleward movement of chromatids in anaphase A during early mitotic divisions in Drosophila embryos.
Dispatch R652
Metaphase
Anaphase
Current Biology
Figure 1. Poleward microtubule flux drives chromosome segregation. Fluorescent speckle microscopy reveals poleward microtubule flux in spindle microtubules as fluorescently tagged tubulin subunits non-uniformly incorporated into spindle microtubules (red) move progressively poleward (dashed line). In metaphase, net microtubule length remains constant as tubulin subunit assembly at plus ends matches disassembly at minus ends (small arrows). At anaphase, chromosomes separate and individual chromatids move poleward under the force of poleward microtubule flux (large arrows) as spindle microtubules primarily disassemble at minus ends at spindle poles.
At 23–25oC, the authors observed variable rates of poleward chromatid movement. Slow phases that closely matched the velocity of poleward microtubule flux were interspersed with fast phases that exceeded the velocity of poleward microtubule flux. During some of the fast phases of chromatid movement, kinetochores labeled with GFP–MeiS332 were observed to overtake and eliminate the speckles on spindle microtubules as the chromatid moved poleward. This indicates that a second mechanism utilizing kinetochore activity coupled to microtubule plus-end disassembly actively pulls chromatids poleward. This kinetochore activity most likely involves the minus-end-directed motor cytoplasmic dynein as previous studies showed that injection of Drosophila embryos with inhibitory dynein antibodies or the dominantly acting p50 subunit of dynactin suppressed only the rapid phase of poleward chromatid movement in anaphase [9]. Thus, as in vertebrate systems, both kinetochore activity and poleward microtubule flux act simultaneously to drive chromatid segregation in Drosophila embryos at 23–25oC. These two different mechanisms driving poleward chromatid movement most likely evolved to provide
redundancy to insure the fidelity of chromosome transmission at each cell division. However, the contribution that each mechanism makes to segregate chromatids differs among experimental systems. In cultured vertebrate cells, kinetochore activity dominates and poleward microtubule flux contributes only 25–30% to poleward chromatid movement [10]. On the other hand, in Drosophila embryos [3], as well as frog egg extracts [6], poleward microtubule flux dominates and kinetochore activity provides little contribution. How does poleward microtubule flux drive chromatid movement in anaphase A? Disassembly of microtubule minus ends at spindle poles appears to be a continuous process in mitosis (Figure 1). However, during metaphase, net microtubule length remains unchanged and no chromosome displacement occurs because the rate of microtubule disassembly at minus ends is exactly matched by the rate of microtubule assembly at plus ends. At anaphase onset, an abrupt change occurs when sister chromatids separate because the addition of tubulin subunits to kinetochore-associated microtubule plus ends ceases (in some cases, kinetochore-associated microtubule plus ends begin actively losing tubulin subunits). Under these conditions, microtubules shorten through subunit loss primarily at minus ends and the microtubules and their associated chromatids are dragged poleward. The molecular details of how poleward microtubule flux is generated are currently not known. Two phenomena lie at the heart of this issue. First is the continuous disassembly of microtubule minus ends at spindle poles and nothing is currently known about the molecules responsible for that process. Second is microtubule translocation poleward. Maddox and colleagues [3] show that all spindle microtubules, both kinetochore and non-kinetochore microtubules, undergo poleward flux. This has been observed in other systems as well and argues against mechanisms for poleward microtubule translocation predicated on the poleward sliding of one group of microtubules against another group of stationary microtubules. The fact that poleward microtubule flux in frog egg extracts is inhibited by non-hydrolyzable ATP analogs fueled speculation that microtubule motors power microtubule translocation [11]. However, other models such as one that combines minus end disassembly with biased microtubule diffusion (a thermal ratchet) cannot be excluded. The field can look forward to rapid progress in the hunt for the molecular mechanism driving poleward microtubule flux in the spindle now that the genetically tractable Drosophila system is available to attack the problem. References 1. Schrader, F. (1953). Mitosis: The movements of chromosomes in cell division, 2nd edn. (Columbia University Press, New York). 2. Gorbsky, G.J. (1992). Chromosome motion in mitosis. Bioessays 14, 73–80. 3. Maddox, P., Desai, A., Oegema, K., Mitchison, T.J. and Salmon, E.D. (2002). Poleward microtubule flux is a major component of spindle dynamics and anaphase A in mitotic Drosophila embryos. Curr. Biol. 12, this issue. 4. Mitchison, T.J. (1989). Poleward microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J. Cell Biol. 109, 637–652.
Current Biology R653
5. Waterman-Storer, C.M., Desai, A., Bulinski, J.C. and Salmon, E.D. (1998). Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Curr. Biol. 8, 1227–1230. 6. Desai, A., Maddox, P.S., Mitchison, T.J. and Salmon, E.D. (1998). Anaphase A chromosome movement and poleward spindle microtubule flux occur at similar rates in Xenopus extract spindles. J. Cell Biol. 141, 703–713. 7. Wilson, P.J., Forer, A. and Leggiadro, C. (1994). Evidence that kinetochore microtubules in crane-fly spermatocytes disassemble during anaphase primarily at the poleward end. J. Cell Sci. 107, 3015–3027. 8. LaFountain, J.R., Oldenbourg, R., Cole, R.W. and Rieder, C.L. (2001). Microtubule flux mediates poleward motion of acentric chromosome fragments during meiosis in insect spermatocytes. Mol. Biol. Cell 12, 4054–4065. 9. Sharp, D.J., Rogers, G.C. and Scholey, J.M. (2000). Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos. Nat. Cell Biol. 2, 922–930. 10. Mitchison, T.J. and Salmon, E.D. (1992). Poleward kinetochore fiber movement occurs during both metaphase and anaphase A in newt lung cell mitosis. J. Cell Biol. 119, 569–582. 11. Sawin, K.E. and Mitchison, T.J. (1991). Poleward microtubule flux in mitotic spindles assembled in vitro. J. Cell Biol. 112, 941–954.