- Email: [email protected]
Flagellar Motility: All Pull Together
Current Biology, Vol. 14, R992–R993, December 14, 2004, ©2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cub.2004.11.019
Flagellar Motility: All Pull Together Kimberly A. Wemmer and Wallace F. Marshall
Eukaryotic flagella produce a swimming force by coordinating thousands of dynein motor proteins. Recent work provides new clues into how this coordination is achieved.
Flagella are microtubule-based structures that propel cells through the surrounding fluid. The internal structure of a flagellum consists of nine parallel doublet microtubules arranged around a central pair of singlet microtubules (Figure 1). Force for propulsion is provided by thousands of dynein motors anchored in rows along one side of each doublet, which can walk along the microtubule of the adjacent doublet. In order to produce coordinated bending of the flagellum, these dynein motors — organized into multi-headed complexes called the inner and outer dynein arms — must produce their power strokes in synchrony, like the oarsmen on an ancient Mediterranean war-galley. But whereas oar-strokes were coordinated by a continuous drum-beat, it is much less clear how flagellar dynein motors are synchronized. One possibility is that the dynein motors can synchronize themselves. An extensive theory exists for spontaneous entrainment of coupled oscillators. This theory shows that if a system of oscillators are connected such that the phase of an oscillator shifts according to the phase difference between itself and the other oscillators, the entire system will spontaneously synchronize under a wide range of coupling parameters, resulting in coherent behavior [1]. This emergent property of coupled-oscillator systems is thought to explain the synchronized flashing of fire-flies and the beating of the heart. Motor proteins are oscillators which undergo coupled cycles of movement and ATP hydrolysis, and it is known that mechanical forces applied to a motor can affect the rate of progress through the ATP hydrolysis cycle. For example, force-dependent oscillatory behavior has been shown for flagellar dyneins [2]. A system of dynein motors mechanically connected via the flagellar microtubules could, therefore, potentially undergo spontaneous entrainment leading to coherent bending movement. This type of model predicts that bending forces applied to a flagellum should alter the activity of dynein arms. In a recent elegant study reported in this issue of Current Biology, Morita and Shingyoji [3] directly applied bending forces to sea urchin sperm flagella. They used the dynein-driven fragmentation of elastase-treated sea urchin flagella as a reporter for dynein arm activity. Wherever dynein arms were activated, the doublets slid apart, providing a visual Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th Street, San Francisco, California 94143, USA. E-mail: [email protected]
Dispatch
indicator of which dynein rows were active. Using this assay, these workers found that an applied bending force activated dynein arms that were inactive in unbent flagella. Their observations support the general idea that force-feedback alters dynein motor activity. But how is a bending force transduced to the dynein arms in a flagellum? One possibility, mentioned above, is that the transduction involves direct mechanical feedback, whereby the dynein arms can sense the applied force. There is substantial evidence, however, that the central pair may play a role in controlling dynein arm activity, which raises the possibility that this structure might regulate dynein in response to bending. The central pair is a complex sub-structure within the flagellum, which contains not only a pair of microtubules, C1 and C2, but also an elaborate set of projections (Figure 1). Studies in sea urchin sperm and the motile alga Chlamydomonas have shown that only those dynein arms located near one side of the C1 microtubule are active, while the rest are inactive [4,5], suggesting that the central pair somehow stimulates dynein activity. The central pair in Chlamydomonas is inherently twisted, and untwists as the flagellum bends [6]. This change in twist might alter the number and position of dynein arms activated by the central pair during the course of a bending cycle, thereby producing a defined wave-form. The central pair is currently thought to interact with the radial spokes and signal the dynein arms through a protein complex called the dynein regulatory complex, located between the spokes and the dynein arms [7]. The molecular mechanism by which the central pair regulates dynein is not known. The presence of proteins homologous to signaling proteins within the spokes and central pair [8] suggests that the central pair uses second messenger molecules located within the spokes or dynein regulatory complex to modulate dynein function. Although rotation of the central pair is likely to play an important role in regulating the flagellar waveform, it is clearly not essential for motility. In some organisms, the central pair does not rotate [9]. In Chlamydomonas, mutants with central pair or radial spoke defects have paralyzed flagella, and this paralysis can be rescued by suppressor mutations in the dynein motor proteins without rescuing the defect in the spokes or central pair ([10] for example). Moreover, in mutants lacking spokes or central pair structures, dynein arm activation is still non-random — only dynein molecules on specific doublets are activated [11]. These results suggest that central pair rotation is less important for generating the waveform than for changing it in response to extrinsic signals, for example during phototaxis [6]. In order for all these interactions to occur in their prescribed manner, cells must assemble the flagellar skeleton properly. This task is not as complex as it might at first appear when the modular structure of the flagellum is taken into account. Each outer doublet consists of a 96 nm repeat structure, which contains a fixed
Current Biology R993
Figure 1. The modular structure of a flagellum. The drawing depicts the repeating structures within a flagellum. Microtubules (dark blue circles) make up the backbone of the structure. Far right: largest magnification, showing the central pair, C1 and C2, with associated projections (1a–2c, light blue) and sheath (gray curves), and a single outer doublet, with its radial spoke (green) and inner (ida) and outer (oda) dynein arms (pink).
oda
ida Radial spoke 1a
2a 2c
1c 1d
C1 1b
C2 2b Sheath
Current Biology
set of dynein arms and radial spokes [12]. Because this basic structure is repeated through the length of the structure, the cell is able to focus on assembling many copies of one small structure over and over rather than a single, large, complicated one. The 96 nm repeat functional module can be further broken down into smaller pieces, such as the radial spoke and the dynein arms, which self-assemble within the cytoplasm, either entirely or in smaller sub-assemblies. They are then added onto the growing structure, rather than building each protein subunit onto the microtubules themselves [13–15]. This kind of organization and building plan would allow the cell to build an intricate structure very simply, by assembling a few pieces that fit well together, rather than trying to place each protein into a specific position along the entire length of the flagellum. The organization of the flagellum as a series of identical repeats poses a problem to the cell, which is how are the number of repeats present in the organelle controlled. This issue of flagellar length control is particularly interesting because the length of a wild-type Chlamydomonas flagellum consistently falls within a precise range. The length of a given flagellum appears to be quite stable, though it has been demonstrated that tubulin subunits at the distal tip are continuously turning over [16]. The constant loss of subunits at the tip is balanced by a constant supply of new subunits brought from the cell body by intraflagellar transport. The rate of particle movement by intraflagellar transport along the microtubules is constant [17] and the particle number is fixed [16], so as the length of the flagellum increases, the rate of particle arrival and delivery of subunits at the tip decreases, reducing the rate of assembly. It has been theorized that the length of the flagellum is achieved when assembly and disassembly are at equilibrium [16]. This flagellar length system is apparently regulated by intracellular signaling pathways [18], providing the cell control over the size of its flagella. These processes put all the pieces together to assemble a subcellular machine of defined size and structure. References 1. Strogatz, S.H. (1994). Nonlinear dynamics and chaos: with applications in physics, biology, chemistry, and engineering. (New York: Addison Wesley).
2. Shingyoji, C., Higuchi, H., Yoshimura, M., Katayama, E., and Yanagida, T. (1998). Dynein arms are oscillating force generators. Nature 393, 711-714. 3. Morita, Y., and Shingyoji, C. (2004). Effects of imposed bending on microtubule sliding in sperm flagella. Curr. Biol. 14, this issue. 4. Nakano, I, Kobayashi, T., Yoshimura, M., and Shingyoji, C. (2003). Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes. J. Cell Sci. 116, 1627-1636. 5. Wargo, M.J., and Smith, E.F. (2003). Asymmetry of the central apparatus defines the location of active microtubule sliding in Chlamydomonas flagella. Proc. Natl. Acad. Sci. U.S.A. 100, 137-142. 6. Mitchell, D.R., and Nakatsugawa, M. (2004). Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella. J. Cell Biol. 166, 709-715. 7. Gardner, L.C., O'Toole, E., Perrone, C.A., Giddings, T., and Porter, M.E. (1994). Components of a ‘dynein regulatory complex’ are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella. J. Cell Biol. 127, 1311-1325. 8. Smith, E.F., and Yang, P. (2004). The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil. Cytoskel. 57, 8-17. 9. Tamm, S.L., and Tamm, S. (1981). Ciliary reversal without rotation of axonemal structures in Ctenophore comb plates. J. Cell Biol. 89, 495-509. 10. Porter, M.E., Knott, J.A., Gardner, L.C., Mitchell, D.R., and Dutcher, S.K. (1994). Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain. J. Cell Biol. 126, 1495-1507. 11. Wargo, M.J., McPeek, M.A., and Smith, E.F. (2004). Analysis of microtubule sliding patterns in Chlamydomonas flagellar axonemes reveals dynein activity on specific doublet microtubules. J. Cell Sci. 117, 2533-2544. 12. Mastronarde, D.N., O'Toole, E.T., McDonald, K.L., McIntosh, J.R., and Porter, M.E. (1992). Arrangement of inner dynein arms in wildtype and mutant flagella of Chlamydomonas. J. Cell Biol. 118, 11451162. 13. Yang, P., Diener, D.R., Rosenbaum, J.L., and Sale, W.S. (2001). Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes. J Cell Biol. 153, 1315-1326. 14. Qin, H., Diener, D.R., Geimer, S., Cole, D.G., and Rosenbaum, J.L. (2004). Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body. J. Cell Biol. 164, 255-266. 15. Fowkes, M.E., and Mitchell, D.R. (1998). The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits. Mol. Biol. Cell 9, 2337-2347. 16. Marshall, W.F., and Rosenabum, J.L. (2001). Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J. Cell Biol. 155, 405-414. 17. Iomini, C., Babaev-Khaimov, V., Sassaroli, M., and Piperno, G. (2001). Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases. J. Cell Biol. 153, 13-24. 18. Wilson, N.F., and Lefebvre, P.A. (2004). Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii. Euk. Cell 3, 1307-1319.
Flagellar Motility: All Pull Together Kimberly A. Wemmer and Wallace F. Marshall
Eukaryotic flagella produce a swimming force by coordinating thousands of dynein motor proteins. Recent work provides new clues into how this coordination is achieved.
Flagella are microtubule-based structures that propel cells through the surrounding fluid. The internal structure of a flagellum consists of nine parallel doublet microtubules arranged around a central pair of singlet microtubules (Figure 1). Force for propulsion is provided by thousands of dynein motors anchored in rows along one side of each doublet, which can walk along the microtubule of the adjacent doublet. In order to produce coordinated bending of the flagellum, these dynein motors — organized into multi-headed complexes called the inner and outer dynein arms — must produce their power strokes in synchrony, like the oarsmen on an ancient Mediterranean war-galley. But whereas oar-strokes were coordinated by a continuous drum-beat, it is much less clear how flagellar dynein motors are synchronized. One possibility is that the dynein motors can synchronize themselves. An extensive theory exists for spontaneous entrainment of coupled oscillators. This theory shows that if a system of oscillators are connected such that the phase of an oscillator shifts according to the phase difference between itself and the other oscillators, the entire system will spontaneously synchronize under a wide range of coupling parameters, resulting in coherent behavior [1]. This emergent property of coupled-oscillator systems is thought to explain the synchronized flashing of fire-flies and the beating of the heart. Motor proteins are oscillators which undergo coupled cycles of movement and ATP hydrolysis, and it is known that mechanical forces applied to a motor can affect the rate of progress through the ATP hydrolysis cycle. For example, force-dependent oscillatory behavior has been shown for flagellar dyneins [2]. A system of dynein motors mechanically connected via the flagellar microtubules could, therefore, potentially undergo spontaneous entrainment leading to coherent bending movement. This type of model predicts that bending forces applied to a flagellum should alter the activity of dynein arms. In a recent elegant study reported in this issue of Current Biology, Morita and Shingyoji [3] directly applied bending forces to sea urchin sperm flagella. They used the dynein-driven fragmentation of elastase-treated sea urchin flagella as a reporter for dynein arm activity. Wherever dynein arms were activated, the doublets slid apart, providing a visual Department of Biochemistry and Biophysics, University of California, San Francisco, 600 16th Street, San Francisco, California 94143, USA. E-mail: [email protected]
Dispatch
indicator of which dynein rows were active. Using this assay, these workers found that an applied bending force activated dynein arms that were inactive in unbent flagella. Their observations support the general idea that force-feedback alters dynein motor activity. But how is a bending force transduced to the dynein arms in a flagellum? One possibility, mentioned above, is that the transduction involves direct mechanical feedback, whereby the dynein arms can sense the applied force. There is substantial evidence, however, that the central pair may play a role in controlling dynein arm activity, which raises the possibility that this structure might regulate dynein in response to bending. The central pair is a complex sub-structure within the flagellum, which contains not only a pair of microtubules, C1 and C2, but also an elaborate set of projections (Figure 1). Studies in sea urchin sperm and the motile alga Chlamydomonas have shown that only those dynein arms located near one side of the C1 microtubule are active, while the rest are inactive [4,5], suggesting that the central pair somehow stimulates dynein activity. The central pair in Chlamydomonas is inherently twisted, and untwists as the flagellum bends [6]. This change in twist might alter the number and position of dynein arms activated by the central pair during the course of a bending cycle, thereby producing a defined wave-form. The central pair is currently thought to interact with the radial spokes and signal the dynein arms through a protein complex called the dynein regulatory complex, located between the spokes and the dynein arms [7]. The molecular mechanism by which the central pair regulates dynein is not known. The presence of proteins homologous to signaling proteins within the spokes and central pair [8] suggests that the central pair uses second messenger molecules located within the spokes or dynein regulatory complex to modulate dynein function. Although rotation of the central pair is likely to play an important role in regulating the flagellar waveform, it is clearly not essential for motility. In some organisms, the central pair does not rotate [9]. In Chlamydomonas, mutants with central pair or radial spoke defects have paralyzed flagella, and this paralysis can be rescued by suppressor mutations in the dynein motor proteins without rescuing the defect in the spokes or central pair ([10] for example). Moreover, in mutants lacking spokes or central pair structures, dynein arm activation is still non-random — only dynein molecules on specific doublets are activated [11]. These results suggest that central pair rotation is less important for generating the waveform than for changing it in response to extrinsic signals, for example during phototaxis [6]. In order for all these interactions to occur in their prescribed manner, cells must assemble the flagellar skeleton properly. This task is not as complex as it might at first appear when the modular structure of the flagellum is taken into account. Each outer doublet consists of a 96 nm repeat structure, which contains a fixed
Current Biology R993
Figure 1. The modular structure of a flagellum. The drawing depicts the repeating structures within a flagellum. Microtubules (dark blue circles) make up the backbone of the structure. Far right: largest magnification, showing the central pair, C1 and C2, with associated projections (1a–2c, light blue) and sheath (gray curves), and a single outer doublet, with its radial spoke (green) and inner (ida) and outer (oda) dynein arms (pink).
oda
ida Radial spoke 1a
2a 2c
1c 1d
C1 1b
C2 2b Sheath
Current Biology
set of dynein arms and radial spokes [12]. Because this basic structure is repeated through the length of the structure, the cell is able to focus on assembling many copies of one small structure over and over rather than a single, large, complicated one. The 96 nm repeat functional module can be further broken down into smaller pieces, such as the radial spoke and the dynein arms, which self-assemble within the cytoplasm, either entirely or in smaller sub-assemblies. They are then added onto the growing structure, rather than building each protein subunit onto the microtubules themselves [13–15]. This kind of organization and building plan would allow the cell to build an intricate structure very simply, by assembling a few pieces that fit well together, rather than trying to place each protein into a specific position along the entire length of the flagellum. The organization of the flagellum as a series of identical repeats poses a problem to the cell, which is how are the number of repeats present in the organelle controlled. This issue of flagellar length control is particularly interesting because the length of a wild-type Chlamydomonas flagellum consistently falls within a precise range. The length of a given flagellum appears to be quite stable, though it has been demonstrated that tubulin subunits at the distal tip are continuously turning over [16]. The constant loss of subunits at the tip is balanced by a constant supply of new subunits brought from the cell body by intraflagellar transport. The rate of particle movement by intraflagellar transport along the microtubules is constant [17] and the particle number is fixed [16], so as the length of the flagellum increases, the rate of particle arrival and delivery of subunits at the tip decreases, reducing the rate of assembly. It has been theorized that the length of the flagellum is achieved when assembly and disassembly are at equilibrium [16]. This flagellar length system is apparently regulated by intracellular signaling pathways [18], providing the cell control over the size of its flagella. These processes put all the pieces together to assemble a subcellular machine of defined size and structure. References 1. Strogatz, S.H. (1994). Nonlinear dynamics and chaos: with applications in physics, biology, chemistry, and engineering. (New York: Addison Wesley).
2. Shingyoji, C., Higuchi, H., Yoshimura, M., Katayama, E., and Yanagida, T. (1998). Dynein arms are oscillating force generators. Nature 393, 711-714. 3. Morita, Y., and Shingyoji, C. (2004). Effects of imposed bending on microtubule sliding in sperm flagella. Curr. Biol. 14, this issue. 4. Nakano, I, Kobayashi, T., Yoshimura, M., and Shingyoji, C. (2003). Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes. J. Cell Sci. 116, 1627-1636. 5. Wargo, M.J., and Smith, E.F. (2003). Asymmetry of the central apparatus defines the location of active microtubule sliding in Chlamydomonas flagella. Proc. Natl. Acad. Sci. U.S.A. 100, 137-142. 6. Mitchell, D.R., and Nakatsugawa, M. (2004). Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella. J. Cell Biol. 166, 709-715. 7. Gardner, L.C., O'Toole, E., Perrone, C.A., Giddings, T., and Porter, M.E. (1994). Components of a ‘dynein regulatory complex’ are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella. J. Cell Biol. 127, 1311-1325. 8. Smith, E.F., and Yang, P. (2004). The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil. Cytoskel. 57, 8-17. 9. Tamm, S.L., and Tamm, S. (1981). Ciliary reversal without rotation of axonemal structures in Ctenophore comb plates. J. Cell Biol. 89, 495-509. 10. Porter, M.E., Knott, J.A., Gardner, L.C., Mitchell, D.R., and Dutcher, S.K. (1994). Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain. J. Cell Biol. 126, 1495-1507. 11. Wargo, M.J., McPeek, M.A., and Smith, E.F. (2004). Analysis of microtubule sliding patterns in Chlamydomonas flagellar axonemes reveals dynein activity on specific doublet microtubules. J. Cell Sci. 117, 2533-2544. 12. Mastronarde, D.N., O'Toole, E.T., McDonald, K.L., McIntosh, J.R., and Porter, M.E. (1992). Arrangement of inner dynein arms in wildtype and mutant flagella of Chlamydomonas. J. Cell Biol. 118, 11451162. 13. Yang, P., Diener, D.R., Rosenbaum, J.L., and Sale, W.S. (2001). Localization of calmodulin and dynein light chain LC8 in flagellar radial spokes. J Cell Biol. 153, 1315-1326. 14. Qin, H., Diener, D.R., Geimer, S., Cole, D.G., and Rosenbaum, J.L. (2004). Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body. J. Cell Biol. 164, 255-266. 15. Fowkes, M.E., and Mitchell, D.R. (1998). The role of preassembled cytoplasmic complexes in assembly of flagellar dynein subunits. Mol. Biol. Cell 9, 2337-2347. 16. Marshall, W.F., and Rosenabum, J.L. (2001). Intraflagellar transport balances continuous turnover of outer doublet microtubules: implications for flagellar length control. J. Cell Biol. 155, 405-414. 17. Iomini, C., Babaev-Khaimov, V., Sassaroli, M., and Piperno, G. (2001). Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases. J. Cell Biol. 153, 13-24. 18. Wilson, N.F., and Lefebvre, P.A. (2004). Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii. Euk. Cell 3, 1307-1319.