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What Doesn’t Kill You Makes You Stronger
Developmental Cell
Spotlight What Doesn’t Kill You Makes You Stronger Andrea K.H. Stavoe1 and Erika L.F. Holzbaur1,* 1Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.11.003
The microtubule-severing proteins spastin and katanin were long thought to destabilize microtubules. Recent work demonstrates that these enzymes inflict nano-damage on the microtubule lattice that is then rapidly repaired by new GTP-tubulin incorporation, for a net stabilization of the polymer, a process that has implications for neurodegenerative disease.
Microtubules are an essential component of the cellular cytoskeleton, providing structural support for the cell as well as serving as tracks for the translocation of organelles during interphase and chromosomes during mitosis. Microtubules form via the head-to-tail polymerization of tubulin dimers into protofilaments that are organized into a 13-protofilament tubule. Key to the biology of microtubules are their dynamics, regulated by GTP hydrolysis in a process termed dynamic instability. GTP-bound tubulin subunits preferentially assemble at microtubule plus ends, leading to a local enrichment of GTP-tubulin forming a ‘‘GTP-cap’’ that favors the continued slow growth of the polymer. However, hydrolysis of the tubulin-bound GTP accelerates once subunits are incorporated into the polymer. Complete loss of the protective GTP-cap at the microtubule plus end induces a switch to rapid disassembly in a process termed catastrophe. Rescue occurs when disassembling microtubules stochastically switch back to resume slow growth. Microtubules are critically important in neurons, where they serve as tracks for transport of organelles over distances of up to 1 m along the axons of motor and sensory neurons. In vivo, microtubule dynamics are modulated by regulatory factors influencing nucleation, stabilization, growth, and shortening, but the underlying mechanisms are still not fully understood. In vitro experiments have demonstrated that AAA (Adenosine triphosphatase Associated with various cellular Activities) ATPase family members spastin, katanin, and fidgetin can sever microtubules. Severing destabilizes the microtubule by exposing GDP-bound subunits, leading to complete disas-
sembly of the polymer over time. Yet, paradoxically, loss of spastin leads to a decrease in microtubule mass in Drosophila and zebrafish neurons (Sherwood et al., 2004; Wood et al., 2006). Furthermore, mutations in spastin are causative for the neurodegenerative disorder hereditary spastic paraplegia (HSP), whereas mutations in katanin cause microcephaly and seizures (Mishra-Gorur et al., 2014; Roll-Mecak and McNally, 2010; Yigit et al., 2016). New insights come from the work of Vemu et al. (2018) that can explain this puzzle of why microtubule mass decreases upon loss of a microtubule-severing enzyme. Roll-Mecak and her group examined the effects of spastin and katanin on microtubules using electron microscopy (EM), as well as total internal reflection (TIRF) microscopy to visualize severing events with singlemolecule resolution. Over prolonged incubation times, microtubules were severed and then disassembled rapidly as previously observed. However, Vemu et al. (2018) noted that shorter incubations of microtubules with spastin or katanin led to extensive nanoscale defects in the polymer—small potholes in the polymer lattice were evident using EM. Only at longer incubation times did these nanoscale defects progress to severing and then frank microtubule disassembly. Remarkably, inclusion of GTP-tubulin in these assays led to local repair of the nanoscale damage—the potholes in the lattice induced by spastin or katanin were repaired by the localized incorporation of new GTP-bound tubulin dimers. These repairs formed ‘‘GTP islands’’ distributed along the length of the polymer. This observation led to the next question: what effect would these GTP
402 Developmental Cell 47, November 19, 2018 ª 2018 Published by Elsevier Inc.
islands have on the overall dynamics of the polymer? Recent work has shown that rescue, the switch from a rapidly disassembling to a slowly growing microtubule, may not be random, but instead occurs preferentially at sites in the lattice enriched in GTP-tubulin (Aumeier et al., 2016; Dimitrov et al., 2008). As expected, the GTP islands resulting from localized repair of spastin- or katanin-mediated damage enhanced rescue by more than 10-fold. Further, Vemu and colleagues (2018) showed that frank severing of the microtubule can also lead to increased microtubule mass, as newly severed ends reinitiate rapid growth via incorporation of GTP-tubulin. Thus, when it comes to the effects of spastin or katanin on a microtubule, what doesn’t kill you makes you stronger. So how does this observation provide new insights into the cellular function of severing enzymes, and more specifically, the observation that mutations in spastin cause HSP? The answer is not yet clear but is beginning to come into focus. Spastin is enriched at synapses (Sherwood et al., 2004). Known HSP-associated mutations in spastin affect its ability to sever microtubules (Roll-Mecak and Vale, 2005) and lead to reduced microtubule density at synapses such as the neuromuscular junction. We now know that alterations in the ratio of GTP-bound to GDP-bound tubulin at microtubule plus ends affect both the stability of the microtubules and the ability of cargos to load or unload from the microtubule (Nirschl et al., 2017). Thus, mutations in spastin or katanin are likely to affect not just the microtubule itself but also the ability of the microtubule track to serve as an effective highway for the high-speed, long-distance trafficking of organelles. In turn,
Developmental Cell
Spotlight defects in this long-distance trafficking are sufficient to cause neurodegeneration (Nirschl et al., 2017). This new work from Vemu et al. (2018) uses both classical (EM) and more recent (TIRF microscopy) techniques to answer an important question in cell biology— what regulates microtubule plasticity— and an important question in neurodegeneration—how might mutations in spastin lead to HSP. The women behind this work, including the first author Annapurna Vemu and the senior author Antoninia Roll-Mecak, are distinguished by their ability to look at a problem with fresh eyes and to use rigorous quantitative biophysical approaches to provide a new and exciting answer. Of course, the work raises many further questions that will require the application of cutting-edge cell biology to the analysis of cytoskeletal and organelle dynamics in vivo. We look forward to these future discoveries.
REFERENCES
paraplegia protein, spastin, severs and disassembles microtubules. Curr. Biol. 15, 650–655.
Aumeier, C., Schaedel, L., Gaillard, J., John, K., Blanchoin, L., and Thery, M. (2016). Self-repair promotes microtubule rescue. Nat. Cell Biol. 18, 1054–1064. Dimitrov, A., Quesnoit, M., Moutel, S., Cantaloube, I., Pous, C., and Perez, F. (2008). Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues. Science 322, 1353–1356. layan, A.O., Schaffer, A.E., Mishra-Gorur, K., C¸ag €mu €s¸, Chabu, C., Henegariu, O., Vonhoff, F., Akgu G.T., Nishimura, S., Han, W., Tu, S., et al. (2014). Mutations in KATNB1 cause complex cerebral malformations by disrupting asymmetrically dividing neural progenitors. Neuron 84, 1226–1239. Nirschl, J.J., Ghiretti, A.E., and Holzbaur, E.L.F. (2017). The impact of cytoskeletal organization on the local regulation of neuronal transport. Nat. Rev. Neurosci. 18, 585–597. Roll-Mecak, A., and McNally, F.J. (2010). Microtubule-severing enzymes. Curr. Opin. Cell Biol. 22, 96–103. Roll-Mecak, A., and Vale, R.D. (2005). The Drosophila homologue of the hereditary spastic
Sherwood, N.T., Sun, Q., Xue, M., Zhang, B., and Zinn, K. (2004). Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS Biol. 2, e429. Vemu, A., Szczesna, E., Zehr, E.A., Spector, J.O., Grigorieff, N., Deaconescu, A.M., and Roll-Mecak, A. (2018). Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation. Science 361, https://doi.org/10.1126/science. aau1504. Wood, J.D., Landers, J.A., Bingley, M., McDermott, C.J., Thomas-McArthur, V., Gleadall, L.J., Shaw, P.J., and Cunliffe, V.T. (2006). The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo. Hum. Mol. Genet. 15, 2763–2771. Yigit, G., Wieczorek, D., Bo¨gershausen, N., Be€ller, J., leggia, F., Mo¨ller-Hartmann, C., Altmu €rnberg, P., and Wollnik, B. (2016). Thiele, H., Nu A syndrome of microcephaly, short stature, polysyndactyly, and dental anomalies caused by a homozygous KATNB1 mutation. Am. J. Med. Genet. A. 170, 728–733.
Developmental Cell 47, November 19, 2018 403
Spotlight What Doesn’t Kill You Makes You Stronger Andrea K.H. Stavoe1 and Erika L.F. Holzbaur1,* 1Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.11.003
The microtubule-severing proteins spastin and katanin were long thought to destabilize microtubules. Recent work demonstrates that these enzymes inflict nano-damage on the microtubule lattice that is then rapidly repaired by new GTP-tubulin incorporation, for a net stabilization of the polymer, a process that has implications for neurodegenerative disease.
Microtubules are an essential component of the cellular cytoskeleton, providing structural support for the cell as well as serving as tracks for the translocation of organelles during interphase and chromosomes during mitosis. Microtubules form via the head-to-tail polymerization of tubulin dimers into protofilaments that are organized into a 13-protofilament tubule. Key to the biology of microtubules are their dynamics, regulated by GTP hydrolysis in a process termed dynamic instability. GTP-bound tubulin subunits preferentially assemble at microtubule plus ends, leading to a local enrichment of GTP-tubulin forming a ‘‘GTP-cap’’ that favors the continued slow growth of the polymer. However, hydrolysis of the tubulin-bound GTP accelerates once subunits are incorporated into the polymer. Complete loss of the protective GTP-cap at the microtubule plus end induces a switch to rapid disassembly in a process termed catastrophe. Rescue occurs when disassembling microtubules stochastically switch back to resume slow growth. Microtubules are critically important in neurons, where they serve as tracks for transport of organelles over distances of up to 1 m along the axons of motor and sensory neurons. In vivo, microtubule dynamics are modulated by regulatory factors influencing nucleation, stabilization, growth, and shortening, but the underlying mechanisms are still not fully understood. In vitro experiments have demonstrated that AAA (Adenosine triphosphatase Associated with various cellular Activities) ATPase family members spastin, katanin, and fidgetin can sever microtubules. Severing destabilizes the microtubule by exposing GDP-bound subunits, leading to complete disas-
sembly of the polymer over time. Yet, paradoxically, loss of spastin leads to a decrease in microtubule mass in Drosophila and zebrafish neurons (Sherwood et al., 2004; Wood et al., 2006). Furthermore, mutations in spastin are causative for the neurodegenerative disorder hereditary spastic paraplegia (HSP), whereas mutations in katanin cause microcephaly and seizures (Mishra-Gorur et al., 2014; Roll-Mecak and McNally, 2010; Yigit et al., 2016). New insights come from the work of Vemu et al. (2018) that can explain this puzzle of why microtubule mass decreases upon loss of a microtubule-severing enzyme. Roll-Mecak and her group examined the effects of spastin and katanin on microtubules using electron microscopy (EM), as well as total internal reflection (TIRF) microscopy to visualize severing events with singlemolecule resolution. Over prolonged incubation times, microtubules were severed and then disassembled rapidly as previously observed. However, Vemu et al. (2018) noted that shorter incubations of microtubules with spastin or katanin led to extensive nanoscale defects in the polymer—small potholes in the polymer lattice were evident using EM. Only at longer incubation times did these nanoscale defects progress to severing and then frank microtubule disassembly. Remarkably, inclusion of GTP-tubulin in these assays led to local repair of the nanoscale damage—the potholes in the lattice induced by spastin or katanin were repaired by the localized incorporation of new GTP-bound tubulin dimers. These repairs formed ‘‘GTP islands’’ distributed along the length of the polymer. This observation led to the next question: what effect would these GTP
402 Developmental Cell 47, November 19, 2018 ª 2018 Published by Elsevier Inc.
islands have on the overall dynamics of the polymer? Recent work has shown that rescue, the switch from a rapidly disassembling to a slowly growing microtubule, may not be random, but instead occurs preferentially at sites in the lattice enriched in GTP-tubulin (Aumeier et al., 2016; Dimitrov et al., 2008). As expected, the GTP islands resulting from localized repair of spastin- or katanin-mediated damage enhanced rescue by more than 10-fold. Further, Vemu and colleagues (2018) showed that frank severing of the microtubule can also lead to increased microtubule mass, as newly severed ends reinitiate rapid growth via incorporation of GTP-tubulin. Thus, when it comes to the effects of spastin or katanin on a microtubule, what doesn’t kill you makes you stronger. So how does this observation provide new insights into the cellular function of severing enzymes, and more specifically, the observation that mutations in spastin cause HSP? The answer is not yet clear but is beginning to come into focus. Spastin is enriched at synapses (Sherwood et al., 2004). Known HSP-associated mutations in spastin affect its ability to sever microtubules (Roll-Mecak and Vale, 2005) and lead to reduced microtubule density at synapses such as the neuromuscular junction. We now know that alterations in the ratio of GTP-bound to GDP-bound tubulin at microtubule plus ends affect both the stability of the microtubules and the ability of cargos to load or unload from the microtubule (Nirschl et al., 2017). Thus, mutations in spastin or katanin are likely to affect not just the microtubule itself but also the ability of the microtubule track to serve as an effective highway for the high-speed, long-distance trafficking of organelles. In turn,
Developmental Cell
Spotlight defects in this long-distance trafficking are sufficient to cause neurodegeneration (Nirschl et al., 2017). This new work from Vemu et al. (2018) uses both classical (EM) and more recent (TIRF microscopy) techniques to answer an important question in cell biology— what regulates microtubule plasticity— and an important question in neurodegeneration—how might mutations in spastin lead to HSP. The women behind this work, including the first author Annapurna Vemu and the senior author Antoninia Roll-Mecak, are distinguished by their ability to look at a problem with fresh eyes and to use rigorous quantitative biophysical approaches to provide a new and exciting answer. Of course, the work raises many further questions that will require the application of cutting-edge cell biology to the analysis of cytoskeletal and organelle dynamics in vivo. We look forward to these future discoveries.
REFERENCES
paraplegia protein, spastin, severs and disassembles microtubules. Curr. Biol. 15, 650–655.
Aumeier, C., Schaedel, L., Gaillard, J., John, K., Blanchoin, L., and Thery, M. (2016). Self-repair promotes microtubule rescue. Nat. Cell Biol. 18, 1054–1064. Dimitrov, A., Quesnoit, M., Moutel, S., Cantaloube, I., Pous, C., and Perez, F. (2008). Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues. Science 322, 1353–1356. layan, A.O., Schaffer, A.E., Mishra-Gorur, K., C¸ag €mu €s¸, Chabu, C., Henegariu, O., Vonhoff, F., Akgu G.T., Nishimura, S., Han, W., Tu, S., et al. (2014). Mutations in KATNB1 cause complex cerebral malformations by disrupting asymmetrically dividing neural progenitors. Neuron 84, 1226–1239. Nirschl, J.J., Ghiretti, A.E., and Holzbaur, E.L.F. (2017). The impact of cytoskeletal organization on the local regulation of neuronal transport. Nat. Rev. Neurosci. 18, 585–597. Roll-Mecak, A., and McNally, F.J. (2010). Microtubule-severing enzymes. Curr. Opin. Cell Biol. 22, 96–103. Roll-Mecak, A., and Vale, R.D. (2005). The Drosophila homologue of the hereditary spastic
Sherwood, N.T., Sun, Q., Xue, M., Zhang, B., and Zinn, K. (2004). Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS Biol. 2, e429. Vemu, A., Szczesna, E., Zehr, E.A., Spector, J.O., Grigorieff, N., Deaconescu, A.M., and Roll-Mecak, A. (2018). Severing enzymes amplify microtubule arrays through lattice GTP-tubulin incorporation. Science 361, https://doi.org/10.1126/science. aau1504. Wood, J.D., Landers, J.A., Bingley, M., McDermott, C.J., Thomas-McArthur, V., Gleadall, L.J., Shaw, P.J., and Cunliffe, V.T. (2006). The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo. Hum. Mol. Genet. 15, 2763–2771. Yigit, G., Wieczorek, D., Bo¨gershausen, N., Be€ller, J., leggia, F., Mo¨ller-Hartmann, C., Altmu €rnberg, P., and Wollnik, B. (2016). Thiele, H., Nu A syndrome of microcephaly, short stature, polysyndactyly, and dental anomalies caused by a homozygous KATNB1 mutation. Am. J. Med. Genet. A. 170, 728–733.
Developmental Cell 47, November 19, 2018 403