- Email: [email protected]
Ciliate Biology: Dynamin Goes Nuclear
Dispatch R923
11.
12.
13.
14.
required for cytokinesis in Drosophila spermatocytes. Curr. Biol. 15, 1401–1406. Logan, M.R., and Mandato, C.A. (2006). Regulation of the actin cytoskeleton by PIP2 in cytokinesis. Biol. Cell. 6, 377–388. Field, S.J., Madson, N., Kerr, M.L., Galbraith, K.A., Kennedy, C.E., Tahiliani, M., Wilkins, A., and Cantley, L.C. (2005). PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis. Curr. Biol. 15, 1407–1412. Janetopoulos, C., Borleis, J., Vazquez, F., Iijima, M., and Devreotes, P. (2005). Temporal and spatial regulation of phosphoinositide signaling mediates cytokinesis. Dev. Cell 4, 467–477. Kumar, A., and Carrera, A.C. (2007). New functions for PI3K in the control of cell division. Cell Cycle 14, 1696–1698.
15. Emoto, K., Inadome, H., Kanaho, Y., Narumiya, S., and Umeda, M. (2005). Local change in phospholipid composition at the cleavage furrow is essential for completion of cytokinesis. J. Biol. Chem. 45, 37901–37907. 16. Schneiter, R., Brugger, B., Amann, C.M., Prestwich, G.D., Epand, R.F., Zellnig, G., Wieland, F.T., and Epand, R.M. (2004). Identification and biophysical characterization of a very-long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes. Biochem. J. 3, 941–949. 17. Giansanti, M.G., Farkas, R.M., Bonaccorsi, S., Lindsley, D.L., Wakimoto, B.T., Fuller, M.T., and Gatti, M. (2004). Genetic dissection of meiotic cytokinesis in Drosophila males. Mol. Biol. Cell. 15, 2509–2522. 18. Janmey, P.A., and Kinnunen, P.K. (2006). Biophysical properties of lipids and dynamic membranes. Trends Cell Biol. 10, 538–546.
Ciliate Biology: Dynamin Goes Nuclear Dynamin and dynamin-related proteins (DRPs) mediate an array of membrane fission processes. A Tetrahymena DRP has adopted a new role, assisting in nuclear differentiation, a finding that further highlights these proteins — and this ciliate — as biological innovators. Douglas L. Chalker Gene duplication is a key path leading to protein neofunctionalization [1]. When genes are duplicated in an organism’s genome, one copy is free to diverge because the other can carry out the original cellular function. It appears that, throughout evolution, existing proteins have been recruited to adopt new roles. In many cases, repeated gene duplication, followed by functional divergence, has led to the generation of large gene families that undertake diverse tasks. The dynamin protein superfamily exemplifies such biological innovation. Dynamins and dynamin-related proteins (DRPs) are relatively large GTPases that are involved in a myriad of processes that require the alteration of membrane structure (see [2]). They play key roles in vesicle scission, organelle division, and cytokinesis. When associated with their target membranes, GTP-stimulated oligomerization and subsequent structural distortion upon GTP hydrolysis allow dynamins to facilitate endocytosis or other vesicular trafficking events. Each member of this superfamily is recruited to a particular membrane-bound compartment and, collectively, they exhibit extensive versatility and sub-specialization. Dynamins are known to work on many cellular membrane systems but,
until now, have not been shown to affect nuclear envelope structure. By studying the DRPs of the ciliated protozoan Tetrahymena thermophila (Figure 1A), Rahaman et al. [3], in a recent issue of Current Biology, found that the Drp6 protein localizes to the developing macronucleus and is required for macronucleus differentiation [3]. Again, the dynamin superfamily shows its aptitude for innovation. Drp6 acts specifically on the macronucleus, one of two functionally distinct nuclei in the unicellular Tetrahymena (Figure 1B). The macronucleus contains the somatic (expressed) genome, while the micronucleus harbors a silent germline copy. Drp6 acts when these nuclei differentiate from one another during conjugation, the sexual phase of the life cycle. Conjugation results in the loss of the parental macronucleus and the creation of a new micronucleus and macronucleus through the fusion of haploid nuclei produced from parental micronuclei that have undergone meiosis (see [4] for details). At the beginning of nuclear differentiation, the precursors of the new micronuclei and macronuclei are identical in size and genome content, but the new macronucleus rapidly enlarges, increasing its diameter 5–10-fold (see the relative size of the nuclei in Figure 1B) in preparation for genome
19. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572. 20. Giansanti, M.G., Bonaccorsi, S., Williams, B., Williams, E.V., Santolamazza, C., Goldberg, M.L., and Gatti, M. (1998). Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev. 12, 396–410.
Biology Department, Boston College, 140 Commonwealth Avenue, Chestnut Hill, Massachusetts 02467, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.08.035
rearrangement and polyploidization. Thus, macronucleus development clearly involves rapid membrane expansion. Rahaman et al. [3] showed that the loss of DRP6 slows or blocks nuclear-membrane expansion, which suggests that Drp6 is involved in this process. Intriguingly, fluorescence recovery after photobleaching (FRAP) experiments showed that the rate of Drp6 assembly/disassembly on the nuclear envelope is developmentally regulated. These observations suggest that Tetrahymena macronucleus development provides a unique and powerful context in which to examine the factors that affect DRP kinetics in vivo. This is just the second example of a dynamin superfamily member with nucleus-related functions. The human MxB protein localizes to the cytoplasmic face of the nuclear envelope and regulates nuclear import [5]. However, there is no evidence that MxB does this by altering nuclear membrane structure, and Rahaman et al. [3] showed that Drp6 does not participate in nuclear protein import [3]. Both MxB and Drp6 appear to have been independently recruited to regulate nuclear functions. DRP6 is one of four similar and adjacent genes (DRP3–6) that likely arose from a series of relatively recent gene duplication events. Intriguingly, DRP6 is the only paralog that encodes a protein that localizes to the nucleus. Thus, recruitment of this DRP to facilitate nuclear differentiation is an obvious biological innovation. Tetrahymena DRP1 is involved in the more conventional dynaminassociated role of clathrin-dependent endocytosis, and it is significantly
Current Biology Vol 18 No 19 R924
A
B
C OA
mac mic
Cp D Protein family Dynamin-related proteins Potassium channels Tubulin tyrosine ligases (tubulin polyglutamylation) Dyneins Karyopherins NIMA-related kinases
Number 8 285 50
Ref [3] [15] [17]
25 >22 39
[18] [19] [20] Current Biology
Figure 1. Tetrahymena are complex and innovative protozoans. The images highlight novel or elaborated characteristics of these organisms: (A) the regularly arrayed cilia, (B) nuclear dimorphism with the micronucleus (mic) and macronucleus (mac) circled, and (C) the membrane skeleton (visualized via a fusion protein comprising the yellow fluorescent protein and the calcium-binding protein TCBP25). The large openings in the cortex indicate the location of the oral apparatus (OA) and the cytoproct (Cp). (D) The table lists several examples of expanded gene families, including the number of related genes encoded in the Tetrahymena genome.
different in sequence from the DRP6 gene family [6]. Future studies of the Tetrahymena DRPs will enhance our understanding of the conserved functions of dynamins, reveal factors affecting their regulation, and perhaps enlarge the repertoire of processes in which members of this superfamily are known to act. This is just the latest example of biological innovation uncovered by researchers using ciliates as model systems. In addition to revealing functional innovations, these organisms have facilitated discoveries that have significantly advanced our understanding of basic scientific principles. Studies with Tetrahymena have produced such major contributions as the first telomere sequence and the discovery of the telomerase enzyme [7], purification of the first microtubule motor protein (dynein) [8], and the revelation that RNAs can be catalytic (self-splicing) [9]. Why has this somewhat unconventional group of organisms been such a fruitful vehicle for novel discoveries? Possibly, researchers have been attracted to ciliates due to their
animal-like qualities. It is easy to see that they are quite complex unicellular organisms. The regular arrays of cilia (Figure 1A) allow them to be highly motile, free-living creatures. They are polarized cells possessing an anterior ‘mouth’ (i.e., oral apparatus) and a posterior ‘anus’ (i.e., the cytoproct) (Figure 1C). To maintain this complex anatomy, ciliates have developed a laminar membrane skeleton, a highly organized, proteinaceous structure that underlies the plasma membrane (Figure 1C) [10]. While such features are innovations, fundamental biology is revealed when scientists examine such structural elaborations, which likely evolved by recruiting existing proteins for their construction or regulation. Genetic researchers have been drawn to the ciliates by their unique nuclear dimorphism. Simple comparison of the ciliates’ germline and somatic genomes has uncovered the novelty of developmentally programmed genome rearrangement. Further study of this unique process uncovered an elementary link between RNA interference and heterochromatin formation [11,12]. More recently, by
examining gene unscrambling in Oxytricha, researchers have found a role for RNA in templating DNA rearrangements that could indicate a more general role for RNA in epigenetic programming events [13]. Creation of such biological innovations may be fostered by the propensity for gene or even genome duplication within the ciliates. The sequencing of the Paramecium genome revealed that it has undergone relatively recent whole-genome duplications such that many genes have two to four paralogs [14]. There is less evidence for whole-genome duplication in Tetrahymena; nevertheless, extensive small-scale gene duplication has occurred as the genome contains w1,600 gene clusters of two or more tandem paralogs [15]. Also, there are ten protein families with greater than 100 members in the Tetrahymena genome. Figure 1D lists some examples of expanded gene families: any of their members may have taken on innovative roles. Whether or not ciliates are more innovative than other model organisms is difficult to measure. There is evidence that ciliates, especially those with highly fragmented somatic genomes, have relatively high rates of protein divergence [16]. But what is again apparent, this time from the studies of the Tetrahymena DRPs, is that ciliates provide a rich resource for uncovering innovative roles for proteins as well as providing unique biology that reveals the fundamental workings of cells. References 1. Prince, V.E., and Pickett, F.B. (2002). Splitting pairs: the diverging fates of duplicated genes. Nat. Rev. Genet. 3, 827–837. 2. Praefcke, G.J., and McMahon, H.T. (2004). The dynamin superfamily: universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell. Biol. 5, 133–147. 3. Rahaman, A., Elde, N.C., and Turkewitz, A.P. (2008). A dynamin-related protein required for nuclear remodeling in Tetrahymena. Curr. Biol. 18, 1227–1233. 4. Collins, K., and Gorovsky, M.A. (2005). Tetrahymena thermophila. Curr. Biol. 15, R317–R318. 5. King, M.C., Raposo, G., and Lemmon, M.A. (2004). Inhibition of nuclear import and cellcycle progression by mutated forms of the dynamin-like GTPase MxB. Proc. Natl. Acad. Sci. USA 101, 8957–8962. 6. Elde, N.C., Morgan, G., Winey, M., Sperling, L., and Turkewitz, A.P. (2005). Elucidation of clathrin-mediated endocytosis in Tetrahymena reveals an evolutionarily convergent recruitment of dynamin. PLoS Genet. 1, e52. 7. Greider, C.W., and Blackburn, E.H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405–413.
Dispatch R925
8. Gibbons, I.R., and Rowe, A.J. (1965). Dynein: A protein with adenosine triphosphatase activity from cilia. Science 149, 424–426. 9. Kruger, K., Grabowski, P.J., Zaug, A.J., Sands, J., Gottschling, D.E., and Cech, T.R. (1982). Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31, 147–157. 10. Honts, J.E., and Williams, N.E. (2003). Novel cytoskeletal proteins in the cortex of Tetrahymena. J. Eukaryot. Microbiol. 50, 9–14. 11. Mochizuki, K., Fine, N.A., Fujisawa, T., and Gorovsky, M.A. (2002). Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in Tetrahymena. Cell 110, 689–699. 12. Taverna, S.D., Coyne, R.S., and Allis, C.D. (2002). Methylation of histone h3 at lysine 9 targets programmed DNA elimination in Tetrahymena. Cell 110, 701–711. 13. Nowacki, M., Vijayan, V., Zhou, Y., Schotanus, K., Doak, T.G., and Landweber, L.F. (2008). RNA-mediated epigenetic programming of a genome-rearrangement pathway. Nature 451, 153–158.
14. Aury, J.M., Jaillon, O., Duret, L., Noel, B., Jubin, C., Porcel, B.M., Segurens, B., Daubin, V., Anthouard, V., Aiach, N., et al. (2006). Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444, 171–178. 15. Eisen, J.A., Coyne, R.S., Wu, M., Wu, D., Thiagarajan, M., Wortman, J.R., Badger, J.H., Ren, Q., Amedeo, P., Jones, K.M., et al. (2006). Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol. 4, e286. 16. Zufall, R.A., McGrath, C.L., Muse, S.V., and Katz, L.A. (2006). Genome architecture drives protein evolution in ciliates. Mol. Biol. Evol. 23, 1681–1687. 17. Janke, C., Rogowski, K., Wloga, D., Regnard, C., Kajava, A.V., Strub, J.M., Temurak, N., van Dijk, J., Boucher, D., van Dorsselaer, A., et al. (2005). Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308, 1758–1762. 18. Wilkes, D.E., Watson, H.E., Mitchell, D.R., and Asai, D.J. (2008). Twenty-five dyneins in
Cell Biology: Watching the First Steps of Podosome Formation Podosomes and invadopodia are actin-rich structures that have come under intense scrutiny over the past several years due to their critical roles in cell migration and invasion. Examination of the initial stages of podosome formation has revealed an important role for the phosphoinositide PI(3,4)P2 in anchoring the scaffold protein Tks5 to the plasma membrane. Marc Symons Podosomes are plasma membrane protrusions that play diverse roles in cell adhesion and migration. These specialized structures are found at the ventral side of a wide range of cells, including osteoclasts, macrophages and endothelial cells [1]. Invasive cancer cells display structures that are similar to podosomes, called invadopodia, that represent the major sites of matrix degradation in these cells [2,3]. The importance of podosomes and invadopodia in many physiological functions has made these structures of burgeoning interest to cell biologists active in fields as diverse as immunology and cancer research. The regulation of podosome structure and function is exceedingly complex. We now know an impressive array of molecular players that are essential for podosome formation [1,4]. A key mediator is the tyrosine kinase c-Src, which is both necessary and sufficient for podosome formation [1,4,5], and several other critical components of podosomes/
invadopodia are Src substrates. Central among these are Tks5, a scaffold protein that binds members of the ADAM family of membrane-spanning proteases [6,7], the Wiskott-Aldrich Syndrome proteins WASp and N-WASp, which stimulate Arp2/3-mediated actin nucleation [8], and cortactin, a protein that stabilizes Arp2/3-mediated actin filament branches [9]. Notably, Tks5, (N-)WASp and a host of other podosome-enriched proteins bind to and are controlled by phosphoinositides, which serve to anchor proteins to various membrane compartments, suggesting that phosphoinositides play an important role in podosome regulation. Although many critical components of podosomes have been identified, the sequence of molecular events that lead to podosome formation is still largely unknown [1]. A recent study by Oikawa et al. [10] provides a new paradigm for dissecting the initial stages of Src-mediated podosome formation and highlights the role of phosphoinositides in this process [10].
Tetrahymena: A re-examination of the multidynein hypothesis. Cell Motil. Cytoskeleton 65, 342–351. 19. Malone, C.D., Falkowska, K.A., Li, A.Y., Galanti, S.E., Kanuru, R.C., Lamont, E.G., Mazzarella, K.C., Micev, A.J., Osman, M.M., Piotrowski, N.K., et al. (2008). Nucleus-specific importin alphas and nucleoporins regulate protein import and nuclear division in the bi-nucleate Tetrahymena thermophila. Eukaryot. Cell 7, 1487–1499. 20. Wloga, D., Camba, A., Rogowski, K., Manning, G., Jerka-Dziadosz, M., and Gaertig, J. (2006). Members of the NIMA-related kinase family promote disassembly of cilia by multiple mechanisms. Mol. Biol. Cell 17, 2799–2810.
Biology Department, Washington University, St. Louis, Missouri 63130, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.07.080
The authors examined the subcellular localization of different species of phosphoinositides using fluorescent versions of specific phosphoinositide-binding pleckstrin homology (PH) domains [11]. They showed that phosphoinositide-3, 4-bisphosphate (PI(3,4)P2) is highly enriched in podosomes that are induced by constitutive activation of Src. PI(3,4,5)P3 is also found in podosomes, although it localizes to lamellipodia and intracellular vesicles as well. Importantly, overexpression of the PI(3,4)P2-binding PH domain of Tapp1 suppresses podosome formation, presumably by sequestering the lipid. In line with this observation, overexpression of the PH domain of Akt, which binds to both PI(3,4)P2 and PI(3,4,5)P3 has a more marked inhibitory effect on podosome formation. Moreover, both PI 3-kinase, the kinase that produces PI(3,4,5)P3 using PI(4,5)P2 as a substrate, and synaptojanin 2, a phosphatase that hydrolyzes PI(3,4,5)P3 to produce PI(3,4)P2, are essential for the formation of podosomes and invadopodia [10,12]. Together, these findings strongly indicate critical roles for both PI(3,4,5)P3 and PI(3,4)P2 in podosome formation. A candidate binding partner of PI(3,4)P2 is Tks5, which uses its PX domain to bind to this phosphoinositide [6]. Of note, PI(4,5)P2 was not detected in podosomes, suggesting that its conversion to PI(3,4,5)P3 is very efficient. To follow the first steps of Src-stimulated podosome formation,
11.
12.
13.
14.
required for cytokinesis in Drosophila spermatocytes. Curr. Biol. 15, 1401–1406. Logan, M.R., and Mandato, C.A. (2006). Regulation of the actin cytoskeleton by PIP2 in cytokinesis. Biol. Cell. 6, 377–388. Field, S.J., Madson, N., Kerr, M.L., Galbraith, K.A., Kennedy, C.E., Tahiliani, M., Wilkins, A., and Cantley, L.C. (2005). PtdIns(4,5)P2 functions at the cleavage furrow during cytokinesis. Curr. Biol. 15, 1407–1412. Janetopoulos, C., Borleis, J., Vazquez, F., Iijima, M., and Devreotes, P. (2005). Temporal and spatial regulation of phosphoinositide signaling mediates cytokinesis. Dev. Cell 4, 467–477. Kumar, A., and Carrera, A.C. (2007). New functions for PI3K in the control of cell division. Cell Cycle 14, 1696–1698.
15. Emoto, K., Inadome, H., Kanaho, Y., Narumiya, S., and Umeda, M. (2005). Local change in phospholipid composition at the cleavage furrow is essential for completion of cytokinesis. J. Biol. Chem. 45, 37901–37907. 16. Schneiter, R., Brugger, B., Amann, C.M., Prestwich, G.D., Epand, R.F., Zellnig, G., Wieland, F.T., and Epand, R.M. (2004). Identification and biophysical characterization of a very-long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes. Biochem. J. 3, 941–949. 17. Giansanti, M.G., Farkas, R.M., Bonaccorsi, S., Lindsley, D.L., Wakimoto, B.T., Fuller, M.T., and Gatti, M. (2004). Genetic dissection of meiotic cytokinesis in Drosophila males. Mol. Biol. Cell. 15, 2509–2522. 18. Janmey, P.A., and Kinnunen, P.K. (2006). Biophysical properties of lipids and dynamic membranes. Trends Cell Biol. 10, 538–546.
Ciliate Biology: Dynamin Goes Nuclear Dynamin and dynamin-related proteins (DRPs) mediate an array of membrane fission processes. A Tetrahymena DRP has adopted a new role, assisting in nuclear differentiation, a finding that further highlights these proteins — and this ciliate — as biological innovators. Douglas L. Chalker Gene duplication is a key path leading to protein neofunctionalization [1]. When genes are duplicated in an organism’s genome, one copy is free to diverge because the other can carry out the original cellular function. It appears that, throughout evolution, existing proteins have been recruited to adopt new roles. In many cases, repeated gene duplication, followed by functional divergence, has led to the generation of large gene families that undertake diverse tasks. The dynamin protein superfamily exemplifies such biological innovation. Dynamins and dynamin-related proteins (DRPs) are relatively large GTPases that are involved in a myriad of processes that require the alteration of membrane structure (see [2]). They play key roles in vesicle scission, organelle division, and cytokinesis. When associated with their target membranes, GTP-stimulated oligomerization and subsequent structural distortion upon GTP hydrolysis allow dynamins to facilitate endocytosis or other vesicular trafficking events. Each member of this superfamily is recruited to a particular membrane-bound compartment and, collectively, they exhibit extensive versatility and sub-specialization. Dynamins are known to work on many cellular membrane systems but,
until now, have not been shown to affect nuclear envelope structure. By studying the DRPs of the ciliated protozoan Tetrahymena thermophila (Figure 1A), Rahaman et al. [3], in a recent issue of Current Biology, found that the Drp6 protein localizes to the developing macronucleus and is required for macronucleus differentiation [3]. Again, the dynamin superfamily shows its aptitude for innovation. Drp6 acts specifically on the macronucleus, one of two functionally distinct nuclei in the unicellular Tetrahymena (Figure 1B). The macronucleus contains the somatic (expressed) genome, while the micronucleus harbors a silent germline copy. Drp6 acts when these nuclei differentiate from one another during conjugation, the sexual phase of the life cycle. Conjugation results in the loss of the parental macronucleus and the creation of a new micronucleus and macronucleus through the fusion of haploid nuclei produced from parental micronuclei that have undergone meiosis (see [4] for details). At the beginning of nuclear differentiation, the precursors of the new micronuclei and macronuclei are identical in size and genome content, but the new macronucleus rapidly enlarges, increasing its diameter 5–10-fold (see the relative size of the nuclei in Figure 1B) in preparation for genome
19. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572. 20. Giansanti, M.G., Bonaccorsi, S., Williams, B., Williams, E.V., Santolamazza, C., Goldberg, M.L., and Gatti, M. (1998). Cooperative interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev. 12, 396–410.
Biology Department, Boston College, 140 Commonwealth Avenue, Chestnut Hill, Massachusetts 02467, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.08.035
rearrangement and polyploidization. Thus, macronucleus development clearly involves rapid membrane expansion. Rahaman et al. [3] showed that the loss of DRP6 slows or blocks nuclear-membrane expansion, which suggests that Drp6 is involved in this process. Intriguingly, fluorescence recovery after photobleaching (FRAP) experiments showed that the rate of Drp6 assembly/disassembly on the nuclear envelope is developmentally regulated. These observations suggest that Tetrahymena macronucleus development provides a unique and powerful context in which to examine the factors that affect DRP kinetics in vivo. This is just the second example of a dynamin superfamily member with nucleus-related functions. The human MxB protein localizes to the cytoplasmic face of the nuclear envelope and regulates nuclear import [5]. However, there is no evidence that MxB does this by altering nuclear membrane structure, and Rahaman et al. [3] showed that Drp6 does not participate in nuclear protein import [3]. Both MxB and Drp6 appear to have been independently recruited to regulate nuclear functions. DRP6 is one of four similar and adjacent genes (DRP3–6) that likely arose from a series of relatively recent gene duplication events. Intriguingly, DRP6 is the only paralog that encodes a protein that localizes to the nucleus. Thus, recruitment of this DRP to facilitate nuclear differentiation is an obvious biological innovation. Tetrahymena DRP1 is involved in the more conventional dynaminassociated role of clathrin-dependent endocytosis, and it is significantly
Current Biology Vol 18 No 19 R924
A
B
C OA
mac mic
Cp D Protein family Dynamin-related proteins Potassium channels Tubulin tyrosine ligases (tubulin polyglutamylation) Dyneins Karyopherins NIMA-related kinases
Number 8 285 50
Ref [3] [15] [17]
25 >22 39
[18] [19] [20] Current Biology
Figure 1. Tetrahymena are complex and innovative protozoans. The images highlight novel or elaborated characteristics of these organisms: (A) the regularly arrayed cilia, (B) nuclear dimorphism with the micronucleus (mic) and macronucleus (mac) circled, and (C) the membrane skeleton (visualized via a fusion protein comprising the yellow fluorescent protein and the calcium-binding protein TCBP25). The large openings in the cortex indicate the location of the oral apparatus (OA) and the cytoproct (Cp). (D) The table lists several examples of expanded gene families, including the number of related genes encoded in the Tetrahymena genome.
different in sequence from the DRP6 gene family [6]. Future studies of the Tetrahymena DRPs will enhance our understanding of the conserved functions of dynamins, reveal factors affecting their regulation, and perhaps enlarge the repertoire of processes in which members of this superfamily are known to act. This is just the latest example of biological innovation uncovered by researchers using ciliates as model systems. In addition to revealing functional innovations, these organisms have facilitated discoveries that have significantly advanced our understanding of basic scientific principles. Studies with Tetrahymena have produced such major contributions as the first telomere sequence and the discovery of the telomerase enzyme [7], purification of the first microtubule motor protein (dynein) [8], and the revelation that RNAs can be catalytic (self-splicing) [9]. Why has this somewhat unconventional group of organisms been such a fruitful vehicle for novel discoveries? Possibly, researchers have been attracted to ciliates due to their
animal-like qualities. It is easy to see that they are quite complex unicellular organisms. The regular arrays of cilia (Figure 1A) allow them to be highly motile, free-living creatures. They are polarized cells possessing an anterior ‘mouth’ (i.e., oral apparatus) and a posterior ‘anus’ (i.e., the cytoproct) (Figure 1C). To maintain this complex anatomy, ciliates have developed a laminar membrane skeleton, a highly organized, proteinaceous structure that underlies the plasma membrane (Figure 1C) [10]. While such features are innovations, fundamental biology is revealed when scientists examine such structural elaborations, which likely evolved by recruiting existing proteins for their construction or regulation. Genetic researchers have been drawn to the ciliates by their unique nuclear dimorphism. Simple comparison of the ciliates’ germline and somatic genomes has uncovered the novelty of developmentally programmed genome rearrangement. Further study of this unique process uncovered an elementary link between RNA interference and heterochromatin formation [11,12]. More recently, by
examining gene unscrambling in Oxytricha, researchers have found a role for RNA in templating DNA rearrangements that could indicate a more general role for RNA in epigenetic programming events [13]. Creation of such biological innovations may be fostered by the propensity for gene or even genome duplication within the ciliates. The sequencing of the Paramecium genome revealed that it has undergone relatively recent whole-genome duplications such that many genes have two to four paralogs [14]. There is less evidence for whole-genome duplication in Tetrahymena; nevertheless, extensive small-scale gene duplication has occurred as the genome contains w1,600 gene clusters of two or more tandem paralogs [15]. Also, there are ten protein families with greater than 100 members in the Tetrahymena genome. Figure 1D lists some examples of expanded gene families: any of their members may have taken on innovative roles. Whether or not ciliates are more innovative than other model organisms is difficult to measure. There is evidence that ciliates, especially those with highly fragmented somatic genomes, have relatively high rates of protein divergence [16]. But what is again apparent, this time from the studies of the Tetrahymena DRPs, is that ciliates provide a rich resource for uncovering innovative roles for proteins as well as providing unique biology that reveals the fundamental workings of cells. References 1. Prince, V.E., and Pickett, F.B. (2002). Splitting pairs: the diverging fates of duplicated genes. Nat. Rev. Genet. 3, 827–837. 2. Praefcke, G.J., and McMahon, H.T. (2004). The dynamin superfamily: universal membrane tubulation and fission molecules? Nat. Rev. Mol. Cell. Biol. 5, 133–147. 3. Rahaman, A., Elde, N.C., and Turkewitz, A.P. (2008). A dynamin-related protein required for nuclear remodeling in Tetrahymena. Curr. Biol. 18, 1227–1233. 4. Collins, K., and Gorovsky, M.A. (2005). Tetrahymena thermophila. Curr. Biol. 15, R317–R318. 5. King, M.C., Raposo, G., and Lemmon, M.A. (2004). Inhibition of nuclear import and cellcycle progression by mutated forms of the dynamin-like GTPase MxB. Proc. Natl. Acad. Sci. USA 101, 8957–8962. 6. Elde, N.C., Morgan, G., Winey, M., Sperling, L., and Turkewitz, A.P. (2005). Elucidation of clathrin-mediated endocytosis in Tetrahymena reveals an evolutionarily convergent recruitment of dynamin. PLoS Genet. 1, e52. 7. Greider, C.W., and Blackburn, E.H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405–413.
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8. Gibbons, I.R., and Rowe, A.J. (1965). Dynein: A protein with adenosine triphosphatase activity from cilia. Science 149, 424–426. 9. Kruger, K., Grabowski, P.J., Zaug, A.J., Sands, J., Gottschling, D.E., and Cech, T.R. (1982). Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31, 147–157. 10. Honts, J.E., and Williams, N.E. (2003). Novel cytoskeletal proteins in the cortex of Tetrahymena. J. Eukaryot. Microbiol. 50, 9–14. 11. Mochizuki, K., Fine, N.A., Fujisawa, T., and Gorovsky, M.A. (2002). Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in Tetrahymena. Cell 110, 689–699. 12. Taverna, S.D., Coyne, R.S., and Allis, C.D. (2002). Methylation of histone h3 at lysine 9 targets programmed DNA elimination in Tetrahymena. Cell 110, 701–711. 13. Nowacki, M., Vijayan, V., Zhou, Y., Schotanus, K., Doak, T.G., and Landweber, L.F. (2008). RNA-mediated epigenetic programming of a genome-rearrangement pathway. Nature 451, 153–158.
14. Aury, J.M., Jaillon, O., Duret, L., Noel, B., Jubin, C., Porcel, B.M., Segurens, B., Daubin, V., Anthouard, V., Aiach, N., et al. (2006). Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444, 171–178. 15. Eisen, J.A., Coyne, R.S., Wu, M., Wu, D., Thiagarajan, M., Wortman, J.R., Badger, J.H., Ren, Q., Amedeo, P., Jones, K.M., et al. (2006). Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol. 4, e286. 16. Zufall, R.A., McGrath, C.L., Muse, S.V., and Katz, L.A. (2006). Genome architecture drives protein evolution in ciliates. Mol. Biol. Evol. 23, 1681–1687. 17. Janke, C., Rogowski, K., Wloga, D., Regnard, C., Kajava, A.V., Strub, J.M., Temurak, N., van Dijk, J., Boucher, D., van Dorsselaer, A., et al. (2005). Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308, 1758–1762. 18. Wilkes, D.E., Watson, H.E., Mitchell, D.R., and Asai, D.J. (2008). Twenty-five dyneins in
Cell Biology: Watching the First Steps of Podosome Formation Podosomes and invadopodia are actin-rich structures that have come under intense scrutiny over the past several years due to their critical roles in cell migration and invasion. Examination of the initial stages of podosome formation has revealed an important role for the phosphoinositide PI(3,4)P2 in anchoring the scaffold protein Tks5 to the plasma membrane. Marc Symons Podosomes are plasma membrane protrusions that play diverse roles in cell adhesion and migration. These specialized structures are found at the ventral side of a wide range of cells, including osteoclasts, macrophages and endothelial cells [1]. Invasive cancer cells display structures that are similar to podosomes, called invadopodia, that represent the major sites of matrix degradation in these cells [2,3]. The importance of podosomes and invadopodia in many physiological functions has made these structures of burgeoning interest to cell biologists active in fields as diverse as immunology and cancer research. The regulation of podosome structure and function is exceedingly complex. We now know an impressive array of molecular players that are essential for podosome formation [1,4]. A key mediator is the tyrosine kinase c-Src, which is both necessary and sufficient for podosome formation [1,4,5], and several other critical components of podosomes/
invadopodia are Src substrates. Central among these are Tks5, a scaffold protein that binds members of the ADAM family of membrane-spanning proteases [6,7], the Wiskott-Aldrich Syndrome proteins WASp and N-WASp, which stimulate Arp2/3-mediated actin nucleation [8], and cortactin, a protein that stabilizes Arp2/3-mediated actin filament branches [9]. Notably, Tks5, (N-)WASp and a host of other podosome-enriched proteins bind to and are controlled by phosphoinositides, which serve to anchor proteins to various membrane compartments, suggesting that phosphoinositides play an important role in podosome regulation. Although many critical components of podosomes have been identified, the sequence of molecular events that lead to podosome formation is still largely unknown [1]. A recent study by Oikawa et al. [10] provides a new paradigm for dissecting the initial stages of Src-mediated podosome formation and highlights the role of phosphoinositides in this process [10].
Tetrahymena: A re-examination of the multidynein hypothesis. Cell Motil. Cytoskeleton 65, 342–351. 19. Malone, C.D., Falkowska, K.A., Li, A.Y., Galanti, S.E., Kanuru, R.C., Lamont, E.G., Mazzarella, K.C., Micev, A.J., Osman, M.M., Piotrowski, N.K., et al. (2008). Nucleus-specific importin alphas and nucleoporins regulate protein import and nuclear division in the bi-nucleate Tetrahymena thermophila. Eukaryot. Cell 7, 1487–1499. 20. Wloga, D., Camba, A., Rogowski, K., Manning, G., Jerka-Dziadosz, M., and Gaertig, J. (2006). Members of the NIMA-related kinase family promote disassembly of cilia by multiple mechanisms. Mol. Biol. Cell 17, 2799–2810.
Biology Department, Washington University, St. Louis, Missouri 63130, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.07.080
The authors examined the subcellular localization of different species of phosphoinositides using fluorescent versions of specific phosphoinositide-binding pleckstrin homology (PH) domains [11]. They showed that phosphoinositide-3, 4-bisphosphate (PI(3,4)P2) is highly enriched in podosomes that are induced by constitutive activation of Src. PI(3,4,5)P3 is also found in podosomes, although it localizes to lamellipodia and intracellular vesicles as well. Importantly, overexpression of the PI(3,4)P2-binding PH domain of Tapp1 suppresses podosome formation, presumably by sequestering the lipid. In line with this observation, overexpression of the PH domain of Akt, which binds to both PI(3,4)P2 and PI(3,4,5)P3 has a more marked inhibitory effect on podosome formation. Moreover, both PI 3-kinase, the kinase that produces PI(3,4,5)P3 using PI(4,5)P2 as a substrate, and synaptojanin 2, a phosphatase that hydrolyzes PI(3,4,5)P3 to produce PI(3,4)P2, are essential for the formation of podosomes and invadopodia [10,12]. Together, these findings strongly indicate critical roles for both PI(3,4,5)P3 and PI(3,4)P2 in podosome formation. A candidate binding partner of PI(3,4)P2 is Tks5, which uses its PX domain to bind to this phosphoinositide [6]. Of note, PI(4,5)P2 was not detected in podosomes, suggesting that its conversion to PI(3,4,5)P3 is very efficient. To follow the first steps of Src-stimulated podosome formation,