- Email: [email protected]
Cytokinesis: Thinking Outside the Cell
Dispatch R119
There might be differences in how apes (including humans) and monkeys form concepts [18], and other animals, including bees, could use yet other solutions towards similar behavioural problems. References 1. Machery, E. (2010). Precis of doing without concepts. Behav. Brain Sci. 33, 195–206. 2. Zentall, T.R., Wasserman, E.A., Lazareva, O.F., Thompson, R.K.R., and Ratterman, M.J. (2008). Concept learning in animals. Comp. Cogn. Behav. Rev. 3, 13–45. 3. Herrnstein, R.J., and Loveland, D.H. (1964). Complex visual concept in the pigeon. Science 146, 549–550. 4. Shettleworth, S.J. (2010). Cognition, Evolution and Behavior (Oxford: Oxford University Press). 5. Penn, D.C., Holyoak, K.J., and Povinelli, D.J. (2008). Darwin’s mistake: explaining the discontinuity between human and nonhuman minds. Behav. Brain Sci. 31, 109–178. 6. Chater, N., and Heyes, C. (1994). Animal concepts: content and discontent. Mind Language 9, 209–246.
7. Tomasello, M., and Call, J. (1997). Primate Cognition (Oxford: Oxford University Press). 8. Savage-Rumbaugh, E.S., Rumbaugh, D.M., Smith, S.T., and Lawson, J. (1980). Reference — the linguistic essential. Science 210, 922–925. 9. Pearce, J.M. (2008). Animal Learning and Cognition (Hove & New York: Psychology Press). 10. Avargue`s-Weber, A., Deisig, N., and Giurfa, M. (2011). Visual cognition in social insects. Annu. Rev. Entomol. 56, 423–443. 11. Avargue`s-Weber, A., Dyer, A.G., and Giurfa, M. (2011). Conceptualization of above and below relationships by an insect. Proc. R. Soc. Lond. B. 10.1098/rspb.2010.1891. 12. Chittka, L., and Raine, N.E. (2006). Recognition of flowers by pollinators. Curr. Opin. Plant Biol. 9, 428–435. 13. Raine, N.E., and Chittka, L. (2007). Flower constancy and memory dynamics in Bumblebees (Hymenoptera: Apidae: Bombus). Entomologia Generalis 29, 179–199. 14. Depy, D., Fagot, J., and Vauclair, J. (1999). Processing of above/below categorical spatial relations by baboons (Papio papio). Behav. Process 48, 1–9. 15. Spinozzi, G., Lubrano, G., and Truppa, V. (2004). Categorization of above and below spatial
Cytokinesis: Thinking Outside the Cell How might the extracellular matrix contribute to cytokinesis? In a recent report, evidence is presented that the conserved extracellular matrix protein hemicentinHIM-4 is required for cytokinesis in worms and mice. Shawn N. Jordan1, Sara Olson2, and Julie C. Canman1,* The extracellular matrix (ECM) functions akin to a cellular exoskeleton, providing structural support and sites of attachment for the cells it surrounds. However, the ECM is much more than just a scaffold. With established roles in cell migration, tissue separation, and cell signal transduction [1], the ECM is clearly a dynamic player in many cellular functions. Cytokinesis is the physical division of one cell into two daughter cells that occurs at the end of the cell cycle. Cytokinesis is accomplished by constriction of a contractile ring composed of filamentous actin and the motor myosin-II (together actomyosin) [2]. In order to divide, a cell must recruit and coordinate a host of regulatory and structural proteins to the division plane, but existing models of cytokinesis do not consider a contribution from secreted extracellular proteins. The secreted ECM protein hemicentin (HMCN1 or fibulin-6 in Homo sapiens, HIM-4 in Caenorhabditis elegans) is of particular interest as it may have an evolutionarily
conserved role in cytokinesis. Hemicentin is a relative of a multigene family of proteins called fibulins, secreted proteins that assimilate into the ECM and form higher order structures, such as elastic fibers [3]. The hemicentin protein contains a single, highly conserved von Willebrand A domain, a long stretch of immunoglobulin repeats, epidermal growth factor domains, and a fibulin-like carboxy-terminal module [4]. In the roundworm C. elegans, hemicentinHIM-4 has been found in cell-matrix adhesion sites known as hemidesmosomes and at multiple connecting junctions throughout the body [5]. Cells surrounding the hermaphroditic worm gonad also secrete hemicentinHIM-4, and functional disruption of the him-4 locus in C. elegans leads to a high incidence of males due to defects in segregation of the X chromosome, resulting in XO male progeny [6]. The him-4 mutants also display pleiotropic defects in motility, adhesion, behavior, and gonad morphology [5]. In a paper published in a recent issue of Current Biology, Xu and Vogel [7] took a closer look at the gonad
relations by tufted capuchin monkeys (Cebus apella). J. Comp. Psychol. 118, 403–412. 16. Quinn, P.C., Polly, J.L., Furer, M.J., Dobson, V., and Narter, D.B. (2002). Young infants’ performance in the object-variation version of the above-below categorization task: a result of perceptual distraction or conceptual limitation? Infancy 3, 323–347. 17. Quinn, P.C., Cummins, M., Kase, J., Martin, E., and Weissman, S. (1996). Development of categorical representations for above and below spatial relations in 3- to 7-month-old infants. Dev. Psychol. 32, 942–950. 18. Thompson, R.K.R., and Oden, D.L. (2000). Categorical perception and conceptual judgments by nonhuman primates: The paleological monkey and the analogical ape. Cogn. Sci. 24, 363–396.
Queen Mary University of London, Research Centre for Psychology, School of Biological and Chemical Sciences, Mile End Road, London E1 4NS, UK. E-mail: [email protected] DOI: 10.1016/j.cub.2010.12.045
morphology defects in him-4 mutants. In the C. elegans gonad, germ cells form as part of a syncytium, with incomplete cytokinetic furrows partitioning single nuclei that share a common cytoplasm [8]. These nuclei are eventually segregated completely as the cell matures into an oocyte and physically separates from the syncytium [8]. HemicentinHIM-4 labeled with green fluorescent protein localized in a ring structure at the base of these incomplete furrows in the gonad. In the absence of hemicentinHIM-4, the gonad in aged worms became disorganized due to the formation of multinucleated germ cells. Temporal analysis revealed that while nascent membrane partitions appeared to have a normal structure, they soon became destabilized and eventually collapsed. The recruitment of hemicentinHIM-4 to the tips of the membrane partitions in the C. elegans gonad was dependent on the highly conserved Rho family guanine nucleotide exchange factor (GEF) ECT-2. During cytokinesis, ECT-2 activates Rho and initiates assembly of the actomyosin ring at the constriction site [2]. In an ECT-2-deficient background, the majority of hemicentinHIM-4 remained in the pseudo-coelomic fluid outside of the gonad. Taken together, these results suggest a role for hemicentinHIM-4 in maintaining the cytokinetic membrane partitions in older worms, and that its
Current Biology Vol 21 No 3 R120
A
B
Mouse embryo
Caenorhabditis elegans gonad
Cytokinetic furrow tips
C
Hemicentin Integrins ?
?
Integrin-associated proteins and kinases
?
Contractile ring Nucleus Ect2
Actomyosin contractility
Ect2
ECM anchorage
Rho family GEF Ect2
Cytokinesis Current Biology
Figure 1. Model for how the ECM protein hemicentinHIM-4 might be contributing to cytokinesis. (A) Section of a syncytial C. elegans gonad and (B) murine embryo with a (C) zoomed view depicting hemicentinHIM-4 localization to the extracellular tips of the cytokinetic furrows and potential linkage to integrins.
recruitment to and/or maintenance at the site of division requires the activity of the intracellular cytokinetic machinery (Figure 1). Notably, only aged worm gonads were affected by hemicentinHIM-4 disruption, and no cytokinesis failures were observed in dividing embryos prior to hatching. Perhaps the weakening of the core cytokinetic machinery over time forces cells to rely more heavily on redundant stabilizing mechanisms, such as those provided by the ECM. This would explain why functional disruption of hemicentinHIM-4 does not perturb cytokinesis until later in life. Association studies in humans have linked mutations in hemicentin to some patients with age-related macular
degeneration (AMD), the most common blinding disorder in the Western world [9]. AMD is frequently associated with the presence of multiple, multinucleated giant cells in the retina [10], which may be indicative of cytokinesis defects resulting from ECM disruption in human cells. Xu and Vogel [7] also showed the role of hemicentin in cytokinesis is conserved. Immuno-fluorescent staining of fixed mitotic one-cell mouse embryos revealed that the two highly conserved hemicentin orthologs, hemicentin-1 and hemicentin-2, both concentrate at the presumptive cleavage furrow and surround the actomyosin contractile ring (Figure 1). Further, double-stranded RNA (dsRNA)-mediated knockdown or
targeted inactivation of the hemicentin-1 gene produced cytokinetic defects in these murine embryos. Knockdown of hemicentin-1 resulted in most embryos arresting before the four-cell stage with multinucleated blastomeres. Targeted inactivation also resulted in multinucleated cells that often had internal polar bodies. Although a role for secreted proteins in cytokinesis has not been previously appreciated, a number of reports have suggested that secreted extracellular proteins may participate in cytokinesis across phylogeny. For example, in budding yeast, septum formation is dependent on synthesis of chitin, a secreted cell-wall component that ultimately separates the mother and daughter cells [11,12]. In C. elegans, simultaneous depletion of the secreted proteoglycans CPG-1 and CPG-2 results in cytokinesis failure and produces multinucleated single-cell embryos [13]. The cytokinesis defects in both yeast chitin synthase mutants and C. elegans proteoglycan mutants is in part indirect due to osmotic sensitivity as cytokinesis can be partially to fully rescued with osmotic support ([12] and Sara Olson, unpublished results). Nevertheless, the cytokinetic defects seen in C. elegans him-4 mutants are not specifically affected by osmotic changes [7], suggesting that hemicentinHIM-4 may be important both structurally and functionally during cell division. How then does the ECM contribute to cytokinesis? Mediation between the ECM and the intercellular space during cytokinesis may be accomplished via integrins, transmembrane receptors important for cell-matrix adhesion and cellular signaling. Integrins are essential to maintain gonad morphology in C. elegans, and disruption of the integrin-linked kinase ILK leads to both mitotic and cytokinetic defects in mammalian cultured cells [14,15]. Further, disruption of integrin function or integrin trafficking in mammalian cells results in cytokinesis failure and formation of multinucleated cells [15,16]. One possibility is that integrins crosslink the ECM to the intracellular cytoskeleton as occurs during interphase, anchoring the cytokinetic contractile machinery to the outside of the cell (Figure 1). This would lend stability and facilitate the maintenance of intact membrane partitions. Undoubtedly, a more complete
Dispatch R121
understanding of cytokinesis will require that we think ‘outside of the cell’ and incorporate a role for the ECM in cell division.
7.
8.
References 1. Hynes, R.O. (2009). The extracellular matrix: not just pretty fibrils. Science 326, 1216–1219. 2. Pollard, T.D. (2010). Mechanics of cytokinesis in eukaryotes. Curr. Opin. Cell Biol. 22, 50–56. 3. Timpl, R., Sasaki, T., Kostka, G., and Chu, M.L. (2003). Fibulins: a versatile family of extracellular matrix proteins. Nat. Rev. Mol. Cell Biol. 4, 479–489. 4. Dong, C., Muriel, J.M., Ramirez, S., Hutter, H., Hedgecock, E.M., Breydo, L., Baskakov, I.V., and Vogel, B.E. (2006). Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules. J. Biol. Chem. 281, 23606–23610. 5. Vogel, B.E., and Hedgecock, E.M. (2001). Hemicentin, a conserved extracellular member of the immunoglobulin superfamily, organizes epithelial and other cell attachments into oriented line-shaped junctions. Development 128, 883–894. 6. Hodgkin, J., Horvitz, H.R., and Brenner, S. (1979). Nondisjunction mutants of the
9.
10.
11.
12.
13.
nematode Caenorhabditis elegans. Genetics 91, 67–94. Xu, X., and Vogel, B.E. (2011). A secreted protein promotes cleavage furrow maturation during cytokinesis. Curr. Biol. 21, 114–119. Hubbard, E.J., and Greenstein, D. (2000). The Caenorhabditis elegans gonad: a test tube for cell and developmental biology. Dev. Dyn. 218, 2–22. Schultz, D.W., Weleber, R.G., Lawrence, G., Barral, S., Majewski, J., Acott, T.S., and Klein, M.L. (2005). HEMICENTIN-1 (FIBULIN-6) and the 1q31 AMD locus in the context of complex disease: review and perspective. Ophthalmic Genet. 26, 101–105. Dastgheib, K., and Green, W.R. (1994). Granulomatous reaction to Bruch’s membrane in age-related macular degeneration. Arch. Ophthalmol. 112, 813–818. Shaw, J.A., Mol, P.C., Bowers, B., Silverman, S.J., Valdivieso, M.H., Duran, A., and Cabib, E. (1991). The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J. Cell Biol. 114, 111–123. Schmidt, M. (2004). Survival and cytokinesis of Saccharomyces cerevisiae in the absence of chitin. Microbiology 150, 3253–3260. Olson, S.K., Bishop, J.R., Yates, J.R., Oegema, K., and Esko, J.D. (2006). Identification of novel chondroitin
Cell–Cell Fusion: A New Function for Invadosomes Podosomes are cytoskeletal-based structures involved in extracellular matrix remodeling and cellular motility. A new study now implicates podosomes in pore formation during myoblast fusion. Bong Hwan Sung and Alissa Weaver* Cell–cell fusion is a highly regulated event that is critical for many physiological and pathological events, including fertilization, muscle development, and immune response. During skeletal muscle development, fusion of muscle cells generates multinucleate and functional muscle fibers and aberrant fusion has been implicated in dystrophic muscle diseases [1,2]. Recently, a number of groups have studied myoblast fusion during body wall muscle formation in Drosophila melanogaster as a genetically tractable in vivo model system to study cell–cell fusion [1]. In a new study published in the Journal of Cell Biology, Sens et al. [3] investigated the role of actin assembly in formation of the fusion pore during Drosophila myoblast fusion. Interestingly, they find that an invasive, actin-rich, podosome-like structure is used by fusion-competent myoblasts (FCMs) to adhere to and fuse with muscle founder cells.
Previously, it was known that actin filaments accumulate transiently at the site of myoblast fusion [4–6], dependent on signaling from heterotypic adhesion molecules and downstream regulators of branched actin assembly, including Rac, SCAR and WASP [7]. Furthermore, both the SCAR and WASP complex activators of the branched-actin-nucleating Arp2/3 complex were known to be essential for myoblast fusion [5,6,8–10]. However, the nature of the fusion structure and the roles of individual actin regulators were poorly understood. To determine whether the prominent actin accumulations at pre-fusion sites were unique to a muscle cell subtype, Sens et al. [3] expressed GFP–actin under the control of FCM-specific or founder-cell-specific promoters and costained for all actin filaments in embryos with fluorescent phalloidin. Interestingly, the large actin foci were exclusively found in FCM cells and were associated with a deformation in the founder cell membrane.
proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2. J. Cell Biol. 173, 985–994. 14. Xu, X., Rongali, S.C., Miles, J.P., Lee, K.D., and Lee, M. (2006). pat-4/ILK and unc-112/Mig-2 are required for gonad function in Caenorhabditis elegans. Exp. Cell Res. 312, 1475–1483. 15. Reverte, C.G., Benware, A., Jones, C.W., and LaFlamme, S.E. (2006). Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis. J. Cell Biol. 174, 491–497. 16. Pellinen, T., Tuomi, S., Arjonen, A., Wolf, M., Edgren, H., Meyer, H., Grosse, R., Kitzing, T., Rantala, J.K., Kallioniemi, O., et al. (2008). Integrin trafficking regulated by Rab21 is necessary for cytokinesis. Dev. Cell 15, 371–385. 1Columbia University, Department of Pathology and Cell Biology, New York, NY 10032, USA. 2University of California at San Diego, Cellular and Molecular Medicine, La Jolla, CA 92093, USA. *E-mail: [email protected]
DOI: 10.1016/j.cub.2010.12.040
Transmission electron microscopy studies showed finger-like FCM cell protrusions apparently invading into the founder cells at the site of cell–cell fusion. Invasive, actin-rich, finger-like protrusions have been well characterized in cells that invade or remodel tissue and are termed invadopodia in cancer cells and podosomes in normal cells (or collectively, invadosomes) [11,12]. However, a role in cell–cell fusion has not been previously described, and their main function is thought to be degradation of extracellular matrix (ECM), in part due to active trafficking of ECM-degrading proteinases to sites of protrusion formation (Figure 1). The myoblast structures observed by Sens et al. [3] seemed to be a potential variation of podosomes, as they were morphologically similar by electron microscopy and even had adhesion ring structures, albeit cell–cell rather than cell–ECM adhesions. If these structures really were podosomes, the new data revealed that podosomes might be more versatile than previously appreciated and also that they are formed in vivo during developmental ‘invasions’. To determine whether the FCM actin-rich protrusions resembled podosomes at the molecular level, Sens et al. [3] manipulated the
There might be differences in how apes (including humans) and monkeys form concepts [18], and other animals, including bees, could use yet other solutions towards similar behavioural problems. References 1. Machery, E. (2010). Precis of doing without concepts. Behav. Brain Sci. 33, 195–206. 2. Zentall, T.R., Wasserman, E.A., Lazareva, O.F., Thompson, R.K.R., and Ratterman, M.J. (2008). Concept learning in animals. Comp. Cogn. Behav. Rev. 3, 13–45. 3. Herrnstein, R.J., and Loveland, D.H. (1964). Complex visual concept in the pigeon. Science 146, 549–550. 4. Shettleworth, S.J. (2010). Cognition, Evolution and Behavior (Oxford: Oxford University Press). 5. Penn, D.C., Holyoak, K.J., and Povinelli, D.J. (2008). Darwin’s mistake: explaining the discontinuity between human and nonhuman minds. Behav. Brain Sci. 31, 109–178. 6. Chater, N., and Heyes, C. (1994). Animal concepts: content and discontent. Mind Language 9, 209–246.
7. Tomasello, M., and Call, J. (1997). Primate Cognition (Oxford: Oxford University Press). 8. Savage-Rumbaugh, E.S., Rumbaugh, D.M., Smith, S.T., and Lawson, J. (1980). Reference — the linguistic essential. Science 210, 922–925. 9. Pearce, J.M. (2008). Animal Learning and Cognition (Hove & New York: Psychology Press). 10. Avargue`s-Weber, A., Deisig, N., and Giurfa, M. (2011). Visual cognition in social insects. Annu. Rev. Entomol. 56, 423–443. 11. Avargue`s-Weber, A., Dyer, A.G., and Giurfa, M. (2011). Conceptualization of above and below relationships by an insect. Proc. R. Soc. Lond. B. 10.1098/rspb.2010.1891. 12. Chittka, L., and Raine, N.E. (2006). Recognition of flowers by pollinators. Curr. Opin. Plant Biol. 9, 428–435. 13. Raine, N.E., and Chittka, L. (2007). Flower constancy and memory dynamics in Bumblebees (Hymenoptera: Apidae: Bombus). Entomologia Generalis 29, 179–199. 14. Depy, D., Fagot, J., and Vauclair, J. (1999). Processing of above/below categorical spatial relations by baboons (Papio papio). Behav. Process 48, 1–9. 15. Spinozzi, G., Lubrano, G., and Truppa, V. (2004). Categorization of above and below spatial
Cytokinesis: Thinking Outside the Cell How might the extracellular matrix contribute to cytokinesis? In a recent report, evidence is presented that the conserved extracellular matrix protein hemicentinHIM-4 is required for cytokinesis in worms and mice. Shawn N. Jordan1, Sara Olson2, and Julie C. Canman1,* The extracellular matrix (ECM) functions akin to a cellular exoskeleton, providing structural support and sites of attachment for the cells it surrounds. However, the ECM is much more than just a scaffold. With established roles in cell migration, tissue separation, and cell signal transduction [1], the ECM is clearly a dynamic player in many cellular functions. Cytokinesis is the physical division of one cell into two daughter cells that occurs at the end of the cell cycle. Cytokinesis is accomplished by constriction of a contractile ring composed of filamentous actin and the motor myosin-II (together actomyosin) [2]. In order to divide, a cell must recruit and coordinate a host of regulatory and structural proteins to the division plane, but existing models of cytokinesis do not consider a contribution from secreted extracellular proteins. The secreted ECM protein hemicentin (HMCN1 or fibulin-6 in Homo sapiens, HIM-4 in Caenorhabditis elegans) is of particular interest as it may have an evolutionarily
conserved role in cytokinesis. Hemicentin is a relative of a multigene family of proteins called fibulins, secreted proteins that assimilate into the ECM and form higher order structures, such as elastic fibers [3]. The hemicentin protein contains a single, highly conserved von Willebrand A domain, a long stretch of immunoglobulin repeats, epidermal growth factor domains, and a fibulin-like carboxy-terminal module [4]. In the roundworm C. elegans, hemicentinHIM-4 has been found in cell-matrix adhesion sites known as hemidesmosomes and at multiple connecting junctions throughout the body [5]. Cells surrounding the hermaphroditic worm gonad also secrete hemicentinHIM-4, and functional disruption of the him-4 locus in C. elegans leads to a high incidence of males due to defects in segregation of the X chromosome, resulting in XO male progeny [6]. The him-4 mutants also display pleiotropic defects in motility, adhesion, behavior, and gonad morphology [5]. In a paper published in a recent issue of Current Biology, Xu and Vogel [7] took a closer look at the gonad
relations by tufted capuchin monkeys (Cebus apella). J. Comp. Psychol. 118, 403–412. 16. Quinn, P.C., Polly, J.L., Furer, M.J., Dobson, V., and Narter, D.B. (2002). Young infants’ performance in the object-variation version of the above-below categorization task: a result of perceptual distraction or conceptual limitation? Infancy 3, 323–347. 17. Quinn, P.C., Cummins, M., Kase, J., Martin, E., and Weissman, S. (1996). Development of categorical representations for above and below spatial relations in 3- to 7-month-old infants. Dev. Psychol. 32, 942–950. 18. Thompson, R.K.R., and Oden, D.L. (2000). Categorical perception and conceptual judgments by nonhuman primates: The paleological monkey and the analogical ape. Cogn. Sci. 24, 363–396.
Queen Mary University of London, Research Centre for Psychology, School of Biological and Chemical Sciences, Mile End Road, London E1 4NS, UK. E-mail: [email protected] DOI: 10.1016/j.cub.2010.12.045
morphology defects in him-4 mutants. In the C. elegans gonad, germ cells form as part of a syncytium, with incomplete cytokinetic furrows partitioning single nuclei that share a common cytoplasm [8]. These nuclei are eventually segregated completely as the cell matures into an oocyte and physically separates from the syncytium [8]. HemicentinHIM-4 labeled with green fluorescent protein localized in a ring structure at the base of these incomplete furrows in the gonad. In the absence of hemicentinHIM-4, the gonad in aged worms became disorganized due to the formation of multinucleated germ cells. Temporal analysis revealed that while nascent membrane partitions appeared to have a normal structure, they soon became destabilized and eventually collapsed. The recruitment of hemicentinHIM-4 to the tips of the membrane partitions in the C. elegans gonad was dependent on the highly conserved Rho family guanine nucleotide exchange factor (GEF) ECT-2. During cytokinesis, ECT-2 activates Rho and initiates assembly of the actomyosin ring at the constriction site [2]. In an ECT-2-deficient background, the majority of hemicentinHIM-4 remained in the pseudo-coelomic fluid outside of the gonad. Taken together, these results suggest a role for hemicentinHIM-4 in maintaining the cytokinetic membrane partitions in older worms, and that its
Current Biology Vol 21 No 3 R120
A
B
Mouse embryo
Caenorhabditis elegans gonad
Cytokinetic furrow tips
C
Hemicentin Integrins ?
?
Integrin-associated proteins and kinases
?
Contractile ring Nucleus Ect2
Actomyosin contractility
Ect2
ECM anchorage
Rho family GEF Ect2
Cytokinesis Current Biology
Figure 1. Model for how the ECM protein hemicentinHIM-4 might be contributing to cytokinesis. (A) Section of a syncytial C. elegans gonad and (B) murine embryo with a (C) zoomed view depicting hemicentinHIM-4 localization to the extracellular tips of the cytokinetic furrows and potential linkage to integrins.
recruitment to and/or maintenance at the site of division requires the activity of the intracellular cytokinetic machinery (Figure 1). Notably, only aged worm gonads were affected by hemicentinHIM-4 disruption, and no cytokinesis failures were observed in dividing embryos prior to hatching. Perhaps the weakening of the core cytokinetic machinery over time forces cells to rely more heavily on redundant stabilizing mechanisms, such as those provided by the ECM. This would explain why functional disruption of hemicentinHIM-4 does not perturb cytokinesis until later in life. Association studies in humans have linked mutations in hemicentin to some patients with age-related macular
degeneration (AMD), the most common blinding disorder in the Western world [9]. AMD is frequently associated with the presence of multiple, multinucleated giant cells in the retina [10], which may be indicative of cytokinesis defects resulting from ECM disruption in human cells. Xu and Vogel [7] also showed the role of hemicentin in cytokinesis is conserved. Immuno-fluorescent staining of fixed mitotic one-cell mouse embryos revealed that the two highly conserved hemicentin orthologs, hemicentin-1 and hemicentin-2, both concentrate at the presumptive cleavage furrow and surround the actomyosin contractile ring (Figure 1). Further, double-stranded RNA (dsRNA)-mediated knockdown or
targeted inactivation of the hemicentin-1 gene produced cytokinetic defects in these murine embryos. Knockdown of hemicentin-1 resulted in most embryos arresting before the four-cell stage with multinucleated blastomeres. Targeted inactivation also resulted in multinucleated cells that often had internal polar bodies. Although a role for secreted proteins in cytokinesis has not been previously appreciated, a number of reports have suggested that secreted extracellular proteins may participate in cytokinesis across phylogeny. For example, in budding yeast, septum formation is dependent on synthesis of chitin, a secreted cell-wall component that ultimately separates the mother and daughter cells [11,12]. In C. elegans, simultaneous depletion of the secreted proteoglycans CPG-1 and CPG-2 results in cytokinesis failure and produces multinucleated single-cell embryos [13]. The cytokinesis defects in both yeast chitin synthase mutants and C. elegans proteoglycan mutants is in part indirect due to osmotic sensitivity as cytokinesis can be partially to fully rescued with osmotic support ([12] and Sara Olson, unpublished results). Nevertheless, the cytokinetic defects seen in C. elegans him-4 mutants are not specifically affected by osmotic changes [7], suggesting that hemicentinHIM-4 may be important both structurally and functionally during cell division. How then does the ECM contribute to cytokinesis? Mediation between the ECM and the intercellular space during cytokinesis may be accomplished via integrins, transmembrane receptors important for cell-matrix adhesion and cellular signaling. Integrins are essential to maintain gonad morphology in C. elegans, and disruption of the integrin-linked kinase ILK leads to both mitotic and cytokinetic defects in mammalian cultured cells [14,15]. Further, disruption of integrin function or integrin trafficking in mammalian cells results in cytokinesis failure and formation of multinucleated cells [15,16]. One possibility is that integrins crosslink the ECM to the intracellular cytoskeleton as occurs during interphase, anchoring the cytokinetic contractile machinery to the outside of the cell (Figure 1). This would lend stability and facilitate the maintenance of intact membrane partitions. Undoubtedly, a more complete
Dispatch R121
understanding of cytokinesis will require that we think ‘outside of the cell’ and incorporate a role for the ECM in cell division.
7.
8.
References 1. Hynes, R.O. (2009). The extracellular matrix: not just pretty fibrils. Science 326, 1216–1219. 2. Pollard, T.D. (2010). Mechanics of cytokinesis in eukaryotes. Curr. Opin. Cell Biol. 22, 50–56. 3. Timpl, R., Sasaki, T., Kostka, G., and Chu, M.L. (2003). Fibulins: a versatile family of extracellular matrix proteins. Nat. Rev. Mol. Cell Biol. 4, 479–489. 4. Dong, C., Muriel, J.M., Ramirez, S., Hutter, H., Hedgecock, E.M., Breydo, L., Baskakov, I.V., and Vogel, B.E. (2006). Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules. J. Biol. Chem. 281, 23606–23610. 5. Vogel, B.E., and Hedgecock, E.M. (2001). Hemicentin, a conserved extracellular member of the immunoglobulin superfamily, organizes epithelial and other cell attachments into oriented line-shaped junctions. Development 128, 883–894. 6. Hodgkin, J., Horvitz, H.R., and Brenner, S. (1979). Nondisjunction mutants of the
9.
10.
11.
12.
13.
nematode Caenorhabditis elegans. Genetics 91, 67–94. Xu, X., and Vogel, B.E. (2011). A secreted protein promotes cleavage furrow maturation during cytokinesis. Curr. Biol. 21, 114–119. Hubbard, E.J., and Greenstein, D. (2000). The Caenorhabditis elegans gonad: a test tube for cell and developmental biology. Dev. Dyn. 218, 2–22. Schultz, D.W., Weleber, R.G., Lawrence, G., Barral, S., Majewski, J., Acott, T.S., and Klein, M.L. (2005). HEMICENTIN-1 (FIBULIN-6) and the 1q31 AMD locus in the context of complex disease: review and perspective. Ophthalmic Genet. 26, 101–105. Dastgheib, K., and Green, W.R. (1994). Granulomatous reaction to Bruch’s membrane in age-related macular degeneration. Arch. Ophthalmol. 112, 813–818. Shaw, J.A., Mol, P.C., Bowers, B., Silverman, S.J., Valdivieso, M.H., Duran, A., and Cabib, E. (1991). The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J. Cell Biol. 114, 111–123. Schmidt, M. (2004). Survival and cytokinesis of Saccharomyces cerevisiae in the absence of chitin. Microbiology 150, 3253–3260. Olson, S.K., Bishop, J.R., Yates, J.R., Oegema, K., and Esko, J.D. (2006). Identification of novel chondroitin
Cell–Cell Fusion: A New Function for Invadosomes Podosomes are cytoskeletal-based structures involved in extracellular matrix remodeling and cellular motility. A new study now implicates podosomes in pore formation during myoblast fusion. Bong Hwan Sung and Alissa Weaver* Cell–cell fusion is a highly regulated event that is critical for many physiological and pathological events, including fertilization, muscle development, and immune response. During skeletal muscle development, fusion of muscle cells generates multinucleate and functional muscle fibers and aberrant fusion has been implicated in dystrophic muscle diseases [1,2]. Recently, a number of groups have studied myoblast fusion during body wall muscle formation in Drosophila melanogaster as a genetically tractable in vivo model system to study cell–cell fusion [1]. In a new study published in the Journal of Cell Biology, Sens et al. [3] investigated the role of actin assembly in formation of the fusion pore during Drosophila myoblast fusion. Interestingly, they find that an invasive, actin-rich, podosome-like structure is used by fusion-competent myoblasts (FCMs) to adhere to and fuse with muscle founder cells.
Previously, it was known that actin filaments accumulate transiently at the site of myoblast fusion [4–6], dependent on signaling from heterotypic adhesion molecules and downstream regulators of branched actin assembly, including Rac, SCAR and WASP [7]. Furthermore, both the SCAR and WASP complex activators of the branched-actin-nucleating Arp2/3 complex were known to be essential for myoblast fusion [5,6,8–10]. However, the nature of the fusion structure and the roles of individual actin regulators were poorly understood. To determine whether the prominent actin accumulations at pre-fusion sites were unique to a muscle cell subtype, Sens et al. [3] expressed GFP–actin under the control of FCM-specific or founder-cell-specific promoters and costained for all actin filaments in embryos with fluorescent phalloidin. Interestingly, the large actin foci were exclusively found in FCM cells and were associated with a deformation in the founder cell membrane.
proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2. J. Cell Biol. 173, 985–994. 14. Xu, X., Rongali, S.C., Miles, J.P., Lee, K.D., and Lee, M. (2006). pat-4/ILK and unc-112/Mig-2 are required for gonad function in Caenorhabditis elegans. Exp. Cell Res. 312, 1475–1483. 15. Reverte, C.G., Benware, A., Jones, C.W., and LaFlamme, S.E. (2006). Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis. J. Cell Biol. 174, 491–497. 16. Pellinen, T., Tuomi, S., Arjonen, A., Wolf, M., Edgren, H., Meyer, H., Grosse, R., Kitzing, T., Rantala, J.K., Kallioniemi, O., et al. (2008). Integrin trafficking regulated by Rab21 is necessary for cytokinesis. Dev. Cell 15, 371–385. 1Columbia University, Department of Pathology and Cell Biology, New York, NY 10032, USA. 2University of California at San Diego, Cellular and Molecular Medicine, La Jolla, CA 92093, USA. *E-mail: [email protected]
DOI: 10.1016/j.cub.2010.12.040
Transmission electron microscopy studies showed finger-like FCM cell protrusions apparently invading into the founder cells at the site of cell–cell fusion. Invasive, actin-rich, finger-like protrusions have been well characterized in cells that invade or remodel tissue and are termed invadopodia in cancer cells and podosomes in normal cells (or collectively, invadosomes) [11,12]. However, a role in cell–cell fusion has not been previously described, and their main function is thought to be degradation of extracellular matrix (ECM), in part due to active trafficking of ECM-degrading proteinases to sites of protrusion formation (Figure 1). The myoblast structures observed by Sens et al. [3] seemed to be a potential variation of podosomes, as they were morphologically similar by electron microscopy and even had adhesion ring structures, albeit cell–cell rather than cell–ECM adhesions. If these structures really were podosomes, the new data revealed that podosomes might be more versatile than previously appreciated and also that they are formed in vivo during developmental ‘invasions’. To determine whether the FCM actin-rich protrusions resembled podosomes at the molecular level, Sens et al. [3] manipulated the