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Cell–Cell Fusion: A New Function for Transmission electron studies showed finger-like FCM cell Invadosomes protrusions apparently invading into the founder cells at the site of cell–cell fusion. are cytoskeletal-based structures involved in extracellular matrix Invasive, -rich, finger-like remodeling and cellular motility. A new study now implicates podosomes in protrusions have been well pore formation during myoblast fusion. characterized in cells that invade or remodel tissue and are termed Bong Hwan Sung and Alissa Weaver* Previously, it was known that actin in cells and filaments accumulate transiently at podosomes in normal cells (or Cell–cell fusion is a highly regulated the site of myoblast fusion [4–6], collectively, invadosomes) [11,12]. event that is critical for many dependent on signaling from However, a role in cell–cell fusion physiological and pathological events, heterotypic adhesion molecules and has not been previously described, including fertilization, muscle downstream regulators of branched and their main function is thought to development, and immune response. actin assembly, including Rac, SCAR be degradation of extracellular matrix During skeletal muscle development, and WASP [7]. Furthermore, both the (ECM), in part due to active trafficking fusion of muscle cells generates SCAR and WASP complex activators of ECM-degrading proteinases to multinucleate and functional muscle of the branched-actin-nucleating sites of protrusion formation (Figure 1). fibers and aberrant fusion has been Arp2/3 complex were known to be The myoblast structures observed implicated in dystrophic muscle essential for myoblast fusion [5,6,8–10]. by Sens et al. [3] seemed to be diseases [1,2]. Recently, a number However, the nature of the fusion a potential variation of podosomes, of groups have studied myoblast fusion structure and the roles of individual as they were morphologically similar during body wall muscle formation in actin regulators were poorly by electron microscopy and even had Drosophila melanogaster as understood. adhesion ring structures, albeit a genetically tractable in vivo model To determine whether the prominent cell–cell rather than cell–ECM system to study cell–cell fusion [1]. actin accumulations at pre-fusion sites adhesions. If these structures really In a new study published in the were unique to a muscle cell subtype, were podosomes, the new data Journal of Cell Biology, Sens et al. [3] Sens et al. [3] expressed GFP–actin revealed that podosomes might be investigated the role of actin assembly under the control of FCM-specific or more versatile than previously in formation of the fusion pore during founder-cell-specific promoters and appreciated and also that they are Drosophila myoblast fusion. costained for all actin filaments in formed in vivo during developmental Interestingly, they find that an invasive, embryos with fluorescent phalloidin. ‘invasions’. actin-rich, -like structure is Interestingly, the large actin foci were To determine whether the FCM used by fusion-competent myoblasts exclusively found in FCM cells and actin-rich protrusions resembled (FCMs) to adhere to and fuse with were associated with a deformation podosomes at the molecular level, muscle founder cells. in the founder . Sens et al. [3] manipulated the Current Biology Vol 21 No 3 R122

A Myoblast podosome B ECM-degrading podosome

N N Fusion- competent myoblast F WASP F M N-WASP M ? WASP ? Sns F Fusogen-containing Duf vesicles

Founder cell M Integrin ring ECM degradation M MMP-containing N Extracellular matrix M M N SCAR M vesicles

Current Biology

Figure 1. Comparison of myoblast podosome with ECM-degrading podosome. (A) Actin assembly is required on both the fusion-competent myoblast and founder cell membranes for initiation of fusion pore formation. Although WASP and SCAR have redundant functions in the formation of actin foci in FCM podosome structures, WASP is essential for invasion of FCM podosomes into founder cells and SCAR induces the formation of a thin actin layer at the pre-fusion site in founder cells. Similar to traditional podosomes (B), myoblast podosomes have an adhesion ring; however, the molecular components are immunoglobulin (Ig) super- family cell–cell adhesion receptors found in the FCM (Sns ring) and founder cells (Duf ring). Fusogen-containing vesicles may be trafficked to podosomes to promote cell fusion. (B) WASP/N-WASP promotes formation of branched-actin-rich, ECM-degrading podosomes. Rings contain- ing and proteins are formed around ECM-degrading podosomes. Matrix metalloproteinases (MMPs) are embedded in vesicles, transported to podosomes, and secreted to induce ECM degradation. N, nucleus.

SCAR and WASP regulators formation of actin-rich, invasive ribosomes and other organelles but of branched-actin assembly and podosomes that protrude into founder not actin. Thus, after fusion pore determined the effects on F-actin cells and allow extensive membrane initiation, rapid disassembly of foci formation and myoblast fusion. contact at the pre-fusion site. actin and pore expansion is likely to Loss of both protein complexes in In founder cells, SCAR promotes occur. scar,sltr double mutants led to loss formation of a thin actin layer at the Overall, the study by Sens et al. [3] of the FCM actin focus, verifying that pre-fusion site and is necessary provides an elegant example of the it is a branched-actin-based structure. for cell fusion. These two actin versatility of actin-based structures However, the actin focus was present structures may allow close enough for cellular invasion processes in vivo. in single scar or sltr mutants, apposition and/or curvature of the Although a previous study had shown suggesting compensation of one membranes for fusion to initiate. that leukocytes use podosomes as branched-actin regulator for the other. They may also serve as docking sites adhesion and invasion structures More interesting, however, was the for vesicles containing fusogenic during transcytosis of endothelial cells finding that, despite continued factors, such as lipid rafts (Figure 1) [16], podosomes had not been formation of the F-actin foci on the FCM [13,14]. Notably, Golgi-derived previously identified as mediators of side of the pre-fusion site, loss of vesicles with electron-dense rims have direct fusion of two cells. Indeed, WASP but not SCAR complexes led been observed to move toward the dogma in the field has been that to defective formation of invasive muscle-cell contact sites in Drosophila podosomes are structures that protrusions and lack of FCM invasion embryos [5]. Moreover, invadopodia in mediate ECM adhesion and into founder cells. By contrast, loss cancer cells have been shown to be degradation. A final novel contribution of SCAR affected actin assembly at dependent on the protein of this paper is the identification of the founder cell membrane side of -1 [15], and both podosomes podosomes in an in vivo setting, the pre-fusion site. As WASP, but not and invadopodia are sites of active evidence for which has so far been SCAR, homologues are known to be vesicle trafficking [11,12]. Since both limited [17,18], potentially due to their essential for podosome formation and WASP and SCAR are essential for small size and transient nature [11,19]. function in mammalian cells, these data myoblast fusion, the authors were not Future studies should shed further light support the concept that the invasive able to dissect further the individual upon the adaptability of these invasive myoblast protrusions are similar or roles of these actin regulators in later structures. identical to podosomes. events. However, they did analyze the Sens et al. [3] suggest that actin structure of the fusion site in wild-type assembly is required on both the FCM embryos by a high-pressure freezing/ References and founder cell membranes for freeze substitution electron 1. Chen, E.H., and Olson, E.N. (2004). Towards a molecular pathway for myoblast fusion initiation of fusion pore formation. microscopy preparation and found in Drosophila. Trends Cell Biol. 14, In FCM cells, WASP activity promotes a single macrofusion pore filled with 452–460. Dispatch R123

2. Volonte, D., Peoples, A.J., and Galbiati, F. WASP and SCAR have distinct roles in matrix degradation by human breast cancer (2003). Modulation of myoblast fusion by activating the Arp2/3 complex during myoblast cells. Cancer Res. 69, 8594–8602. caveolin-3 in dystrophic skeletal muscle cells: fusion. J. Cell Sci. 121, 1303–1313. 16. Carman, C.V., Sage, P.T., Sciuto, T.E., de la implications for Duchenne muscular dystrophy 9. Massarwa, R., Carmon, S., Shilo, B.Z., and Fuente, M.A., Geha, R.S., Ochs, H.D., and limb-girdle muscular dystrophy-1C. Mol. Schejter, E.D. (2007). WIP/WASp-based Dvorak, H.F., Dvorak, A.M., and Springer, T.A. Biol. Cell 14, 4075–4088. actin-polymerization machinery is essential (2007). Transcellular diapedesis is initiated by 3. Sens, K.L., Zhang, S., Jin, P., Duan, R., for myoblast fusion in Drosophila. Dev. Cell 12, invasive podosomes. Immunity 26, 784–797. Zhang, G., Luo, F., Parachini, L., and Chen, E.H. 557–569. 17. Quintavalle, M., Elia, L., Condorelli, G., and (2010). An invasive podosome-like structure 10. Schafer, G., Weber, S., Holz, A., Bogdan, S., Courtneidge, S.A. (2010). MicroRNA control promotes fusion pore formation during Schumacher, S., Muller, A., Renkawitz-Pohl, R., of podosome formation in vascular smooth myoblast fusion. J. Cell Biol. 191, 1013–1027. and Onel, S.F. (2007). The Wiskott-Aldrich muscle cells in vivo and in vitro. J. Cell Biol. 4. Kesper, D.A., Stute, C., Buttgereit, D., syndrome protein (WASP) is essential for 189, 13–22. Kreiskother, N., Vishnu, S., Fischbach, K.F., myoblast fusion in Drosophila. Dev. Biol. 304, 18. Rottiers, P., Saltel, F., Daubon, T., Chaigne- and Renkawitz-Pohl, R. (2007). Myoblast fusion 664–674. Delalande, B., Tridon, V., Billottet, C., in Drosophila melanogaster is mediated 11. Gimona, M., Buccione, R., Courtneidge, S.A., Reuzeau, E., and Genot, E. (2009). TGFbeta- through a fusion-restricted myogenic-adhesive and Linder, S. (2008). Assembly and biological induced endothelial podosomes mediate structure (FuRMAS). Dev. Dyn. 236, 404–415. role of podosomes and invadopodia. Curr. basement membrane collagen degradation in 5. Kim, S., Shilagardi, K., Zhang, S., Hong, S.N., Opin. Cell Biol. 20, 235–241. arterial vessels. J. Cell Sci. 122, 4311–4318. Sens, K.L., Bo, J., Gonzalez, G.A., and 12. Weaver, A.M. (2006). Invadopodia: specialized 19. Destaing, O., Saltel, F., Geminard, J.C., Chen, E.H. (2007). A critical function for the cell structures for cancer invasion. Clin. Exp. Jurdic, P., and Bard, F. (2003). Podosomes actin in targeted exocytosis of Metastasis 23, 97–105. display actin turnover and dynamic prefusion vesicles during myoblast fusion. Dev. 13. Martens, S., and McMahon, H.T. (2008). self-organization in expressing Cell 12, 571–586. Mechanisms of membrane fusion: disparate actin-green fluorescent protein. Mol. Biol. Cell 6. Richardson, B.E., Beckett, K., Nowak, S.J., and players and common principles. Nat. Rev. Mol. 14, 407–416. Baylies, M.K. (2007). SCAR/WAVE and Arp2/3 Cell Biol. 9, 543–556. are crucial for cytoskeletal remodeling at the 14. Mukai, A., Kurisaki, T., Sato, S.B., site of myoblast fusion. Development 134, Kobayashi, T., Kondoh, G., and Hashimoto, N. Department of Cancer Biology, Vanderbilt 4357–4367. (2009). Dynamic clustering and dispersion of University Medical Center, Nashville, 7. Rochlin, K., Yu, S., Roy, S., and Baylies, M.K. lipid rafts contribute to fusion competence of (2010). Myoblast fusion: when it takes more to myogenic cells. Exp. Cell Res. 315, 3052–3063. TN 37232, USA. make one. Dev. Biol. 341, 66–83. 15. Yamaguchi, H., Takeo, Y., Yoshida, S., *E-mail: [email protected] 8. Berger, S., Schafer, G., Kesper, D.A., Holz, A., Kouchi, Z., Nakamura, Y., and Fukami, K. Eriksson, T., Palmer, R.H., Beck, L., Klambt, C., (2009). Lipid rafts and caveolin-1 are required Renkawitz-Pohl, R., and Onel, S.F. (2008). for invadopodia formation and extracellular DOI: 10.1016/j.cub.2010.12.024

Neuroscience: What We Cannot aspects of the complex neuronal ensemble responses one aims to Model, We Do Not Understand explain. Instead of trying to predict the detailed spiking activity of every single neuron as done in many other studies To understand computations in neuronal circuits, a model of a small patch (for example [2,3]), Rasch et al. [1] of cortex has been developed that can describe the firing regime in the defined a ‘firing regime’ that is visual system remarkably well. characterized by several properties of the neuronal responses. These William S. Anderson1 of neurons. This synergy of theory properties included the firing rate, and Gabriel Kreiman2,3,4,* and neurophysiology is beautifully distribution of interspike intervals, illustrated in recent work by Rasch et al. variability in spike counts over time, Circuits of neurons in the brain are [1]. These authors took a courageous degree of burst firing and degree very complicated: because of the approach using computational models of synchronization in the network. multiple non-linearities, different to describe the activity in a local 5 x The authors use these inter-related types of neurons, complex dendritic 5 mm patch of neocortex with an properties to define the state of the geometries, diverse connectivity impressive set of 35,000 neurons network. patterns and dependencies on learning and w4 million synapses. They focused Another important aspect that the and development, the cerebral cortex on primary visual cortex, one of the theorist must consider when thinking and other neuronal circuits constitute most studied parts of cortex and the about such network models is the the most complex systems ever first stage in the hierarchical cascade large number of parameters that arise studied by science. Perhaps not of processes that convert the retinal as a consequence of the complexity in surprisingly, the computational input into our visual perceptions. The the circuitry. The modeler needs to power that emerges from such Logothetis lab used multiple microwire make decisions about the number and circuits is astounding; neuronal electrodes to measure the activity of type of neurons, their distribution and networks are responsible for neurons in primary visual cortex of connectivity, the type of ionic diverse cognitive phenomena anesthetized monkeys while the channels they are embedded with and such as seeing, smelling, monkeys watched a natural scene their corresponding characteristics. remembering, planning and so on. movie. The authors then ‘presented’ Some of these decisions may be To understand how function the same movie to their model to constrained by experimental data; emerges from ensembles of neurons explore its fidelity and quantitatively others may require more guesswork. and their interactions, we need compare the computational output Parameters are our enemies. It is a rigorous interplay of theoretical and the neurophysiological one. extremely difficult from work and experimental approaches To compare the circuit in silico a computational viewpoint to capable of listening to the activity and in vivo, one must consider what systematically characterize the