What is the mechanism of cortical force generation at the IS?

Studies of spindle positioning in yeast and C. elegans, and MTOC repositioning in migrating fibroblasts, have identified two mechanisms for MT pulling at the cortex in cells (Gundersen, 2002; Burakov et al., 2003; Manneville and Etienne-Manneville, 2006; Tanaka and Desai, 2008). The first mechanism involves the pulling force driven by MT

depolymerization coupled with MT plus end-attachment to the cortex. This may be the major mechanism for asymmetric spindle positioning in the C. elegans embryo, where depolymerizing MT ends are held at the cortex via cortical attachment factors (Gundersen, 2002; Kaltschmidt and Brand, 2002; Manneville and Etienne-Manneville, 2006). The second mechanism involves the pulling force driven by the minus end-directed movement of

cortically anchored dynein motors along MTs. Studies in budding yeast show that

translocation of the nucleus and spindle into the bud neck occurs through such ‘MT capture and sliding’ mechanism, driven by dynein localized at the bud cortex via the cortical receptor, Num1 (Adames and Cooper, 2000; Gundersen, 2002). However, recent in vitro results by Laan et al., indicate that dynein can also be involved in MT depolymerization-mediated force transduction, as cortical dynein bound on microfabricated barriers were shown to capture

MT plus ends and trigger MT depolymerization, resulting in a strong pulling force on MTs (Laan et al., 2012).

Currently, the mechanism of cortical force generation during MTOC repositioning in T cells is not yet known. Since the two mechanisms of MT pulling (MT depolymerization vs. dynein pulling) require distinct configurations of cortically associated MTs (end-on vs. lateral), we will perform high resolution imaging of MTs at the IS using structured illumination

microscopy to test the dominant mechanism of MT pulling during T cell activation. Moreover, live-imaging of cortically associated dynein and MTs will allow us to observe MT plus-end capture or MT sliding events at the IS. Lastly, we plan to perform laser ablation on cortically associated MTs to test whether net shrinking is observed (indicative of MT

depolymerization-driven pulling) or whether net MT sliding is observed (indicative of dynein- driven pulling) in ablated MTs at the IS.

VII. References

Adames, N.R., and Cooper, J.A. (2000). Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. J Cell Biol 149, 863-874.

Burakov, A., Nadezhdina, E., Slepchenko, B., and Rodionov, V. (2003). Centrosome positioning in interphase cells. J Cell Biol 162, 963-969.

Carlier, M.F., Criquet, P., Pantaloni, D., and Korn, E.D. (1986). Interaction of cytochalasin D with actin filaments in the presence of ADP and ATP. J Biol Chem 261, 2041-2050.

Chen, Q., Nag, S., and Pollard, T.D. (2012). Formins filter modified actin subunits during processive elongation. J Struct Biol 177, 32-39.

Chesarone, M.A., DuPage, A.G., and Goode, B.L. (2010). Unleashing formins to remodel the actin and microtubule cytoskeletons. Nat Rev Mol Cell Biol 11, 62-74.

Chhabra, E.S., and Higgs, H.N. (2007). The many faces of actin: matching assembly factors with cellular structures. Nat Cell Biol 9, 1110-1121.

Combs, J., Kim, S.J., Tan, S., Ligon, L.A., Holzbaur, E.L., Kuhn, J., and Poenie, M. (2006). Recruitment of dynein to the Jurkat immunological synapse. Proc Natl Acad Sci U S A 103, 14883-14888.

DeMond, A.L., Mossman, K.D., Starr, T., Dustin, M.L., and Groves, J.T. (2008). T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation. Biophys J 94, 3286-3292.

Gomez, T.S., Kumar, K., Medeiros, R.B., Shimizu, Y., Leibson, P.J., and Billadeau, D.D. (2007). Formins regulate the actin-related protein 2/3 complex-independent polarization of the centrosome to the immunological synapse. Immunity 26, 177-190.

Gundersen, G.G. (2002). Evolutionary conservation of microtubule-capture mechanisms. Nat Rev Mol Cell Biol 3, 296-304.

Hartman, N.C., and Groves, J.T. (2011). Signaling clusters in the cell membrane. Curr Opin Cell Biol 23, 370-376.

Hartman, N.C., Nye, J.A., and Groves, J.T. (2009). Cluster size regulates protein sorting in the immunological synapse. Proc Natl Acad Sci U S A 106, 12729-12734.

Kaizuka, Y., Douglass, A.D., Varma, R., Dustin, M.L., and Vale, R.D. (2007). Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells. Proc Natl Acad Sci U S A 104, 20296-20301.

Kaltschmidt, J.A., and Brand, A.H. (2002). Asymmetric cell division: microtubule dynamics and spindle asymmetry. J Cell Sci 115, 2257-2264.

Laan, L., Pavin, N., Husson, J., Romet-Lemonne, G., van Duijn, M., Lopez, M.P., Vale, R.D., Julicher, F., Reck-Peterson, S.L., and Dogterom, M. (2012). Cortical dynein controls

microtubule dynamics to generate pulling forces that position microtubule asters. Cell 148, 502-514.

Lee, K.H., Dinner, A.R., Tu, C., Campi, G., Raychaudhuri, S., Varma, R., Sims, T.N., Burack, W.R., Wu, H., Wang, J., Kanagawa, O., Markiewicz, M., Allen, P.M., Dustin, M.L.,

Chakraborty, A.K., and Shaw, A.S. (2003). The immunological synapse balances T cell receptor signaling and degradation. Science 302, 1218-1222.

Manneville, J.B., and Etienne-Manneville, S. (2006). Positioning centrosomes and spindle poles: looking at the periphery to find the centre. Biol Cell 98, 557-565.

Pollard, T.D., and Borisy, G.G. (2003). Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465.

Ponti, A., Machacek, M., Gupton, S.L., Waterman-Storer, C.M., and Danuser, G. (2004). Two distinct actin networks drive the protrusion of migrating cells. Science 305, 1782-1786. Stinchcombe, J.C., Majorovits, E., Bossi, G., Fuller, S., and Griffiths, G.M. (2006).

Centrosome polarization delivers secretory granules to the immunological synapse. Nature 443, 462-465.

Tanaka, T.U., and Desai, A. (2008). Kinetochore-microtubule interactions: the means to the end. Curr Opin Cell Biol 20, 53-63.

Vardhana, S., Choudhuri, K., Varma, R., and Dustin, M.L. (2010). Essential role of ubiquitin and TSG101 protein in formation and function of the central supramolecular activation cluster. Immunity 32, 531-540.

Yokosuka, T., Sakata-Sogawa, K., Kobayashi, W., Hiroshima, M., Hashimoto-Tane, A., Tokunaga, M., Dustin, M.L., and Saito, T. (2005). Newly generated T cell receptor

microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76. Nat Immunol 6, 1253-1262.

Yu, C.H., Wu, H.J., Kaizuka, Y., Vale, R.D., and Groves, J.T. (2010). Altered actin centripetal retrograde flow in physically restricted immunological synapses. PLoS One 5, e11878.