@article {67989, title = {Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.}, journal = {Small Methods}, year = {2022}, month = {2022 Jun 01}, pages = {e2200149}, abstract = {

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

}, issn = {2366-9608}, doi = {10.1002/smtd.202200149}, author = {Colin-York, Huw and Heddleston, John and Wait, Eric and Karedla, Narain and DeSantis, Michael and Khuon, Satya and Chew, Teng-Leong and Sbalzarini, Ivo F and Fritzsche, Marco} } @article {64831, title = {Cytoskeletal actin patterns shape mast cell activation.}, journal = {Communications Biology}, volume = {2}, year = {2019}, month = {2019}, pages = {93}, abstract = {

Activation of immune cells relies on a dynamic actin cytoskeleton. Despite detailed knowledge of molecular actin assembly, the exact processes governing actin organization during activation remain elusive. Using advanced microscopy, we here show that Rat Basophilic Leukemia (RBL) cells, a model mast cell line, employ an orchestrated series of reorganization events within the cortical actin network during activation. In response to IgE antigen-stimulation of FCε receptors (FCεR) at the RBL cell surface, we observed symmetry breaking of the F-actin network and subsequent rapid disassembly of the actin cortex. This was followed by a reassembly process that may be driven by the coordinated transformation of distinct nanoscale F-actin architectures, reminiscent of self-organizing actin patterns. Actin patterns co-localized with zones of Arp2/3 nucleation, while network reassembly was accompanied by myosin-II activity. Strikingly, cortical actin disassembly coincided with zones of granule secretion, suggesting that cytoskeletal actin patterns contribute to orchestrate RBL cell activation.

}, issn = {2399-3642}, doi = {10.1038/s42003-019-0322-9}, author = {Colin-York, Huw and Li, Dong and Korobchevskaya, Kseniya and Chang, Veronica T and Betzig, Eric and Eggeling, Christian and Fritzsche, Marco} } @article {64852, title = {Cytoskeletal control of antigen-dependent T cell activation.}, journal = {Cell Reports}, volume = {26}, year = {2019}, month = {2019 Mar 19}, pages = {3369-3379.e5}, abstract = {

Cytoskeletal actin dynamics is essential for T\ cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T\ cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T\ cell system expressing a specific T\ cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T\ cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T\ cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus\ be a mechanism that allows T\ cells to adjust the lengthscale and timescale of T\ cell receptor signaling.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2019.02.074}, author = {Colin-York, Huw and Javanmardi, Yousef and Skamrahl, Mark and Kumari, Sudha and Chang, Veronica T and Khuon, Satya and Taylor, Aaron and Chew, Teng-Leong and Betzig, Eric and Moeendarbary, Emad and Cerundolo, Vincenzo and Eggeling, Christian and Fritzsche, Marco} } @article {48607, title = {Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation.}, journal = {Science Advances}, volume = {3}, year = {2017}, month = {2017 Jun 21}, pages = {e1603032}, abstract = {

T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

}, doi = {10.1126/sciadv.1603032}, author = {Fritzsche, Marco and Fernandes, Ricardo A and Chang, Veronica T and Colin-York, Huw and Clausen, Mathias P and Felce, James H and Galiani, Silvia and Erlenk{\"a}mper, Christoph and Santos, Ana M and Heddleston, John M and Pedroza-Pacheco, Isabela and Waithe, Dominic and de la Serna, Jorge Bernadino and Lagerholm, B Christoffer and Liu, Tsung-Li and Chew, Teng-Leong and Betzig, Eric and Davis, Simon J and Eggeling, Christian} }