Prof Pablo Saez

UKE Hamburg

Signal integration during leukocyte chemotaxis in complex microenvironments

Migration of leukocytes, including dendritic cells (DCs), is a key process that determines the outcome of the immune response. To efficiently migrate, DCs quickly adapt to the dynamic properties of the micro-environment. This adaptation occurs due to high cellular plasticity, which allows fast integration of chemical and physical cues. Combining live imaging with new microfluidic devices that mimic 3D interstitial DC chemotaxis inside collagen gels, and 2D microchannels, we analyzed the role of microtubules. We found that microtubules are required for directionality only when the microenvironment is complex enough to require decision making between branches. Upon danger signal detection DCs undergo a maturation process and switch from slow/random migration to a fast/persistent mode, optimizing their locomotion through complex interstitial tissues to reach the lymph vessels. This phenomenon requires cytoskeleton re-organization and non-muscular MyosinIIA (MyoII) Ca2+-dependent contractility. In addition, DC maturation increases the expression of CCR7, a chemokine receptor that enables DC chemotaxis. However, how cytoskeleton polarity contributes to couple directionality and speed during chemotaxis remains unclear. Here, we evaluated how increasing the geometrical complexity of the microenvironment affects DC chemotaxis, revealing new essential mechanisms required for 3D migration. By combining live cell imaging with new microfluidic devices, we evaluated how DCs resolve branching to advance in an irregular landscape. To do that, we screened the impact of cytoskeletal components in DC directionality and speed. In 3D gels, we found that disruption of acto-myosin contractility (i.e. low dose of Latrunculin A, MyoII inhibition, ROCK inhibition) specifically decreased DC speed, but did not affect directionality. On the other hand, disruption of microtubule dynamics (i.e. treatment with nocodazole or taxol) diminished the directionality of DCs undergoing chemotaxis. Importantly, the effect of nocodazole over directionality was independent of the effect of microtubule depletion on cell contractility, as nocodazole-treated MyoIIA knock-out DCs still presented a reduced directionality. Noticeably, the effect of nocodazole over directionality was lost in 2D confined environments, where DCs displayed no branching and faced no obstacles. In conclusion we showed that cell branching around physical obstacles introduces a specific constrain, which makes microtubules essential for directional migration. We also found that cell speed and directionality rely on distinct molecular mechanisms during guided migration through complex microenvironments.

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