Cellular locomotion using environmental topography.

Eukaryotic cells migrate by coupling the intracellular force of the actin cytoskeleton to the environment. While force coupling is usually mediated by transmembrane adhesion receptors, especially those of the integrin family, amoeboid cells such as leukocytes can migrate extremely fast despite very low adhesive forces1. Here we show that leukocytes cannot only migrate under low adhesion but can also transmit forces in the complete absence of transmembrane force coupling. When confined within three-dimensional environments, they use the topographical features of the substrate to propel themselves. Here the retrograde flow of the actin cytoskeleton follows the texture of the substrate, creating retrograde shear forces that are sufficient to drive the cell body forwards. Notably, adhesion-dependent and adhesion-independent migration are not mutually exclusive, but rather are variants of the same principle of coupling retrograde actin flow to the environment and thus can potentially operate interchangeably and simultaneously. As adhesion-free migration is independent of the chemical composition of the environment, it renders cells completely autonomous in their locomotive behaviour.

IgM's exit route.

In this issue of JEM, Thierry et al. (https://doi.org/10.1084/jem.20180344) demonstrate that, once secreted by freshly activated plasmablasts, IgM leaves the lymph node via the microarchitecture of the fibroblastic reticular cell conduit. This work demonstrates how the very peculiar stromal compartment of lymphatic organs optimizes the systemic distribution of immune effectors.

Micro-engineered "pillar forests" to study cell migration in complex but controlled 3D environments.

Cells migrating in multicellular organisms steadily traverse complex three-dimensional (3D) environments. To decipher the underlying cell biology, current experimental setups either use simplified 2D, tissue-mimetic 3D (e.g., collagen matrices) or in vivo environments. While only in vivo experiments are truly physiological, they do not allow for precise manipulation of environmental parameters. 2D in vitro experiments do allow mechanical and chemical manipulations, but increasing evidence demonstrates substantial differences of migratory mechanisms in 2D and 3D. Here, we describe simple, robust, and versatile "pillar forests" to investigate cell migration in complex but fully controllable 3D environments. Pillar forests are polydimethylsiloxane-based setups, in which two closely adjacent surfaces are interconnected by arrays of micrometer-sized pillars. Changing the pillar shape, size, height and the inter-pillar distance precisely manipulates microenvironmental parameters (e.g., pore sizes, micro-geometry, micro-topology), while being easily combined with chemotactic cues, surface coatings, diverse cell types and advanced imaging techniques. Thus, pillar forests combine the advantages of 2D cell migration assays with the precise definition of 3D environmental parameters.

Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments.

The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.

Dendritic Cells Interpret Haptotactic Chemokine Gradients in a Manner Governed by Signal-to-Noise Ratio and Dependent on GRK6.

Navigation of cells along gradients of guidance cues is a determining step in many developmental and immunological processes. Gradients can either be soluble or immobilized to tissues as demonstrated for the haptotactic migration of dendritic cells (DCs) toward higher concentrations of immobilized chemokine CCL21. To elucidate how gradient characteristics govern cellular response patterns, we here introduce an in vitro system allowing to track migratory responses of DCs to precisely controlled immobilized gradients of CCL21. We find that haptotactic sensing depends on the absolute CCL21 concentration and local steepness of the gradient, consistent with a scenario where DC directionality is governed by the signal-to-noise ratio of CCL21 binding to the receptor CCR7. We find that the conditions for optimal DC guidance are perfectly provided by the CCL21 gradients we measure in vivo. Furthermore, we find that CCR7 signal termination by the G-protein-coupled receptor kinase 6 (GRK6) is crucial for haptotactic but dispensable for chemotactic CCL21 gradient sensing in vitro and confirm those observations in vivo. These findings suggest that stable, tissue-bound CCL21 gradients as sustainable "roads" ensure optimal guidance in vivo.

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse.

B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR-antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR-antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells.

How B cells capture, process and present antigens: a crucial role for cell polarity.

B cells are key components of the adaptive immune response. Their differentiation into either specific memory B cells or antibody-secreting plasma cells is a consequence of activation steps that involve the processing and presentation of antigens. The engagement of B cell receptors by surface-tethered antigens leads to the formation of an immunological synapse that coordinates cell signalling events and that promotes antigen uptake for presentation on MHC class II molecules. In this Review, we discuss membrane trafficking and the associated molecular mechanisms that are involved in antigen extraction and processing at the B cell synapse, and we highlight how B cells use cell polarity to coordinate the complex events that ultimately lead to efficient humoral responses.

Polarized secretion of lysosomes at the B cell synapse couples antigen extraction to processing and presentation.

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as a central mechanism that couples Ag extraction to Ag processing and presentation.