During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments 1-3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell 4,5. Most mesenchymal and epithelial cells and some, but not all, cancer cells actively generate their migratory path using pericellular tissue proteolysis 6. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion 7 , raising Reprints and permissions information is available at http://www.nature.com/reprints.

Although much is known about the physiological framework of T cell motility, and numerous rate-limiting molecules have been identified through loss-of-function approaches, an integrated functional concept of T cell motility is lacking. Here, we used in vivo precision morphometry together with analysis of cytoskeletal dynamics in vitro to deconstruct the basic mechanisms of T cell migration within lymphatic organs. We show that the contributions of the integrin LFA-1 and the chemokine receptor CCR7 are complementary rather than positioned in a linear pathway, as they are during leukocyte extravasation from the blood vasculature. Our data demonstrate that CCR7 controls cortical actin flows, whereas integrins mediate substrate friction that is sufficient to drive locomotion in the absence of considerable surface adhesions and plasma membrane flux.

Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymerization within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resolution. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.

Cd8a and Cd8b1 coreceptor gene (Cd8) expression is tightly controlled during T-cell development by the activity of five Cd8 enhancers (E8 I -E8 V ). Here we demonstrate a unique transcriptional program regulating CD8 expression during CD8 + effector T-cell differentiation. The Cd8 enhancer E8 I and Runx/core-binding factor-β (CBFβ) complexes were required for the establishment of this regulatory circuit, because E8 I -, Runx3-, or CBFβ-deficient CD8 + T cells down-regulated CD8α expression during activation. This finding correlated with enhanced repressive histone marks at the Cd8a promoter in the absence of E8 I , and the down-regulation of CD8α expression could be blocked by treating E8 I -, Runx3-, or CBFβ-deficient CD8 + T cells with the histone deacetylase inhibitor trichostatin A. Moreover, Runx/CBFβ complexes bound the Cd8ab gene cluster in activated CD8 + T cells, suggesting direct control of the Cd8a locus. However, CD8 + effector T cells maintained high levels of CD8α when CBFβ was conditionally deleted after activation. Thus, our data suggest an E8 I -and Runx3/CBFβ-dependent epigenetic programming of the Cd8a locus during T-cell activation, leading to Runx/ CBFβ complex-independent maintenance of CD8α expression in effector T cells.epigenetic marks | transcriptional control | cytotoxic T lymphocytes T he expression of the CD4 and CD8 coreceptors is linked with the functional phenotype of mature T cells. On conventional T cells, CD8 usually consist of CD8α and CD8β heterodimers (encoded by the closely linked Cd8a and Cd8b1 genes, respectively), and the expression of the Cd8 genes during T-cell development is regulated by the activity of at least five different cisregulatory elements (1). The first Cd8 enhancer identified, designated E8 I , is active in mature CD8 single-positive thymocytes and in CD8 + T cells, and in innate-like CD8αα + intraepithelial lymphocyte (IEL) of the gut (2, 3). The generation of E8 I -deficient mice revealed that E8 I is essential for CD8αα expression in γδTCR (T-cell receptor) IEL, while CD8 expression on conventional T cells was not impaired (4, 5). The Cd8 enhancer E8 II directs expression of a reporter transgene in double-positive (DP) thymocytes and CD8 + T cells (4), while E8 II -deficient mice have normal CD8 expression (6). Combined deletion of E8 I and E8 II leads to variegated expression of CD8 in DP thymocytes (6), and subsequent studies showed that CD8 variegation correlates with an epigenetic "off" state (7). A similar variegation phenotype is also observed in mice lacking the Cd8 enhancer E8 V (8). Another enhancer, E8 III , is active in DP thymocytes (4), and combined deletion of E8 II and E8 III resulted in a mild CD8 variegation phenotype in DP thymocytes, but E8 II ,E8 III -deficient mice have normal levels of CD8 on peripheral T cells (9). Taken together, these studies revealed a complex network of cis-regulatory elements, and link Cd8 enhancer functions with chromatin

Summary When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes.

The organization of microtubule arrays in immune cells is critically important for a properly operating immune system. Leukocytes are white blood cells of hematopoietic origin, which exert effector functions of innate and adaptive immune responses. During these processes the microtubule cytoskeleton plays a crucial role for establishing cell polarization and directed migration, targeted secretion of vesicles for T cell activation and cellular cytotoxicity as well as the maintenance of cell integrity. Considering this large spectrum of distinct effector functions, leukocytes require flexible microtubule arrays, which timely and spatially reorganize allowing the cells to accommodate their specific tasks. In contrast to other specialized cell types, which typically nucleate microtubule filaments from non-centrosomal microtubule organizing centers (MTOCs), leukocytes mainly utilize centrosomes for sites of microtubule nucleation. Yet, MTOC localization as well as microtubule organization and dynamics are highly plastic in leukocytes thus allowing the cells to adapt to different environmental constraints. Here we summarize our current knowledge on microtubule organization and dynamics during immune processes and how these microtubule arrays affect immune cell effector functions. We particularly highlight emerging concepts of microtubule involvement during maintenance of cell shape and physical coherence.

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Cell production and differentiation for the acquisition of specific functions are key features of living systems. The dynamic network of cellular microtubules provides the necessary platform to accommodate processes associated with the transition of cells through the individual phases of cytogenesis. Here, we show that the plant hormone cytokinin fine‐tunes the activity of the microtubular cytoskeleton during cell differentiation and counteracts microtubular rearrangements driven by the hormone auxin. The endogenous upward gradient of cytokinin activity along the longitudinal growth axis in Arabidopsis thaliana roots correlates with robust rearrangements of the microtubule cytoskeleton in epidermal cells progressing from the proliferative to the differentiation stage. Controlled increases in cytokinin activity result in premature re‐organization of the microtubule network from transversal to an oblique disposition in cells prior to their differentiation, whereas attenuated hormone perception delays cytoskeleton conversion into a configuration typical for differentiated cells. Intriguingly, cytokinin can interfere with microtubules also in animal cells, such as leukocytes, suggesting that a cytokinin‐sensitive control pathway for the microtubular cytoskeleton may be at least partially conserved between plant and animal cells.