Cytoskeletal function in the immune system

This article introduces a series of reviews covering The Cytoskeleton appearing in Volume 256 of Immunological Reviews. Study of cytoskeletal function in the immune response was born just over 30 years ago, when several pioneering laboratories noticed the polarization of actin filaments and the microtubule array, together with associated secretory organelles, toward the interface formed between cytotoxic cells and their targets 1-4. After a turbulent youth spent observing life in wonder, experimenting with drugs, and questioning whether it all really matters, the field has clearly come of age. Indeed, the cytoskeletal biology of leukocytes has become one of the most exciting, fruitful, and challenging areas in molecular immunology. While the cytoskeleton is important for all cell types, it plays a special role in hematopoietic lineage cells, which are unusually nimble and versatile. Most cells of the immune system are exquisitely sensitive to chemotactic cues. They can move across surfaces using adhesive forces so small that they are difficult to measure, or move through dense tissue in amoeboid fashion in the total absence of integrins. Lymphocytes can polarize within just minutes of immunoreceptor engagement, a process that is essential for directed secretion of lytic granules and cytokines, and one that can determine cell fate. Phagocytes can take up foreign material in multiple ways, destroying pathogens and directing antigenic material to appropriate processing compartments. Finally, the cytoskeleton plays a central role in signal transduction, by segregating signaling components in resting cells, bringing them together to promote signal transduction, and finally driving signal attenuation by directing internalization and degradation of signaling intermediates. The unique and complex roles played by the cytoskeleton in hematopoietic cells may help to explain the existence of hematopoietic-specific isoforms of ubiquitously expressed proteins (e.g. WASp versus N-WASp, HS1 versus cortactin, Hem-1 versus Sra-1, and L-plastin versus T-plastin). This interesting question, however, remains largely untested. What is clear is that mutations in cytoskeletal proteins lead to immunodeficiency on one hand and autoimmunity on the other, and that pathogens subvert immune cells by manipulating cytoskeletal dynamics. However, this weakness is also an opportunity, as cytoskeletal regulatory proteins present attractive new therapeutic targets. In this volume of Immunological Reviews, experts in the field review our current understanding of cytoskeletal function in lymphocytes, natural killer (NK) cells, neutrophils, macrophages, dendritic cells, and hematopoietic progenitor cells. In addition, they address the role of cytoskeletal function in immunodeficiency, autoimmunity, and viral infection. This far-ranging set of reviews reveals the breadth and intellectual complexity that now characterizes the field (Fig. 1). Progress in this field started with T cells, and the T-cell literature continues to be the largest and most highly developed. In keeping with this, many reviews in this volume focus on T-cell biology. In the first set of these, the authors address the role of individual proteins that control specific aspects of actin dynamics. Matalon et al. 5 focus on Wiskott–Aldrich syndrome protein (WASp), the protein that is defective in the severe immunodeficiency disorder Wiskott–Aldrich syndrome. WASp is almost certainly the best studied cytoskeletal protein in immune cells, yet lessons continue to be learned. In addition to reviewing the literature regarding WASp's role in promoting T-cell activation and migration, Matalon et al. 5 discuss other aspects of WASp function, including its ability to carry out epigenetic reprogramming in the nucleus. Finally, they review new studies showing the mechanisms through which WASp is degraded as part of signal termination. Samstag and colleagues 6 review regulation of cofilin, a protein that plays a central role in actin remodeling through its ability to both depolymerize and sever actin filaments, thus freeing barbed ends and actin monomer for new filament assembly. They address well-established regulatory pathways such as phosphorylation and interaction with inositol lipids, as well as the functions of cofilin in the nucleus. Finally, this article describes newly identified pathways involving regulation of cofilin by the cellular redox milieu, highlighting the significance of redox changes during T-cell activation. Morley 7 reviews the growing literature on L-plastin, an actin-bundling protein regulated by calcium and phosphorylation that plays a key role in T-cell activation and migration. Like cofilin, L-plastin has been implicated in costimulatory signaling in T cells. Moreover, it seems poised to play an important role in integrin function. And finally, Parameswaran and Gupta 8 cover the literature on ezrin and moesin in lymphocytes. ERM proteins link the actin cytoskeleton to cell surface proteins in a binary fashion, regulated by phosphorylation and binding to inositol lipids. This simple activity can be used for complex functions such as modulating cortical rigidity, providing molecular fences to dampen signaling in resting cells, and organizing specialized membrane domains such as the distal pole complex or structures associated with pathogen entry. Taken together, these reviews reveal a nuanced, mechanistic view of how the regulated activity of individual proteins shapes the T-cell response. Other recent work in the field has sought to elucidate higher order mechanisms through which cytoskeletal events mesh with T-cell signaling and effector function. One example of this has already been mentioned, the analysis of redox effects on cofilin activity 6. In a similar vein, Babich and I 9 review the many pathways that link actin remodeling with Ca2+ signaling pathways. We discuss mechanisms through which cytoskeletal remodeling promotes both Ca2+ release from endoplasmic reticulum stores and calcium release-activated calcium channel activation, as well as reciprocal mechanisms through which Ca2+ modulates the function of cytoskeletal proteins such as L-plastin. Another important link involves common inositol lipid intermediates. Lipid second messengers are intimately involved with many aspects of cytoskeletal remodeling in T cells, and Huse et al. 10 address the mechanisms through which they dictate the site of microtubule organizing center (MTOC) reorientation. Using an innovative photoactivation technique, their group has shown that local production of diacylglycerol and consequent activation of novel PKCs direct the site of MTOC reorientation, in a process that is driven by the coordinate activity of cytoplasmic dynein and myosin II. The topic of MTOC reorientation is also addressed by Ritter et al. 11, who review the mechanisms through which dynein interacts with SLP-76 and ADAP at the T-cell cortex, and the role of other proteins, such as IQ-GAP and formins, in mediating cross-talk between actin and microtubules. Ritter et al. 11 also discuss the role of the actin cortical network as ‘barrier versus carrier’ during lytic granule secretion. The topic of cross-talk between actin and microtubules is also reviewed by Soares et al. 12. In addition to discussing the role of IQ-GAP, they describe their work showing a novel role for the ERM protein ezrin, in conjunction with the polarity protein Dlg1, in organizing the radial microtubule network at the immunological synapse. In addition, they address an emerging topic in the field, the role of regulated vesicle traffic in immunological synapse function. Taken together, this set of reviews show that significant progress has been made in understanding how cytoskeletal elements work with one another, with lipid and ion second messengers, and with protein trafficking pathways during T-cell activation and effector function. The next set of reviews comes from investigators who are tackling the complexity of cytoskeletal networks head-on. Roybal et al. 13 use a systems approach to review the spatiotemporal organization of over 60 signaling molecules and cytoskeletal elements at the immunological synapse. By studying T cell–B cell conjugates in this way, the group has identified proteins associated with a relatively stable interface center and a more dynamic actin-supported lamellar region. In addition, they describe evidence that costimulatory signaling and actin dynamics create a highly interdigitated T cell–APC interface and discuss the implications for T-cell signaling. Beemiller and Krummel 14 deal with similar biology in a very different way. They put forth the interesting idea that the T-cell actin meshwork and cytoplasm functions as a poroelastic network that controls the movement of TCR and other molecules through nonequilibrium hydraulic pressure, similar to water in a sponge. This model has the potential to explain several longstanding conundrums in the field, such as how microclusters of signaling molecules can be driven by actin flow without any evidence for direct attachment. Clearly, it will be interesting and important to try to reconcile findings reviewed elsewhere in this volume in light of this new model. The biophysical theme continues in the third and final review in this group. Chen and Zhu 15 review the biomechanical regulation of T-cell function and address cytoskeletal function in controlling cortical rigidity, adhesive catch bonds, and mechanotransduction. The systems biology and biophysics approaches that are covered in this set of reviews clearly represent major growth areas for the molecular immunology. Such studies require us to develop new tools and learn new vocabularies, but given the highly dynamic nature of hematopoietic cells, our ability to manipulate them, and the wealth of knowledge about their function, the next decade is likely to see great progress in these new areas. Although this volume is admittedly T-cell centric, our understanding of cytoskeletal function in other hematopoietic lineages has also grown, revealing mechanisms similar to those defined in T cells as well as important cell type–specific mechanisms. As previously noted, the review on ERM proteins by Parameswaran and Gupta 8 deals with both T and B cells. In addition, two reviews specifically address actin cytoskeletal function in B cells. First, Song et al. 16 provide an overview of actin function in modulating BCR signaling. They review the role of actin in antigen gathering and discuss positive feedback loops through which coupling of actin with lipid rafts, membrane proteins, and adapter proteins can amplify BCR signaling by allowing coclustering of BCR with complexes containing BCR, CD19, CD21, and CD81. In addition, they describe actin-dependent negative regulation of BCR signaling via mobility barriers, recruitment of negative regulatory signaling molecules, and receptor endocytosis. These themes parallel those raised by Matalon et al. 5 in T cells, and interestingly, Song et al. 16 describe evidence that WASp and N-WASP function differentially in promoting and attenuating B-cell activation. Next, Santos-Argumedo et al. 17 address the role of class I myosins. In some respects, these one-headed myosins function like ERM proteins, linking the cell surface to the cortical cytoskeleton. Like ERM proteins, class I myosins can control membrane tension and organize microvilli and other membrane protrusions, including those associated with cell migration. However, as discussed by Santos-Argumedo et al. 17, these proteins have also been implicated in regulating multiple other processes, including vesicular traffic to and from the immunological synapse. Lagrue et al. 18 provide an overview of cytoskeletal function in NK cells. They discuss cytoskeletal function during the initial steps NK cell adhesion and spreading and the special role of nanotubes and integrin signaling. One feature that distinguishes NK cells from T cells is the involvement of both activating and inhibitory signals, and Lagrue et al. 18 discuss how the actin cytoskeleton functions to integrate these signals by controlling dynamics of signaling proteins at the NK-IS. Finally, the authors turn to the role of actin and microtubules in directing membrane trafficking, including the formation of signaling endosomes and the secretion of lytic granules and cytokines. The next three articles in this volume address cytoskeletal protein function in antigen-presenting cells and hematopoietic precursors. Rougerie et al. 19 review the mechanisms through which macrophages utilize similar actin regulatory pathways to direct phagocytosis and chemotaxis. They discuss the function of individual actin regulatory proteins in discrete steps of phagocytosis and make the important point that this depends on the phagocytic receptor involved. In migrating macrophages, many of the same proteins are responsible for generating cell protrusions, adhesive contacts (particularly podosomes), and myosin-dependent contractility. In their review, Heuzé et al. 20 describe amoeboid movement of DCs through confined three-dimensional environments. This ‘flowing and squeezing’ process is driven by actin polymerization-based protrusions in the front of the cell and actomyosin-based contractility of the rear, again raising biophysical issues about force generation and hydrostatic pressure. One of the central mysteries in APC biology is how membrane traffic and migration can be coordinated by highly overlapping cytoskeletal regulatory pathways. In DCs, the literature on this topic typically emphasizes on the maturation-dependent switch between antigen uptake and migration. However, Heuzé et al. 20 discuss work from their group showing that membrane trafficking and migration are also mechanistically coupled. One of the major ways in which the actin cytoskeleton is reprogrammed during DC maturation is via differential regulation of Rho family GTPases. In the final review in this group, Nayak et al. 21 address the range and complexity of Rho GTPase function in hematopoietic stem cells. These fast acting switches can direct actin protrusion, myosin contractility, and microtubule dynamics. In hematopoietic stem cells, they regulate proliferation and survival as well as homing, retention, and migration. Thus, they are prime targets for therapeutic intervention. Given the central role played by the cytoskeleton in the development and function of every immune cell lineage, it comes as no surprise that defects in cytoskeletal proteins result in aberrant immune responses. Thus, the final series of reviews addresses the role of cytoskeletal function in autoimmunity and immunodeficiency. First, Shelef et al. 22 address cytoskeletal control of neutrophil migration, rounding out the list of cell types covered in this volume. Here, however, the group focuses on lessons learned from the zebrafish system and the connection with human autoimmune diseases such as rheumatoid arthritis, lupus, and vasculitis. In addition to reviewing key molecules that regulate neutrophil function in autoimmunity, Shelef et al. 22 address their finding in the zebrafish system that neutrophils undergo ‘reverse migration’, away from a wound, a novel process that may deliver antigen to lymphoid organs and that yields ‘wound-sensitized’ neutrophils that may influence disease progression by interacting with other immune cells. On the other side of the balance sheet, defects in actin regulatory proteins such as WASp have long been known to result in primary immunodeficiency. Muolding et al. 23 review the literature on immune defects arising from mutations in the WAS gene. This literature has mushroomed and, like the field as a whole, has grown in both breadth and depth. Over 300 disease-causing mutations in the WASp protein are now known, with loss-of-function mutations resulting in phenotypes ranging from thrombocytopenia to severe immunodeficiency complicated by autoimmunity and malignancy, and activating mutations leading to X-linked neutropenia, an entirely distinct disease. In keeping with the broad involvement of WASp in actin remodeling, multiple defects have been documented in nearly every hematopoietic lineage. Moulding et al. 23 also discuss primary immunodeficiency diseases linked to other cytoskeletal proteins, a topic that is also addressed by Lagure et al. 18. As we move toward whole-genome sequencing, more such diseases are sure to be identified. And finally, Spear et al. 24 address the role of the cytoskeleton in acquired immunodeficiency. They review actin regulatory pathways that regulate HIV infection, a topic also addressed by Soares et al. 12 Importantly, Spear et al. 24 focus on novel therapeutic strategies for combatting HIV based on targeting key actin regulatory proteins needed for HIV entry into cells. Of course, many of the proposed therapeutic targets could also interfere with infection by other pathogens, as well as with inflammatory and autoimmune diseases. This important effort shows that the field is now poised to make clinical use of the basic science lessons learned about cytoskeletal function in infection and immunity. The 20 articles in this volume of Immunological Reviews show that the study of cytoskeletal function in the immune response truly has come of age. As investigators have brought to bear new imaging technologies, molecular biological techniques, biophysical methods, and systems biology approaches, the field has certainly become more complicated. But as we come to understand and appreciate that complexity, we have found that cytoskeletal dynamics in hematopoietic cells are even more intricate and elegant than the pioneers of 30 years ago could have imagined. The author would like to thank all of the contributors to this volume for their thoughtful reviews, and Justina Stadanlick for expert editorial assistance. The author declares no conflict of interest.