The systems that refine actomyosin forces during motility remain poorly understood. Septins assemble on the T cell cortex and are enriched at the mid-zone in filaments. Septin knockdown causes membrane blebbing, excess leading edge protrusions, and lengthening of the trailingedge uropod. The associated loss of rigidity permits motility, but cells become uncoordinated and poorly persistent. This also relieves a previously unrecognized restriction to migration through small pores. Pharmacologically rigidifying cells counteracts this effect, and relieving cytoskeletal rigidity synergizes with septin-depletion. These data suggest that septins tune actomyosin forces during motility, and likely regulate lymphocyte trafficking in confined tissues.

Septins assemble on the cortex and restore normal cell shape by retracting aberrantly protruding membranes and promoting cortical contraction during amoeboid motility.

Parasite Ag-specific T cell unresponsiveness and diminished IFN-γ production are immunologic hallmarks of patent infection with lymph-dwelling filarial nematodes. Although this diminished responsiveness is directed primarily against the intravascular microfilarial (MF) parasite stage and mediated in part by reduced APC function, the mechanisms involved are not fully understood. In this report, we demonstrate that human dendritic cells (DC) exposed to live MF up-regulate both the cell surface and gene expression of CD54 (ICAM-1). Moreover, live MF result in a 3-fold increase in DC death compared with MF-unexposed DC, primarily due to apoptosis. Notably, microarray and real-time RT-PCR data indicate that live MF concurrently up-regulate mRNA expression of proinflammatory molecules such as IL-8, RANTES, IL-1α, TNF-α, and IL-β in DC, the presence of which is also detected at the protein level, while inhibiting the production of IL-12 (p40 and p70) and IL-10. Soluble excretory-secretory products from live MF diminished IL-12 and IL-10 production and induced DC death, although to a lesser degree. Moreover, exposure of DC to live MF resulted in a decrease in the ability of DC to promote CD4+ T cell production of IFN-γ and IL-5. Our findings clearly suggest that the interaction between live MF and DC is complex but contributes to the hyporesponsiveness and parasite persistence associated with the MF+ state in the infected human. These data further suggest that MF induce an orchestrated response in APC that leads to a diminished capacity to function appropriately, which in turn has significant consequences for CD4+ T cells.

The cortex is the outermost region of the cell, comprising all of the elements from the plasma membrane to the cortical actin cytoskeleton that cooperate to maintain the cell's shape and topology. In eukaryotes without cell walls, this cortex governs the contact between their plasma membranes and the environment and thereby influences cell shape, motility, and signaling. It is therefore of considerable interest to understand how cells control their cortices, both globally and with respect to small subdomains. Here we review the current understanding of this control, including the regulation of cell shape by balances of outward hydrostatic pressure and cortical tension. The actomyosin cytoskeleton is the canonical regulator of cortical rigidity and indeed many would consider the cortex to comprise the actin cortex nearly exclusively. However, this actomyosin array is intimately linked to the membrane, for example via ERM and PIP2 proteins. Additionally, the lipid membrane likely undergoes rigidification by other players, such as Bin-Amphiphysin-Rvs proteins. Recent data also indicates that the septin cytoskeleton may play a formidable and more direct role in stabilization of membranes, particularly in contexts where cells receive limited external stabilization from their environments. Here, we review how septins may play this role, drawing on their physical form, their ability to directly bind and modify membranes and actomyosin, and their interactions with vesicular machinery. Deficiencies and alterations in the nature of the septin cytoskeleton may thus be relevant in multiple disease settings. V C 2010 WileyLiss, Inc.Key Words: septin, cortical tension, cell motility, actin IntroductionT he rigidity of the cortex is a fundamental property of all cells, and in metazoans, where the nature of each contact between a cell and its neighbor or substrate is critical to complex biology, cortical rigidity plays particularly interesting and important roles. Specialized cellular function is supported by the regulation of interactions between cortical elements, which ensures mechanical stability and flexibility of the cortex. For instance, stable contacts between cells, such as tight junctions, can be encouraged by long-lived, highly rigid cortical structures [Van Itallie et al., 2009]. Alternatively, locally rigid regions can be dynamically coordinated to promote motility. At the simplest level, a critical difference between stable and motile cells is the rapidity with which their membranes are able to locally contort and promote access between receptors and ligands. This deformability may be grossly referred to as ''cortical rigidity''-the importance of which also extends to include the regulation of cell shape, cytokinesis, development, and the response to a changing external environment. Although cortical rigidity and the cortex is often used to invoke features of the actin cytoskeleton, this review aims not to restrict only to that framework but to consider the cortex as a sum of actin as well as other participants. A...

Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P2 in T. brucei. We find a low steady state level of PI(3,5)P2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P2 pool leading to rapid cell death. TbFab1 and PI(3,5)P2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P2 metabolism, the PI(3,5)P2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology.

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4+ T cell responses. In Fyn-deficient mice, CD4+ T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10+CD4+ T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323–339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10+ Fyn-deficient CD4+ T cells, was virtually indistinguishable from the response of DO11.10+ wild-type CD4+ T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4+ Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10+ Fyn-deficient CD4+ T cells were more potent cytokine-producing cells than DO11.10+ wild-type CD4+ T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4+ T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4+ T cells.

T cell proliferation is initiated by T cell antigen receptor (TCR) triggering and/or by soluble growth factors. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and -independent pathways. Septin-deficient T cells failed cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was independent of septins when cell-cell contacts, such as those from antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and co-stimulatory signals. We could differentiate cytokine- versus antigen-driven expansion in vivo and thus demonstrate that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation.

Antibody Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies. Measurement of antibody‐dependent cellular cytotoxicity (ADCC) is critical for understanding the Fc effector function during monoclonal antibody development. This article covers two cell‐based ADCC bioassays which can quantitatively measure the antibody potency in ADCC. Basic Protocol 1 describes the ADCC reporter bioassay using engineered ADCC effector cells which measures the FcγRIIIa‐mediated luciferase reporter activation upon the binding of antibody‐coated target cells. Basic Protocol 2 describes the PBMC ADCC bioassay using primary peripheral blood mononuclear cells (PBMC) as effector cells and engineered HiBiT target cells in an assay that measures the release of HiBiT from target cells upon antibody‐mediated target lysis. Optimization of several key assay parameters including cell handling, effector:target (E:T) ratios, assay plate, and plate reader requirement, and how these parameters impact assay performance are discussed. © 2021 Promega Corporation. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ADCC reporter bioassay using engineered ADCC bioassay effector cells Basic Protocol 2: PBMC ADCC bioassay using primary PBMC and engineered HiBiT target cells