Subunit 1 of the prefoldin chaperone complex is required for lymphocyte development and function

Prefoldin is a hexameric chaperone that facilitates posttranslational folding of actins and other cytoskeletal proteins by the Tcp1-containing ring complex chaperonin, TriC. The present study characterized mice with a null mutation in Pfdn1, which encodes the first subunit of the Prefoldin complex. Pfdn1-deficient mice displayed phenotypes characteristic of defects in cytoskeletal function, including manifestations of ciliary dyskinesia, neuronal loss, and defects in B and T cell development and function. B and T cell maturation was markedly impaired at relatively early stages, namely at the transitions from pre-pro-B to pre-B cells in the bone marrow and from CD4-CD8- double-negative to CD4+CD8+ double-positive T cells in the thymus. In addition, mature B and T lymphocytes displayed cell activation defects upon Ag receptor cross-linking accompanied by impaired Ag receptor capping in B cells. These phenotypes illustrate the importance of cytoskeletal function in immune cell development and activation.     

Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor

The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.     

Compartment resolved reference proteome map from highly purified naïve,activated, effector,and memory CD8+ murine immune cells

Differentiation of CD8⁺ T lymphocytes into effector and memory cells is key for an adequate immune response and relies on complex interplay of pathways that convey signals from the cell surface to the nucleus. In this study, we investigated the proteome of four cytotoxic T‐cell subtypes; naïve, recently activated effector, effector, and memory cells. Cells were fractionated into membrane, cytosol, soluble nuclear, chromatin‐bound, and cytoskeletal compartments. Following LC‐MS/MS analysis, identified peptides were analyzed via MaxQuant. Compartment fractionation and gel‐LC‐MS separation resulted in 2399 proteins identified in total. Comparison between the different subsets resulted in 146 significantly regulated proteins for naïve and effector cells, followed by 116 for activated, and 55 for memory cells. Besides Granzyme B signaling (for activated and/ or effector cells vs. naïve cells), the most prominent changes occurred in the TCA cycle and aspartate degradation. These changes suggest that correct balancing of metabolism is key for differentiation processes. All MS data have been deposited in the ProteomeXchange with identifier PXD001065 ( http://proteomecentral.proteomexchange.org/dataset/PXD001065 ).     

The brush border cytoskeleton is not static: in vivo turnover of proteins

The shape and stability of intestinal epithelial cell microvilli are maintained by a cytoskeletal core composed of a bundle of actin filaments with several associated proteins. The core filaments are intimately associated with the overlying plasma membrane, in which there occur rapid turnover of proteins and constant incorporation of new membrane. Previous work has shown that starvation or inhibition of protein synthesis results in modulation of microvillar length, which indicates that there may be cytoskeletal protein turnover. We demonstrate herein, by means of in vivo pulse labeling with radioactive amino acids, that turnover of brush border cytoskeletal proteins occurs in mature absorptive cells. Turnover of cytoskeletal proteins appears to be quite slow relative to membrane protein turnover, which suggests that the turnover of these two microvillar compartments is not coupled. We thus conclude that cytoskeletal protein turnover may be a factor used to maintain normal length and stability of microvilli and that the cytoskeleton cannot be considered a static structure.     

Cutting edge: Rac GTPases sensitize activated T cells to die via Fas

In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.     

The effect of free fatty acids on spectrin organization in lymphocytes.

Previous studies have shown that cis unsaturated free fatty acids (uFFAs) are able to cause alterations in the normal distribution pattern of certain cytoskeletal proteins in lymphocytes, including tubulin, actin, alpha-actinin, and myosin. The cytoskeletal protein spectrin naturally possesses a marked heterogeneity of distribution among resting T and B lymphocytes isolated from all murine lymphoid organs. In some cells, spectrin is observed in a ring-like staining pattern at the periphery of the cell, reflecting a likely association with the cell membrane; in other cells, spectrin is found within the cytoplasm as a large single aggregate or in several smaller aggregates. Addition of uFFA to freshly isolated murine lymphocytes causes disruption in the latter pattern of spectrin organization. Following short-term incubation (15 min) of tissue-derived lymphocytes (from spleen, thymus, and lymph node) and 1 microgram/mL uFFA (oleic [18:1 cis], linoleic [18:2 cis, cis], arachidonic [20:4], or elaidic [18:1 trans] acid) there is a loss of cytoplasmic aggregates of spectrin and a concomitant increase in cells in which spectrin is diffusely distributed. This effect is not seen when two saturated FFAs (sFFAs) were used. When using DO11.10 cells, a T-cell hybridoma in which nearly all cells constitutively express a cytoplasmic aggregate of spectrin, a similar effect was observed, but greater concentrations (10-20 micrograms/mL) of FFA were needed to obtain the same effect. Addition of calcium to the incubation buffer substantially blocks spectrin reorganization. In several disease states, serum levels of FFA are observed to be excessively high; our data support the hypothesis that cytoskeletal reorganization in lymphocytes may be related to the altered immune function frequently observed in these conditions.     

Augmentation of interleukin-2 release by cytochalasins

Augmentation of mitogen-induced release of the T cell lymphokine interleukin-2 (IL-2) occurred using several cytochalasins in coculture with a T cell lymphoma line (JM) or with purified T cells. When added concurrently with mitogen, cytochalasins had no apparent negative effect either on the ability of mitogen to signal IL-2 production or on the total amount produced. Use of cytochalasins that affect submembranous actin in peripheral lymphocytes established a link between cytoskeletal alterations and lymphokine release, although direct membrane perturbation cannot be excluded. These data indicate that an intracellular pool of IL-2 may accumulate in both T cell lines and in purified peripheral T cells and that maintenance of that pool may be affected by alterations of the cytoskeletal elements. Cytochalasins not only increased IL-2 release, but could substitute for phorbol myristic acetate (PMA) in supporting mitogen-signaled IL-2 production. We interpret these data also to indicate that cytoskeletal attachment to the surface molecules that signal IL-2 production is not needed for the activation.     

A simple guide to biochemical approaches for analyzing protein-lipid interactions

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein-lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein-lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.     

Diversity in immune-cell interactions: states and functions of the immunological synapse

The contact-dependent exchange of signals between epithelial and neuronal cells results from close membrane-membrane appositions, which are stabilized for years by polarized adhesion, cytoskeletal assemblies and extracellular scaffold proteins. By contrast, owing to a lack of scaffold proteins, interactions between immune cells such as T lymphocytes and antigen-presenting cells (APCs) comprise a spectrum of structurally diverse and short-lived interaction modes that last from minutes to hours. Signals exchanged between T cells and APCs are generated in a specific contact region, termed the "immunological synapse", that coordinates cytoskeletal dynamics with the T-cell receptor (TCR), the engagement of accessory receptors and membrane-proximal signaling. Recent data shed light on the different physical and molecular interaction modes that occur between T cells and APCs, including their dynamics and transition stages, and their consequences for signaling, activation and T-cell effector function.     

Antigen processing for presentation to CD4+ T cells.

The immune system surveys the organism for the presence of foreign or abnormal structures. An important role in the immune response is assumed by T lymphocytes that recognize foreign antigen while tolerating self-proteins. T lymphocytes can recognize only peptide fragments that are presented to them by molecules of the major histocompatibility complex (MHC). Antigen processing for presentation to T cells involves distinct cellular compartments where peptides and MHC molecules interact. Whereas class I MHC molecules (recognized by CD8+ cytotoxic T cells) acquire peptides in an early biosynthetic compartment, class II molecules (recognized by CD4+ helper T cells) acquire peptides most efficiently in an endocytic compartment. It has emerged recently that the class II processing compartment can be fed not only from the outside with exogenous antigen but also from endogenous sources, including membrane-associated and cytosolic proteins. The potential sources of proteins that can trigger a helper T cell response during viral infections and that can induce self-tolerance are thus much wider than previously anticipated.     

Cutting edge: integration of human T lymphocyte cytoskeleton by the cytolinker plectin.

Chemokine-induced polarization of lymphocytes involves the rapid collapse of vimentin intermediate filaments (IFs) into an aggregate within the uropod. Little is known about the interactions of lymphocyte vimentin with other cytoskeletal elements. We demonstrate that human peripheral blood T lymphocytes express plectin, an IF-binding, cytoskeletal cross-linking protein. Plectin associates with a complex of structural proteins including vimentin, actin, fodrin, moesin, and lamin B in resting peripheral blood T lymphocytes. During chemokine-induced polarization, plectin redistributes to the uropod associated with vimentin and fodrin; their spatial distribution indicates that this vimentin-plectin-fodrin complex provides a continuous linkage from the nucleus (lamin B) to the cortical cytoskeleton. Overexpression of the plectin IF-binding domain in the T cell line Jurkat induces the perinuclear aggregation of vimentin IFs. Plectin is therefore likely to serve as an important organizer of the lymphocyte cytoskeleton and may regulate changes of lymphocyte cytoarchitecture during polarization and extravasation.     

Intracellular pathways of CD1 antigen presentation

Each of the human CD1 proteins takes a different route through secretory and endocytic compartments before finally arriving at the cell surface, where these proteins present glycolipid antigens to T cells. Recent studies have shown that adaptor-protein complexes and CD1-associated chaperones control not only CD1 trafficking, but also the development and activation of CD1-restricted T cells. This indicates that CD1 proteins, similar to MHC class I and II molecules, selectively acquire certain antigens in distinct cellular subcompartments. Here, we summarize evidence supporting the hypothesis that CD1 proteins use separate, but parallel, pathways to survey endosomal compartments differentially for lipid antigens.     

The T-cell receptor: just another hypothesis.

Functional activities of T and B lymphocytes and the kinetics of hematopoietic stem cells were studied in mice with inoculated or spontaneous tumors. The development and growth of the tumor inhibited B cells and helper T cells, while the activity of killer T cells and spleen suppressor cells was markedly enhanced. The processes of stem cell migration from the bone marrow were considerably intensified and altered in tumor-bearing mice. Data were obtained suggesting that helper T cells and killer T cells represent nonidentical compartments within the population of thymus-dependent lymphocytes. Immunosuppression during tumor bearing is probably due to an impairment of T lymphocytes cooperating in immune responses, B-lymphocytes and their precursors.     

Relationship between membrane lipid mobility and spectrin distribution in lymphocytes.

We have previously established that T and B lymphocytes in situ are remarkably heterogeneous with respect to the cytoskeletal protein spectrin. Since in erythrocytes spectrin is known to play an important role in the regulation of membrane fluidity, lipid organization and lateral mobility of membrane proteins, we have sought to determine if the heterogeneous patterns of spectrin distribution that we have observed are related to possible differences in membrane lipid organization in these various subsets. To this end, we have utilized a fluorescent pyrene-labelled phospholipid as a probe of the lipid lateral mobility and have examined two related T cell systems maintained in vitro, DO.11.10 cells and a spontaneously arising variant, DO.11.10V. In these (and other cloned in vitro systems) we have previously observed that the cells homogeneously express one of the kinds of spectrin distribution patterns observed in situ. Thus the uniformity of staining of these systems permits us to address whether the various patterns of spectrin distribution may be predictive of differences in membrane lipid properties. Here we show that in cells in which there is little or nor spectrin at the plasma membrane (DO.11.10) that the lipids in the plasma membrane are considerably less mobile than in its related variant in which spectrin is diffusely distributed within the cell and at the plasma membrane. From this and previous results, we conclude that differences in the distribution of the cytoskeletal protein spectrin among lymphocytes may be a useful parameter in helping to predict the status of membrane lipid organization.     

Moesin Interacts with the Cytoplasmic Region of Intercellular Adhesion Molecule-3 and Is Redistributed to the Uropod of T Lymphocytes during Cell Polarization

During activation, T lymphocytes become motile cells, switching from a spherical to a polarized shape. Chemokines and other chemotactic cytokines induce lymphocyte polarization with the formation of a uropod in the rear pole, where the adhesion receptors intercellular adhesion molecule-1 (ICAM-1), ICAM-3, and CD44 redistribute. We have investigated membrane–cytoskeleton interactions that play a key role in the redistribution of adhesion receptors to the uropod. Immunofluorescence analysis showed that the ERM proteins radixin and moesin localized to the uropod of human T lymphoblasts treated with the chemokine RANTES (regulated on activation, normal T cell expressed, and secreted), a polarization-inducing agent; radixin colocalized with arrays of myosin II at the neck of the uropods, whereas moesin decorated the most distal part of the uropod and colocalized with ICAM-1, ICAM-3, and CD44 molecules. Two other cytoskeletal proteins, β-actin and α-tubulin, clustered at the cell leading edge and uropod, respectively, of polarized lymphocytes. Biochemical analysis showed that moesin coimmunoprecipitates with ICAM-3 in T lymphoblasts stimulated with either RANTES or the polarization- inducing anti–ICAM-3 HP2/19 mAb, as well as in the constitutively polarized T cell line HSB-2. In addition, moesin is associated with CD44, but not with ICAM-1, in polarized T lymphocytes. A correlation between the degree of moesin–ICAM-3 interaction and cell polarization was found as determined by immunofluorescence and immunoprecipitation analysis done in parallel. The moesin–ICAM-3 interaction was specifically mediated by the cytoplasmic domain of ICAM-3 as revealed by precipitation of moesin with a GST fusion protein containing the ICAM-3 cytoplasmic tail from metabolically labeled Jurkat T cell lysates. The interaction of moesin with ICAM-3 was greatly diminished when RANTES-stimulated T lymphoblasts were pretreated with the myosin-disrupting drug butanedione monoxime, which prevents lymphocyte polarization. Altogether, these data indicate that moesin interacts with ICAM-3 and CD44 adhesion molecules in uropods of polarized T cells; these data also suggest that these interactions participate in the formation of links between membrane receptors and the cytoskeleton, thereby regulating morphological changes during cell locomotion.     

Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes.

How intracellular cytoskeletal and signaling proteins connect and communicate with the extracellular matrix (ECM) is a fundamental question in cell biology. Recent biochemical, cell biological, and genetic studies have revealed important roles of cytoplasmic integrin-linked kinase (ILK) and its interactive proteins in these processes. Cell adhesion to ECM is an important process that controls cell shape change, migration, proliferation, survival, and differentiation. Upon adhesion to ECM, integrins and a selective group of cytoskeletal and signaling proteins are recruited to cell matrix contact sites where they link the actin cytoskeleton to the ECM and mediate signal transduction between the intracellular and extracellular compartments. In this review, we discuss the molecular activities and cellular functions of ILK, a protein that is emerging as a key component of the cell-ECM adhesion structures.     

Cytoskeleton changes and impaired motility of monocytes at modelled low gravity

Summary. Investigations performed in space have shown that gravity changes affect important cellular mechanisms like proliferation, differentiation, genetic expression, cytoskeletal architecture, and motility in lymphocytes, monocytes, and other mammalian cells. In particular, a dramatic depression of the mitogenic in vitro activation of human peripheral blood lymphocytes was observed at low gravity. The hypothesis of the present work is that a reduced interaction between T lymphocytes and monocytes, essential for the second signalling pathway, might be one of the reasons for the observed depression of the in vitro activation of human lymphocytes. Cell motility and with it a continuous rearrangement of the cytoskeletal network within the cell is essential for cell-to-cell contacts. Whereas nonactivated lymphocytes in suspension are highly motile at low gravity, no data are available so far on the motility of adherent monocytes. It thus can be argued that impaired monocyte locomotion and cytoskeletal changes could be responsible for a reduced interaction of monocytes with T lymphocytes. In this study, the locomotion ability of J-111 cells, an adherent monocyte cell line, attached to colloidal gold particles on coverslips and exposed to modelled low gravity in the random positioning machine was found to be severely reduced compared with that of controls and the structures of actin, tubulin, and vinculin were affected. Correspondence and reprints: Space Biology Group, Swiss Federal Institute of Technology, Technopark, Technoparkstrasse 1, 8005 Zürich, Switzerland.     

B cell receptors and complement receptors target the antigen to distinct intracellular compartments

The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells. This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation.     

The truncated form of the Epstein-Barr virus latent-infection membrane protein expressed in virus replication does not transform rodent fibroblasts.

The gene encoding the Epstein-Barr virus membrane protein LMP, expressed in latent infection, is known to induce morphologic changes and some loss of contact inhibition in NIH 3T3 cells as well as profound loss of contact inhibition and of anchorage dependence in Rat-1 cells. Another form of LMP (D1LMP), deleted for the amino terminus and first four putative transmembrane domains of LMP, was recently shown to be expressed late in Epstein-Barr virus replication. We now demonstrate that D1LMP has no transformation-associated phenotypic effect in Rat-1 cells and does not significantly affect LMP-induced Rat-1 cell transformation. LMP activity and D1LMP inactivity in inducing anchorage-independent growth are not restricted to Rat-1 cells, but are also evident in BALB/c 3T3 cells. In both cell types, loss of contact inhibition and anchorage independence are acutely evident after LMP expression. Although newly transfected polyclonal Rat-1 or BALB/c cells have a lower agar cloning efficiency than established LMP-expressing clones, this is attributable, at least in part, to their lower average LMP expression, since among clones of transfected cells, higher cloning efficiencies correlated with higher levels of LMP. LMP is bound to the vimentin cytoskeletal network in rodent fibroblasts as it is in transformed lymphocytes, whereas D1LMP showed no detectable cytoskeletal binding, suggesting that cytoskeletal association may be integral to LMP-mediated cell transformation. LMP association with the cytoskeleton in latently infected, growth-transformed lymphocytes and LMP-transformed rodent fibroblasts, correlated with the lack of both rodent cell-transforming activity and cytoskeletal association of D1LMP supports working hypothesis that cytoskeletal association is important in LMP transforming activity.     

Age-related defects in moesin/ezrin cytoskeletal signals in mouse CD4 T cells

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.