A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.

Association between biomechanical alterations and migratory ability of semaphorin-3A-treated thymocytes.

BACKGROUND: Class 3 semaphorins are soluble proteins involved in cell adhesion and migration. Semaphorin-3A (Sema3A) was initially shown to be involved in neuronal guidance, and it has also been reported to be associated with immune disorders. Both Sema3A and its receptors are expressed by most immune cells, including monocytes, macrophages, and lymphocytes, and these proteins regulate cell function. Here, we studied the correlation between Sema3A-induced changes in biophysical parameters of thymocytes, and the subsequent repercussions on cell function. METHODS: Thymocytes from mice were treated in vitro with Sema3A for 30min. Scanning electron microscopy was performed to assess cell morphology. Atomic force microscopy was performed to further evaluate cell morphology, membrane roughness, and elasticity. Flow cytometry and/or fluorescence microscopy were performed to assess the F-actin cytoskeleton and ROCK2. Cell adhesion to a bovine serum albumin substrate and transwell migration assays were used to assess cell migration. RESULTS: Sema3A induced filopodia formation in thymocytes, increased membrane stiffness and roughness, and caused a cortical distribution of the cytoskeleton without changes in F-actin levels. Sema3A-treated thymocytes showed reduced substrate adhesion and migratory ability, without changes in cell viability. In addition, Sema3A was able to down-regulate ROCK2. CONCLUSIONS: Sema3A promotes cytoskeletal rearrangement, leading to membrane modifications, including increased stiffness and roughness. This effect in turn affects the adhesion and migration of thymocytes, possibly due to a reduction in ROCK2 expression. GENERAL SIGNIFICANCE: Sema3A treatment impairs thymocyte migration due to biomechanical alterations in cell membranes.

Crinophagy in Thyroid Follicular and Parafollicular Cells of Male Obese Zucker Rat.

Comparison between lean (Fa/?) and obese (fa/fa) young adult male Zucker rat thyroids reveals that obese rats display larger clusters of parafollicular cells than the lean ones with a lesser blood supply. Fa/? thyroid typically shows single or "twin" C cells in follicles; fa/fa parafollicular cells appear with three functional aspects. Crinophagy is found in the fa/fa C cells amassing numerous aberrant calcitonin-containing vesicles among which lysosomes build these autophagic bodies by capturing vesicle contents, other organelles and, fusing with each other, increase their size. Other C cells contain many secretory vesicles but show few or no crinophagic structures. Another parafollicular cell type is revealed with scant organelles and highly contrasted secretory vesicles, different from calcitonin. Hypercalcemia of fa/fa rats corresponds to increased C cells population with accrued calcitonin production but a low calcitonin plasma level - verified by others - is likely caused by crinophagy of the altered vesicles. In addition, the T thyrocytes of fa/fa rats exhibit crinophagy bodies; this can confirm their hypothyroidism. Possibly, the known leptin mutation along with other unknown paracrine secretions alter both T and C thyrocytes' functions of the fa/fa rats, allowing high intracellular calcium and lower pH favoring autophagocytosis. Other longitudinal, interdisciplinary studies should further clarify the complex paracrine interactions existing between these endocrine structures because this animal model could be useful to understand human defects, such as the metabolic syndrome that involves obesity, cardiovascular, renal, hepatic, non-insulin dependent diabetes mellitus (NIDDM), hypothyroidism defects, as well as the etiology of thyroid medullary tumors.

Mitochondrial dysfunction in ataxia-telangiectasia.

Ataxia-telangiectasia mutated (ATM) plays a central role in DNA damage responses, and its loss leads to development of T-cell malignancies. Here, we show that ATM loss also leads to intrinsic mitochondrial abnormalities in thymocytes, including elevated reactive oxygen species, increased aberrant mitochondria, high cellular respiratory capacity, and decreased mitophagy. A fraction of ATM protein is localized in mitochondria, and it is rapidly activated by mitochondrial dysfunction. Unexpectedly, allelic loss of the autophagy regulator Beclin-1 significantly delayed tumor development in ATM-null mice. This effect was not associated with rescue of DNA damage signaling but rather with a significant reversal of the mitochondrial abnormalities. These data support a model in which ATM plays direct roles in modulating mitochondrial homeostasis and suggest that mitochondrial dysfunction and associated increases in mitochondrial reactive oxygen species contribute to the cancer-prone phenotype observed in organisms lacking ATM. Thus, ataxia-telangiectasia should be considered, at least in part, as a mitochondrial disease.

Bioactivity of alginetin, a caramelization product of pectin: Cytometric analysis of rat thymic lymphocytes using fluorescent probes.

Alginetin is the major product formed from pentoses and hexurionic acids. Alginetin is producted by cooking process of food including pection, a naturally-occurring polysacharride found in many plants. However, the biological interaction and toxicity of alginetin are not known at all. The aim of the present study was to investigate the cellular actions of alginetin on rat thymic lymphocytes. The effects of alginetin on the cell were examined using flow cytometry with fluorescent probes. Alginetin increased cellular content of non-protein thiols ([NPT]i) and elevated intracellular Zn2+ levels ([Zn2+]i). Chelation of intracellular Zn2+ reduced the effect of alginetin on [NPT]i, and chelation of external Zn2+ almost completely diminished alginetin-induced elevation of [Zn2+]i, indicating that alginetin treatment increased Zn2+ influx. Increased [NPT]i and [Zn2+]i levels in response to alginetin were positively correlated. Alginetin protected cells against oxidative stress induced by hydrogen peroxide and Ca2+ overload by calcium ionophore. It is considered that the increases in [NPT]i and [Zn2+]i are responsible for the cytoprotective activity of alginetin because NPT attenuates oxidative stress and Zn2+ competes with Ca2+. Alginetin may be produced during manufacturing of jam, which may provide additional health benefits of jam.

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation.

Plasma membrane damage and cell death during processes such as necroptosis and apoptosis result from cues originating intracellularly. However, death caused by pore-forming agents, like bacterial toxins or complement, is due to direct external injury to the plasma membrane. To prevent death, the plasma membrane has an intrinsic repair ability. Here, we found that repair triggered by pore-forming agents involved TMEM16F, a calcium-activated lipid scramblase also mutated in Scott's syndrome. Upon pore formation and the subsequent influx of intracellular calcium, TMEM16F induced rapid "lipid scrambling" in the plasma membrane. This response was accompanied by membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice exhibited compromised control of infection by Listeria monocytogenes associated with a greater sensitivity of neutrophils to the pore-forming Listeria toxin listeriolysin O (LLO). Thus, the lipid scramblase TMEM16F is critical for plasma membrane repair after injury by pore-forming agents.

RORγt limits the amount of the cytokine receptor γc through the prosurvival factor Bcl-xL in developing thymocytes.

The cytokine receptor subunit γc provides critical signals for T cell survival and differentiation. We investigated the molecular mechanism that controls the cell surface abundance of γc during T cell development in the thymus. We found that the amount of γc was low on CD4+CD8+ double-positive (DP) thymocytes before their positive selection to become mature T cells. The transcription factor RORγt was abundant in immature DP thymocytes, and its loss resulted in an increase in the abundance of surface γc, specifically on preselection DP cells. Rather than directly repressing expression of the gene encoding γc, RORγt acted through the antiapoptotic protein Bcl-xL to reduce the abundance of surface γc, which resulted in decreased cytokine signaling and was associated with inhibition of cell metabolism and mitochondrial biogenesis. Accordingly, overexpression of Bcl-xL in RORγt-deficient thymocytes restored the amount of surface γc to that present on normal preselection DP cells. Together, these data highlight a previously unappreciated role for RORγt and Bcl-xL in limiting γc abundance at the cell surface and reveal a signaling circuit in which survival factors control cytokine signaling by limiting the abundance and surface distribution of a receptor subunit shared by several cytokines.

Thymic Nurse Cells Participate in Heterotypic Internalization and Repertoire Selection of Immature Thymocytes; Their Removal from the Thymus of Autoimmune Animals May be Important to Disease Etiology.

Thymic nurse cells (TNCs) are specialized epithelial cells that reside in the thymic cortex. The initial report of their discovery in 1980 showed TNCs to contain up to 200 thymocytes within specialized vacuoles in their cytoplasm. Much has been reported since that time to determine the function of this heterotypic internalization event that exists between TNCs and developing thymocytes. In this review, we discuss the literature reported that describes the internalization event and the role TNCs play during T cell development in the thymus as well as why these multicellular complexes may be important in inhibiting the development of autoimmune diseases.

Elemental mapping by electron energy loss spectroscopy in biology.

Over the past decades there have been significant advances in transmission electron microscopy for biological applications, including in energy filtering and spectrum imaging, which are techniques based on the principles of electron energy loss spectroscopy. These imaging modalities allow quantitative mapping of specific chemical elements with high sensitivity and spatial resolution. This chapter describes the experimental and computational procedures for elemental mapping in two dimensions as well as a more recent extension to three dimensions, which can reveal quantitative distributions of elements in cells on a macromolecular scale.

Accumulation of tissue macrophages and depletion of resident macrophages in the diabetic thymus in response to hyperglycemia-induced thymocyte apoptosis.

AIMS: We investigated the dynamics and morphology of thymus macrophages in response to thymus involution caused by hyperglycemia. Thymus is an organ affected early and dramatically after the onset of diabetes, losing most of the thymocyte populations but diabetes's impact on the components of the thymus stroma is largely unknown. METHODS: Rats were injected with streptozotocin and thymus weight, body weight, and glycemia were measured at various time points. The dynamics and morphology of macrophages in the diabetic thymus were investigated by histology, immunohistochemistry, qPCR, electron microscopy and flow cytometry. RESULTS: In hyperglycemic animals the involuting thymus is gradually infiltrated by tissue macrophages (ED1-positive) and depleted of resident macrophages (ED2-positive). While ED1 positive macrophages are scattered in both cortex and medulla the ED2 positive ones are limited to the cortex and cortico-medullary junction. CD4+CD11b+macrophages also accumulate. The TUNEL reaction that detects the degradation of the DNA from apoptotic thymocytes in the macrophages is enhanced. The thymic macrophages enlarge and accumulate lipid vacuoles and apoptotic bodies. qPCR measurements of the expression of macrophage markers showed a persistent increase in the diabetic thymus after the injection of streptozotocin. CONCLUSIONS: Thymus involutes rapidly and persistently after the onset of hyperglycemia because of the elevated apoptosis in the thymocytes. Tissue macrophages accumulate in the thymus and the resident macrophages decrease. This results in an overall increase in macrophage activity in the diabetic thymus in response to the elevated apoptosis of thymocytes produced by hyperglycemia.

Proteomic characterization of thymocyte-derived microvesicles and apoptotic bodies in BALB/c mice.

Several studies have characterized exosomes derived from different cell sources. In this work we set the goal of proteomic characterization of two less studied populations of membrane vesicles, microvesicles (100-800 nm) and apoptotic bodies (> 800 nm) released by thymus cells of BALB/c mice. The vesicles were isolated by the combination of differential centrifugation and gravity driven multistep filtration of the supernatant of thymus cell cultures. The size distribution of vesicle preparations was determined by transmission electron microscopy. Proteins were released from the vesicles, digested in solution, and analyzed using nano-HPLC/MS(MS). Ingenuity pathway analysis was used to identify functions related to membrane vesicle proteins. In apoptotic bodies and microvesicles we have identified 142 and 195 proteins, respectively. A striking overlap was detected between the proteomic compositions of the two subcellular structures as 108 proteins were detected in both preparations. Identified proteins included autoantigens implicated in human autoimmune diseases, key regulators of T-cell activation, molecules involved in known immune functions or in leukocyte rolling and transendothelial transmigration. The presence and abundance of proteins with high immunological relevance within thymocyte-derived apoptotic bodies and microvesicles raise the possibility that these subcellular structures may substantially modulate T-cell maturation processes within the thymus.