Lymphocyte Membrane Research Articles

Horse IgG- and ostrich IgY-F(ab′)2 groups have different affinities for mice erythrocytes and lymphocytes. Implications for avian immunoglobulin therapeutic usefulness

We used high sensitivity and resolution fluorescence microscopy to study the interaction of ostrich IgY, horse F(ab′)2 and horse IgG with mice lymphocyte and erythrocyte plasma membrane. The immunoglobulins were labeled with fluorescein isotiocyanate (FITC). Our results show an interaction of IgY with lymphocyte plasma membrane which does not result in endocytosis of the labeled protein. Less IgG and its F(ab′)2 fraction bind to lymphocytes, and this binding seems to be followed by endocytosis producing a diffuse cytoplasmic fluorescence in most lymphocytes exposed to FITC-IgG or FITC-F(ab′)2. Cytoplasmic fluorescence resembling FITC was not observed in lymphocytes exposed to FITC-IgY. Receptors in the erythrocyte membrane also differentiate between avian and horse Ig; while erythrocytes exposed to horse Igs became intensely fluorescent for at least 5 h, no erythrocyte labeling occurred when FITC-IgY was used. Our results suggest that IgY may be a stronger activator of adaptive immunity than horse IgG in mammals. Adaptive immunity against IgY is detrimental to its IV therapeutic use in humans and other mammals.

Progesterone effects on lymphocytes may be mediated by membrane progesterone receptors

Luteal cell-induced proliferation of T lymphocytes devoid of the nuclear progesterone receptor (PGR) is inhibited by progesterone. Functional effects of progesterone on bovine lymphocytes and the expression of membrane progesterone receptors (mPRs) alpha (PAQR7), beta (PAQR8), gamma (PAQR5), and progesterone receptor membrane component 1 (PGRMC1) mRNA were analyzed in corpus luteum (CL) and lymphocytes. Progesterone and a cell-impermeable progesterone conjugate caused a dose-dependent decrease in IL2 receptor α-subunit (IL2RA) mRNA and an increase in interleukin 2 (IL2) mRNA concentrations in cultured PBMCs. In luteal tissues, concentrations of PAQR7 and PAQR8 mRNA were lower in CL collected on day 11 compared with day 18, whereas PGRMC1 and PGR mRNA were greater on day 11 than on day 18. The mRNA of all three PAQRs and PGRMC1 were detected in bovine T lymphocytes, but not in B cells/monocytes. Progesterone increased intracellular Ca++ and reduced the phosphorylation of zeta-chain-associated protein kinase 70 (Zap70). A specific, saturable, and single progesterone binding site with a steroid specificity characteristic of mPRs was demonstrated by saturation and competitive binding assays using T lymphocyte membranes, and PAQR7 receptors were localized on the plasma membranes by immunofluorescence. Thus, progesterone induces specific and rapid functional effects on T lymphocytes in the absence of PGR. The mPRs are potential intermediaries of the cell-surface actions of progesterone because they are expressed in lymphocytes, the actions of progesterone are mimicked by a cell-impermeable form of progesterone, and specific, saturable progesterone binding, which is characteristic of mPRs, is present on lymphocyte membranes.

Receptor-Independent Interaction of Bacterial Lipopolysaccharide with Lipid and Lymphocyte Membranes; the Role of Cholesterol

Lipopolysaccharide (LPS) is a major constituent of bacterial outer membranes where it makes up the bulk of the outer leaflet and plays a key role as determinant of bacterial interactions with the host. Membrane-free LPS is known to activate T-lymphocytes through interactions with Toll-like receptor 4 via multiprotein complexes. In the present study, we investigate the role of cholesterol and membrane heterogeneities as facilitators of receptor-independent LPS binding and insertion, which underpin bacterial interactions with the host in symbiosis, pathogenesis and cell invasion. We use fluorescence spectroscopy to investigate the interactions of membrane-free LPS from intestinal Gram-negative organisms with cholesterol-containing model membranes and with T-lymphocytes. LPS preparations from Klebsiella pneumoniae and Salmonella enterica were found to bind preferentially to mixed lipid membranes by comparison to pure PC bilayers. The same was observed for LPS from the symbiote Escherichia coli but with an order of magnitude higher dissociation constant. Insertion of LPS into model membranes confirmed the preference for sphimgomyelin/cholesterol-containing systems. LPS insertion into Jurkat T-lymphocyte membranes reveals that they have a significantly greater LPS-binding capacity by comparison to methyl-β-cyclodextrin cholesterol-depleted lymphocyte membranes, albeit at slightly lower binding rates.

ROS induction and structural modification in human lymphocyte membrane under the influence of carbon nanotubes

To assess the potential risks of using the artificial nanostructures the structural state of the human lymphocyte membrane and lipid peroxidation under the influence of multi-walled carbon nanotubes with metal impurities was studied. The ability of carbon nanotubes to induce the formation of reactive oxygen species in cells was examined. A dose-dependent increase in reactive oxygen species formation in lymphocytes, which was not registered in cells pre-incubated with N-acetylcystein, after exposure to carbon nanotubes was shown. The addition of iron chelator deferoxamine to carbon nanotubes has also resulted in a decrease of reactive oxygen species. The mechanism of the activation of lipid peroxidation under the influence of carbon nanotubes and a structural modification of human lymphocyte membranes were discussed.

MGluR1 interacts with cystic fibrosis transmembrane conductance regulator and modulates the secretion of IL-10 in cystic fibrosis peripheral lymphocytes

Cystic fibrosis (CF) is caused by the mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. CFTR dysfunction in T cells could lead directly to aberrant immune responses. The action of glutamate on the secretion of IL-8 and IL-10 by lymphocytes derived from healthy subjects and cystic CF patients, as well as the expression of metabotropic glutamate receptor subtype 1 (mGluR1) in the membrane fractions of lymphocytes was investigated. Our results have shown that CF-derived T-cells in the presence of IL-2 produce more IL-8 and IL-10, than T-cell from healthy control. However, only in normal lymphocytes a significant increase (144%) in the IL-10 secretion during exposure to high concentration of glutamate (10−4M) was detected. Glutamate-dependent secretion of IL-10 was not inhibited either by NMDA-receptor (NMDAR), or by AMPA-receptor (AMPAR) antagonist. Only mGluR1 antagonist, LY367385, strongly decreases the production of IL-10. Furthermore, the content of mGluR1, as well as cystic fibrosis transmembrane conductance regulator-associated ligand (CAL), Na+/H+ exchanger regulatory factor 1 (NHERF-1), was analyzed in plasma membrane of lymphocytes after immunoprecipitation of CFTR. We have found that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with metabotropic mGluR1, but the level of surface exposed mGluR1 in CF-lymphocytes was much lower than in normal cells. Besides, our results have shown that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with NHERF-1 and CAL; however in lymphocytes with CFTR mutation the amount of cell-surface expressed CFTR-CAL complex was greatly decreased. We have concluded that CFTR and mGluR1 could compete for binding to CAL, which in turn downregulates the post-synthetic trafficking of mGluR1 and decreases the synthesis of IL-10.

Effects of chronic kidney disease on blood cells membrane properties

Chronic kidney disease (CKD) is progressive loss of renal function associated among others with increased intracellular calcium concentration. The purpose of this study was to identify the effects of CKD on cell membrane properties such as human red blood cell Ca2+ ATPase activity, lymphocyte plasma membrane P2X7 receptor expression and function. This could help us in elucidating the origin of increased calcium concentration in blood cells. We found out Ca2+ ATPase activity is decreased in early stage CKD patients resulting in altered calcium removal from cytoplasm. By means of flow cytometry we assessed that P2X7 receptor expression on lymphocyte membrane is 1.5 fold increased for CKD patients. Moreover, we detected an increased uptake of ethidium bromide through this receptor in CKD at basal conditions. It means CKD lymphocyte membranes contain more receptors which are more permeable thus allowing increased calcium influx from extracellular milieu. Finally, we can state alterations in blood cell membranes are closely linked to CKD and may be responsible for intracellular calcium accumulation.

Constitutively active ezrin increases membrane tension, slows migration, and impedes endothelial transmigration of lymphocytes in vivo in mice

ERM (ezrin, radixin moesin) proteins in lymphocytes link cortical actin to plasma membrane, which is regulated in part by ERM protein phosphorylation. To assess whether phosphorylation of ERM proteins regulates lymphocyte migration and membrane tension, we generated transgenic mice whose T-lymphocytes express low levels of ezrin phosphomimetic protein (T567E). In these mice, T-cell number in lymph nodes was reduced by 27%. Lymphocyte migration rate in vitro and in vivo in lymph nodes decreased by 18% to 47%. Lymphocyte membrane tension increased by 71%. Investigations of other possible underlying mechanisms revealed impaired chemokine-induced shape change/lamellipod extension and increased integrin-mediated adhesion. Notably, lymphocyte homing to lymph nodes was decreased by 30%. Unlike most described homing defects, there was not impaired rolling or sticking to lymph node vascular endothelium but rather decreased migration across that endothelium. Moreover, decreased numbers of transgenic T cells in efferent lymph suggested defective egress. These studies confirm the critical role of ERM dephosphorylation in regulating lymphocyte migration and transmigration. Of particular note, they identify phospho-ERM as the first described regulator of lymphocyte membrane tension, whose increase probably contributes to the multiple defects observed in the ezrin T567E transgenic mice.

Role of Membrane Oxidation in Controlling the Activity of Secretory Phospholipase A2 Toward Apoptotic Lymphoma Cells

The membranes of healthy lymphocytes normally resist hydrolysis by extracellular phospholipase A2 (sPLA2). However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIA sPLA2 isozyme (hGIIa). This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential, BODIPY, suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by glucocorticoid or thapsigargin. When oxidative potential was high, the activity of hGIIa sPLA2 was enhanced more than 10-fold compared to any other condition favorable to hydrolysis by other sPLA2 isoforms. Moreover, confocal microscopy with BODIPY and the vital stain propidium iodide verified that sPLA2 preferentially attacked oxidized cells. Direct oxidation of cell membranes with either of two oxidizing agents (TBHP and AAPH) also stimulated hydrolysis by sPLA2. Both agents induced externalization of phosphatidylserine, although only TBHP caused a reduction in the apparent order of membrane lipids. These results demonstrated that membrane oxidation strongly stimulates sPLA2 activity, especially that of the hGIIa isoform. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted.

Dynamics of lymphocyte membrane in Chernobyl clean-up workers with type 2 diabetes mellitus

Objective: Diabetes mellitus is one of the most crippling diseases that man has seen and its prevalence has risen dramatically over the past two decades. Currently there are over the 150 million diabetics worldwide and this number is likely to increase to 300 million or more by the year 2025. Diabetes mellitus increases the risk of many disorders including cardiovascular diseases. Understanding the molecular properties of diabetic progression is a big challenge in systems-biology era. Methods: The 3-aminobenzanthrone derivative ABM, developed at the Daugavpils University, Latvia, has been previously shown as a potential biomarker for determination of the immune state of patients with different pathologies. The aim of this study was to determine the several aspects of membrane alterations in the group of Chernobyl clean-up workers with diabetes mellitus in relation with its common group without diabetes mellitus and humans having no professional contact with radioactivity. The following parameters were examined: (1) the spectral characteristics of ABM in cell suspension (e.g. anisotropy index); (2) quantitative parameters of protein/lipid interaction in liposomes prepared from phosphatidylcholine and its mixtures with cardiolipin and cholesterol. Results: Screening of the individuals with diabetes mellitus 25-26 years after the work in Chernobyl revealed two groups of patients differing in structural and functional membrane properties, first of all on the lipid/protein interrelations and conformations of membrane proteins. The revealed structural modifications of membranes are dependent on radiation-induced factors. Concomitant diseases (diabetes mellitus, cardiovascular diseases) reinforce radiation induced effects. Conclusion: ABM is a sensitive probe of membrane architecture alterations, and can be used to elucidate the changes in membrane systems. Significant differences in membrane dynamics exist between control (donors), and diabetics and non-diabetics groups of Chernobyl clean-up workers

Fluorescent biomarker ABM: properties and estimation of immune state of patients with different pathologies

Patients with cancer (namely advanced cancer) exhibit poorly functionating immune system. It is now widely accepted that dynamics of changes, along the certain types of alterations in structures of lipid/protein themselves of immune system cells and blood plasma, plays a critical role in the maintenance of the immune status of organism. Biomarkers for prediction of disease outcome are of great interest in human medicine. The fluorescent probe ABM was used to characterize lymphocyte membranes and blood plasma albumin of cancer patients suffering from advanced cancer with wide metastasis and intoxication. The aim of these studies was to evaluate the potential applicability of ABM fluorescence measures as a standard tool in the analyses of host immune status and for a clinical interpretation of alterations in albumin per se and in lymphocyte functional activity in cancer patients receiving palliative care. We registered probe ABM spectral parameters in patients lymphocytes, blood plasma, and ABM auto-fluorescence in plasma. The fluorescence intensity of ABM in blood plasma and lymphocyte suspension differed from control values and showed specific differences in patient groups in accordance (correlation) with survival rate. A significant decrease in ABM fluorescence in plasma could be explained, in part, by a diminished binding capacity of the albumin of these patients. The lymphocyte distribution among the subsets of patients also differed. Interestingly, the ABM fluorescence in the cell suspension and blood plasma was also found to correlate with select immunological parameters (CD4+/CD8+ ratios, lymphocyte counts, etc). Results strongly correlated with changes in ABM spectral characteristics and both clinical and pathological estimates of patient immune state. ABM spectroscopy appears to be useful for clinicians to monitor the course of certain diseases (e.g. gastrointestinal cancers).

Rat lymphocytes express NMDA receptors that take part in regulation of cytokine production

Incubation of rat lymphocytes with homocysteine (HC) or homocysteic acid (HCA) was found to increase the stationary levels of free radicals in lymphocytes, the effect of both ligands being mediated by ionotropic receptors activated by N-methyl-D-aspactic acid (NMDA), the expression of which on rat lymphocyte membranes was earlier demonstrated. In agreement with these data, increase of free radicals in the lymphocyte cytoplasm is preceded by an increase in the intracellular calcium levels, activation of protein kinase C, nicotinamide adenine dinucleotide phosphate oxidase and/or nitric oxide synthase. Both HC and HCA increase the production of IFN-γ and TNF-α by lymphocytes and antagonist of NMDA receptors; MK-801 prevents this effect. The data presented show that rat lymphocyte membrane contains functionally active NMDA receptors, which regulate cytokine accumulation.

HMGB1 and Cord Blood: Its Role as Immuno-Adjuvant Factor in Innate Immunity

In newborn the innate immune system provides essential protection during primary infections before the generation of an appropriate adaptive immune response that is initially not fully operative. Innate immune response is evoked and perpetuated by molecules derived from microorganisms or by the damage/death of host cells. These are collectively known as damage-associated molecular-pattern (DAMP) molecules. High-mobility group box 1 protein (HMGB1) or amphoterin, which previously was considered to be only a nuclear factor, has been recently identified as a DAMP molecule. When it is actively secreted by inflammatory cells or passively released from necrotic cells, HMGB1 mediates the response to infection, injury and inflammation, inducing dendritic cells maturation and T helper-1-cell responses. To characterize the role of HMGB1 in the innate and immature defense mechanisms in newborns, human cord blood (CB) mononuclear cells, in comparison to adult peripheral blood (PB) mononuclear cells, have been analyzed for its expression. By flow cytometry and western blot analysis, we observed that in CB and PB cells: i) HMGB1 is expressed on cell surface membranes of myeloid dendritic cell precursors, mostly, and lymphocytes (gamma/delta and CD4+ T cells) to a lesser extent; ii) different pro-inflammatory stimuli or molecules that mimic infection increased cell surface expression of HMGB1 as well as its secretion into extracellular environment; iii) the treatment with synthetic molecules such as aminobisphosphonates (ABs), identified to be γδ T cell antigens, triggered up-regulation of HMGB1 expression on mononuclear cells, as well γδ T lymphocytes, inducing its secretion. The modulation of its secretion and the HMGB1-mediated migration of monocytes indicated HMGB1 as regulator of immune response in an immature system, like CB, through engagement of γδ T lymphocytes and myeloid dendritic cell precursors, essential components of innate immunity. In addition, the increased HMGB1 expression/secretion triggered by ABs, previously characterized for their immuno-modulating and immune-adjuvant capabilities, indicated that immunomodulation might represent a new therapeutical approach for neonatal and adult pathologies.

AFM detection of mitogen‐induced morphological changes in Human B lymphocyte

B-lymphocyte activation plays an important role in humoral immune system, and its process has been studied well in vivo and in vitro. However, the ultrastructure and adhesion property changes remain unclear. In this study, changes in the morphology and mechanical properties of human peripheral blood B lymphocytes were first studied by atomic force microscopy (AFM). B lymphocytes were treated with the mitogen, pokeweed mitogen (PWM), and Staphylococcus aureus Cowan strain I (SAC) for 24 hr. After B lymphocyte is stimulated by the mitogen, the cell height, diameter, and volume are changed in different degree. The ultrastructure of the B lymphocytes membrane obviously displayed proteins gathering, corresponding with larger changes of average roughness and mean height of particles on cell membrane. Meanwhile, we detected the adhesion force of B lymphocytes after being stimulated by PWM and SAC. We found that the treated cells had a higher adhesion force of 304.16 ± 60.30 pN (PWM) and 249.63 ± 58.03 pN (SAC) than that of control group (104.28 ± 21.77 pN). Therefore, our results could provide new information to further understand the B-lymphocyte activation process and their structure-function analyses.

Higher Serum DPP-4 Enzyme Activity and Decreased Lymphocyte CD26 Expression in Type 1 Diabetes

Dipeptidyl peptidase-4 (DPP-4) is involved in the metabolism of peptide hormones, T-cell activation and proliferation. In type 1 diabetes mellitus (T1DM) β-cell destruction involves a number of dysregulated T-cells. Our aim was to assess the serum DPP-4 activity and the lymphocyte membrane bound CD26 expression in patients with type 1 diabetes and healthy controls. Ninety-eight (T1DM: 48, F/M = 20/28, mean age: 34.4y; control: 50, F/M = 39/11 mean age: 32,4y) individuals were included. Fasting serum DPP-4 enzymatic activity, plasma glucose (FPG), CD26 expression on CD3+, CD4+ and CD8+ lymphocytes, HbA1c and body mass index (BMI) were assessed. ICA and GAD antibodies were assessed in the T1DM group. DPP-4 enzymatic activity was determined by kinetic enzyme assay, ICA and GAD were assessed by ELISA. Determination of the CD26 expression on CD3+, CD4+ and CD8+ lymphocytes was performed by flow-cytometric analysis. We found higher serum DPP-4 activity (Mean: T1DM: 30.069U/L, control: 22.62U/L, p < 0.0001) and decreased CD26 lymphocyte expression on all lymphocyte subpopulations in T1DM. Fasting serum DPP-4 activity was independent from the ICA or GAD status of patients with T1DM. Here we first present that the serum DPP-4 activity is increased and the lymphocyte membrane bound CD26 expression is decreased in type 1 diabetes. Decreased lymphocyte membrane bound CD26 expression in T1DM might be a novel part of the T-lymphocyte regulatory dysfunction observed in type 1 diabetes mellitus. These results might provide some basis for the clinical implication of DPP-4 inhibition in patients with T1DM.

Study of zinc-induced changes in lymphocyte membranes using atomic force microscopy, luminescence, and light scattering methods

Changes in the surface structure of lymphocyte membranes exposed to various concentrations of zinc ions are studied. It is found by atomic force microscopy that increasing the concentration of zinc ions leads to a reduction in the correlation length of the autocorrelation function of the roughness profile of a lymphocyte compared to control samples; this may indicate the existence of fine structure in the membrane surface. Fluorescence markers are used to observe a reduction in the microviscosity of the lipids in the outer monolayer of the lipid bilayer after lymphocytes are exposed to Zn ions, as well as the exposure of phosphatidylserine on the surface membrane, and the oxidation of HS-groups of membrane proteins. Calculations of the absorption coefficients of lymphocytes modified with zinc reveal the existence of absorption bands owing to the formation of metal-protein complexes and zinc oxide nanoparticles. These results indicate significant changes in the structural and functional state of lymphocyte membranes exposed to zinc ions.

Effect of cholesterol on the functional activity of proteins responsible for the resistance of human lymphocytes to xenobiotics

The influence of agents modifying cholesterol in plasma membranes on the functional activity of transporter proteins (P-glycoprotein (P-gp) and the multidrug resistance protein 1 (MRP1)) in human lymphocytes has been studied. It was shown that changes in lateral distribution of cholesterol using the polyene antibiotic filipin, which disturb the structure and function of glycolipid microdomains in plasma membranes of lymphocytes lead to a decrease in the transport activity of both P-gp and MRP1. It was found that the treatment of human lymphocytes with the cyclic oligosaccharide methyl-beta-cyclodextrine, which leads to cholesterol depletion and reduction of lipid bilayer microviscosity in membranes of these cells, also decreases the functional activity of these proteins. It was concluded that the transport activity of P-gp and MRP1 depends on the modification of cholesterol in the membranes of human lymphocytes, i.e., is closely associated with the level of cholesterol and its lateral distribution.

CD38 expression as response of hematopoietic system to cancer

Erythrocyte and lymphocyte NAD(+) glycohydrolase levels were previously found to be elevated in cancer patients. These results were confirmed in an animal model. The administration of live Ehrlich ascites tumor cells to BALB/c mice led to increases in erythrocyte and lymphocyte NAD(+) glycohydrolase, along with tumor development. Serum samples, ascites fluid from mice with developed tumors, serum samples from cancer patients and Ehrlich cell supernatants had a similar stimulatory effect when administered to mice or when incubated with peripheric lymphocytes in culture. These increases were accompanied by the appearance of an anti-CD38 reactive band of 45 kDa in SDS-PAGE/Western blot analyses of erythrocyte ghost and lymphocyte membrane proteins. The results, supported by flow cytometry data, support previous clinical findings that an enhancement in CD38 expression occurs in the hematopoietic system during proliferative processes. Moreover, they suggest that CD38 expression is triggered at least in part by a certain cytokine(s) secreted by cancer cells. Finally, the results emphasize the prospective use of CD38 expression as a marker of tumor development and progression.

Effects of Cisplatin on Lymphocyte Structure and Functions in Mice with Ehrlich Ascitic Carcinoma

The effects of cisplatin on [Ca2+]in, pHin, NAD(P)H level, and lymphocyte membrane microviscosity were studied in mice with Ehrlich ascitic carcinoma. The level of free [Ca2+]in in lymphocytes from mice with Ehrlich ascitic carcinoma was 4-fold reduced compared to that in intact animals on day 13 of tumor development, while [H+]in level was elevated. Cisplatin caused no changes in the level of free Ca2+, but reduced cytosol acidification. Lymphocyte membrane fluidity in mice with tumors was increased in the lipid bilayer and in the protein-lipid contact zone and did not depend on cisplatin treatment. The level of NAD(P)H was low in mice with tumors, but increased sharply after cisplatin treatment. It seems that functional activity of lymphocytes decreased at the stage of well-developed tumor, which promoted inhibition of the lymphocyte defense properties. Cisplatin did not modify the structure and functions of lymphocytes and presumably even improved their energy status.

Abstract 1678: Visualization of thymidine kinase 1 on the surface of cancer cell lines: A potential diagnostic marker and therapeutic target

Abstract Thymidine Kinase 1(TK1) is a salvage pathway enzyme that has been shown to be elevated in many different cancer types, including breast, prostate, and colon. Previous research in our lab using plasma membrane separation has shown an increase in TK1 levels associated with the plasma membrane of cancer cells when compared to normal lymphocytes. After membrane separation the plasma membrane of normal lymphocytes showed a TK1 activity of 1,012 CPM/mg protein/hr(N=20) whereas those of cancer cells showed: Jurkat 34,096(N=28), Raji 70,195(N=20), HL60 46,217(N=34), and MDA-MB-435 50,359(N=24). In this study we sought to further investigate this association by visualizing the surface staining of fluorescently tagged antibodies to TK1. Using a primary rabbit anti-TK1 antibody and a secondary goat anti-rabbit antibody conjugated with a FITC fluorochrome we were able to stain the cells and identify TK1 bound to the cell surface. Using a fluorescent microscope we were able to visualize the fluorescent staining of TK1 in various cancer cell lines, including: Raji, Jurkat, HL60, MDA-MB-435, H358, and MCF-7. We compared these results to our control of stained normal lymphocytes, which showed significantly greater levels of staining in cancer cells. Our results further confirm the presence of membrane associated TK1, and provide further evidence of TK1's usefulness as a diagnostic marker and potential therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1678. doi:10.1158/1538-7445.AM2011-1678

Myosin 1c participates in B cell cytoskeleton rearrangements, is recruited to the immunological synapse and contributes to antigen presentation. (100.48)

Abstract Myosin 1c (Myo1c) is a member of the unconventional class I myosins of vertebrates, which directly link the plasma membrane with the microfilament cortical web. Although this molecular motor has been implicated in cell functions such as cytoskeleton organization, cell motility, nuclear transcription and endocytosis; its role in the hematopoietic cells is largely unknown. We found that Myo1c is abundantly expressed in murine B lymphocytes and is preferentially located at the plasma membrane, especially in peripheral processes such as microvilli. It was observed not only that this motor concentrates at the growing membrane protrusions generated during B cell spreading, but also that is actively recruited to the immune synapse. Interestingly, Myo1c was detected in the lipid rafts of B cells and showed a strong colocalization with MHC-II, particularly after crosslinking of these molecules. By transfecting a dominant negative form of Myo1c, we also detected alterations in spreading and antigen presentation ability of these cells. All these data suggest that Myo1c could be involved in the B lymphocyte cytoskeleton dynamics and membrane protein anchoring and/or sorting, during cell-cell contacts, including those related with CD4+ T cell activation.