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By admin, on May 9th, 2012
Int J Biochem Cell Biol. 2012 May 4;
Hoozemans JJ, Scheper W
The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signalling pathways involved in protein quality control in the ER and tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.
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ENDOPLASMIC RETICULUM: The Unfolded Protein Response is Tangled in Neurodegeneration.
By admin, on May 9th, 2012
PLoS One. 2012; 7(4): e35204
Ben-Dov N, Korenstein R
The different pathways of endocytosis share an initial step involving local inward curvature of the cell's lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH≤3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell's surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake.
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Enhancement of cell membrane invaginations, vesiculation and uptake of macromolecules by protonation of the cell surface.
By admin, on May 9th, 2012
PLoS One. 2012; 7(4): e35204
Ben-Dov N, Korenstein R
The different pathways of endocytosis share an initial step involving local inward curvature of the cell's lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH≤3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell's surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake.
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Enhancement of cell membrane invaginations, vesiculation and uptake of macromolecules by protonation of the cell surface.
By admin, on May 4th, 2012
Cold Spring Harb Perspect Biol. 2012 May 2;
McCaffrey LM, Macara IG
A key function of signal transduction during cell polarization is the creation of spatially segregated regions of the cell cortex that possess different lipid and protein compositions and have distinct functions. Polarity can be initiated spontaneously or in response to signaling inputs from adjacent cells or soluble factors and is stabilized by positive-feedback loops. A conserved group of proteins, the Par proteins, plays a central role in polarity establishment and maintenance in many contexts. These proteins generate and maintain their distinct locations in cells by actively excluding one another from specific regions of the plasma membrane. The Par signaling pathway intersects with multiple other pathways that control cell growth, death, and organization.
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Signaling Pathways in Cell Polarity.
By admin, on April 28th, 2012
PLoS One. 2012; 7(4): e35892
Ye S, Tan L, Yang R, Fang B, Qu S, Schulze EN, Song H, Ying Q, Li P
Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains.We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice.Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.
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Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains.
By admin, on April 28th, 2012
PLoS One. 2012; 7(4): e35892
Ye S, Tan L, Yang R, Fang B, Qu S, Schulze EN, Song H, Ying Q, Li P
Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains.We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice.Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.
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Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains.
By admin, on April 25th, 2012
Channels (Austin). 2012 Jan 1; 6(1):
Fu LY, Cummins TR, Moczydlowski EG
Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K+ current, a phenomenon known as inward rectification characteristic of a major class of KIR K+ channels. We previously described block of heterologously expressed voltage-gated Na+ channels (NaV) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal NaV channels. In this study, we compared the sensitivity of four different cloned mammalian NaV isoforms to PAs to investigate whether PA block is a common feature of NaV channel pharmacology. We find that outward Na+ current of muscle (NaV1.4), heart (NaV1.5), and neuronal (NaV1.2, NaV1.7) NaV isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac NaV1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the NaV1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term NaV channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.
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Sensitivity of cloned muscle, heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo.
By admin, on April 25th, 2012
Channels (Austin). 2012 Jan 1; 6(1):
Fu LY, Cummins TR, Moczydlowski EG
Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K+ current, a phenomenon known as inward rectification characteristic of a major class of KIR K+ channels. We previously described block of heterologously expressed voltage-gated Na+ channels (NaV) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal NaV channels. In this study, we compared the sensitivity of four different cloned mammalian NaV isoforms to PAs to investigate whether PA block is a common feature of NaV channel pharmacology. We find that outward Na+ current of muscle (NaV1.4), heart (NaV1.5), and neuronal (NaV1.2, NaV1.7) NaV isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac NaV1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the NaV1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term NaV channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.
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Sensitivity of cloned muscle, heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo.
By admin, on April 21st, 2012
Plant Cell . 2012 Apr 18; Lung SC, Chuong SD Although Toc159 is known to be one of the key GTPase receptors for selective recognition of chloroplast preproteins, the mechanism for its targeting to the chloroplast surface remains unclear. To compare the targeting of these GTPase receptors, we identified two Toc159 isoforms and a Toc34 from Bienertia sinuspersici, a single-cell C(4) species with dimorphic chloroplasts in individual chlorenchyma cells.
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A Transit Peptide-Like Sorting Signal at the C Terminus Directs the Bienertia sinuspersici Preprotein Receptor Toc159 to the Chloroplast Outer Membrane.
By admin, on April 13th, 2012
PLoS One. 2012; 7(4): e35048
Narayan SB, Master SR, Sireci AN, Bierl C, Stanley PE, Li C, Stanley CA, Bennett MJ
Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein.
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Short-chain 3-hydroxyacyl-coenzyme a dehydrogenase associates with a protein super-complex integrating multiple metabolic pathways.
By admin, on April 13th, 2012
BMC Genomics. 2012 Apr 11; 13(1): 134
Palgi M, Greco D, Lindstrom R, Auvinen P, Heino TI
ABSTRACT: BACKGROUND: MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by endoplasmic reticulum (ER) stress and unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. RESULTS: The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the ER stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. CONCLUSIONS: We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome ER stress especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons.
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Gene expression analysis of Drosophila Manf mutants reveals perturbations in membrane traffic and major metabolic changes.
By admin, on April 13th, 2012
BMC Genomics. 2012 Apr 11; 13(1): 134
Palgi M, Greco D, Lindstrom R, Auvinen P, Heino TI
ABSTRACT: BACKGROUND: MANF and CDNF are evolutionarily conserved neurotrophic factors that specifically support dopaminergic neurons. To date, the receptors and signalling pathways of this novel MANF/CDNF family have remained unknown. Independent studies have showed upregulation of MANF by endoplasmic reticulum (ER) stress and unfolded protein response (UPR). To enlighten the role of MANF in multicellular organism development we carried out a microarray-based analysis of the transcriptional changes induced by the loss and overexpression of Drosophila Manf. RESULTS: The most dramatic change of expression was observed with genes coding membrane transport proteins and genes related to metabolism. When evaluating in parallel the ultrastructural data and transcriptome changes of maternal/zygotic and only zygotic Manf mutants, the ER stress and membrane traffic alterations were evident. In Drosophila Manf mutants the expression of several genes involved in Parkinson's disease (PD) was altered as well. CONCLUSIONS: We conclude that besides a neurotrophic factor, Manf is an important cellular survival factor needed to overcome ER stress especially in tissues with high secretory function. In the absence of Manf, the expression of genes involved in membrane transport, particularly exocytosis and endosomal recycling pathway was altered. In neurodegenerative diseases, such as PD, correct protein folding and proteasome function as well as neurotransmitter synthesis and uptake are crucial for the survival of neurons. The degeneration of dopaminergic neurons is the hallmark for PD and our work provides a clue on the mechanisms by which the novel neurotrophic factor MANF protects these neurons.
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Gene expression analysis of Drosophila Manf mutants reveals perturbations in membrane traffic and major metabolic changes.
By admin, on April 7th, 2012
Commun Integr Biol. 2012 Jan 1; 5(1): 88-93
Verweij FJ, Middeldorp JM, Pegtel DM
Tight control of intracellular signaling is essential for developmental processes such as cell differentiation, migration but also for maintaining tissue homeostasis. Disruption in the control of these signaling pathways can result in cell death (apoptosis), anergy or uncontrolled cell proliferation and growth leading to cancer. In multicellular organisms, timely termination of signaling is thus equally important as initiation. Known pathways for downregulating membrane receptor-mediated signaling are mediated via specialized endosomal organelles known as lysosomes and proteosomes that degrade such proteins in the cytoplasm. An alternative pathway for attenuating receptor-mediated signaling was recently discovered independently by the group of M. Caplan and our own group.1,2 It appears that apart from the classical protein degradation machineries, the release of signaling proteins also effectively restricts signaling of at least two major signal transduction routes; the canonical Wnt/β-catenin and NFκB pathways. Expelling proteins from the cell, rather than coordinated degradation in lysosomes may involve defined protein modifications, such as ubiquitination, myristyolation, and/or palmitoylation, but little experimental data are currently available. Although the secretion of proteins via exosomes starts by accumulation within multivesicular bodies (MVBs), a key distinction with degredatory MVBs is that exosome-producing MVBs seem to preferentially fuse with the plasmamembrane (Fig. 1). Here we discuss the latest developments in the biology of exosomes and their unexpected effect on intracellular signal transduction.
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Intracellular signaling controlled by the endosomal-exosomal pathway.
By admin, on April 7th, 2012
Commun Integr Biol. 2012 Jan 1; 5(1): 88-93
Verweij FJ, Middeldorp JM, Pegtel DM
Tight control of intracellular signaling is essential for developmental processes such as cell differentiation, migration but also for maintaining tissue homeostasis. Disruption in the control of these signaling pathways can result in cell death (apoptosis), anergy or uncontrolled cell proliferation and growth leading to cancer. In multicellular organisms, timely termination of signaling is thus equally important as initiation. Known pathways for downregulating membrane receptor-mediated signaling are mediated via specialized endosomal organelles known as lysosomes and proteosomes that degrade such proteins in the cytoplasm. An alternative pathway for attenuating receptor-mediated signaling was recently discovered independently by the group of M. Caplan and our own group.1,2 It appears that apart from the classical protein degradation machineries, the release of signaling proteins also effectively restricts signaling of at least two major signal transduction routes; the canonical Wnt/β-catenin and NFκB pathways. Expelling proteins from the cell, rather than coordinated degradation in lysosomes may involve defined protein modifications, such as ubiquitination, myristyolation, and/or palmitoylation, but little experimental data are currently available. Although the secretion of proteins via exosomes starts by accumulation within multivesicular bodies (MVBs), a key distinction with degredatory MVBs is that exosome-producing MVBs seem to preferentially fuse with the plasmamembrane (Fig. 1). Here we discuss the latest developments in the biology of exosomes and their unexpected effect on intracellular signal transduction.
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Intracellular signaling controlled by the endosomal-exosomal pathway.
By admin, on April 7th, 2012
J Vis Exp. 2012;
Crites TJ, Chen L, Varma R
Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)(1). Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules(2). The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation(2). TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors (3-4). Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways. One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously(5-6). They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface (7-8). This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands (9-11) as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ(12-16).
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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins.
By admin, on April 7th, 2012
J Vis Exp. 2012;
Crites TJ, Chen L, Varma R
Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)(1). Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules(2). The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation(2). TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors (3-4). Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways. One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously(5-6). They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface (7-8). This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands (9-11) as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ(12-16).
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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins.
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