The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

Termination of TCR-mediated activation signals is regulated by CrkII-dependent Cbl-mediated ubiquitination and degradation of C3G.

Crk adaptor proteins are key players in signal transduction from multiple cell surface receptors, including the T cell antigen receptor (TCR). The involvement of CrkII in the early stages of T cell activation is well documented, but little is known about its role during the termination of the activation response. We substantiated findings showing that CrkII utilizes its SH3N and SH2 domains to constitutively associate with C3G and transiently with Cbl in resting and TCR/CD3-stimulated T cells, respectively. Association of CrkII with Cbl peaks within 1 min post-TCR/CD3 stimulation, and involves the formation of multiple CrkII-containing complexes of different molecular mass. Ubiquitination of C3G commences at ∼5 min post TCR/CD3 stimulation concomitantly with its degradation. This entire process conversely correlates with the levels of expression of CrkII and is dependent on the presence of the CrkII-bound Cbl protein. The data suggest that CrkII functions as a scaffold that brings Cbl into close proximity with C3G in TCR/CD3-stimulated T cells and that tyrosine phosphorylation and activation of Cbl promotes C3G ubiquitination and degradation. We suggest that this mechanism contributes to the termination of the TCR/CD3-induced activation signal and helps tune the length and intensity of T cell-mediated immune responses.

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

Loss of CD47 alters CD8+ T cell activation in vitro and immunodynamics in mice.

CD47 has established roles in the immune system for regulating macrophage phagocytosis and lymphocyte activation, with growing evidence of its cell-intrinsic regulatory roles in natural killer and CD8+ T cells. CD47 limits antigen-dependent cytotoxic activities of human and murine CD8+ T cells, but its role in T cell activation kinetics remains unclear. Using in vitro and in vivo models, we show here that CD47 differentially regulates CD8+ T cell responses to short- versus long-term activation. Although CD47 was not required for T cell development in mice and early activation in vitro, short-term stimuli elevated pathogen-reactive gene expression and enhanced proliferation and the effector phenotypes of Cd47-deficient relative to Cd47-sufficient CD8+ T cells. In contrast, persistent TCR stimulation limited the effector phenotypes of Cd47 -/- CD8+ T cells and enhanced their apoptosis signature. CD8+ T cell expansion and activation in vivo induced by acute lymphocytic choriomeningitis virus (LCMV) infection did not differ in the absence of CD47. However, the frequency and effector phenotypes of Cd47-/- CD8+ T cells were constrained in chronic LCMV-infected as well as in mice bearing B16 melanoma tumors. Therefore, CD47 regulates CD8+ T cell activation, proliferation, and fitness in a context-dependent manner.

A centrifugal microfluidic cross-flow filtration platform to separate serum from whole blood for the detection of amphiphilic biomarkers.

The separation of biomarkers from blood is straightforward in most molecular biology laboratories. However, separation in resource-limited settings, allowing for the successful removal of biomarkers for diagnostic applications, is not always possible. The situation is further complicated by the need to separate hydrophobic signatures such as lipids from blood. Herein, we present a microfluidic device capable of centrifugal separation of serum from blood at the point of need with a system that is compatible with biomarkers that are both hydrophilic and hydrophobic. The cross-flow filtration device separates serum from blood as efficiently as traditional methods and retains amphiphilic biomarkers in serum for detection.

Design and implementation of a J-coupled spectrometer for multidimensional structure and relaxation detection at low magnetic fields.

In recent years, it has been realized that low and ultra-low field (mT-nT magnetic field range) nuclear magnetic resonance spectroscopy can be used for molecular structural analysis. However, spectra are often hindered by lengthy acquisition times or require large sample volumes and high concentrations. Here, we report a low field (50 µT) instrument that employs a linear actuator to shuttle samples between a 1 T prepolarization field and a solenoid detector in a laboratory setting. The current experimental setup is benchmarked using water and 13C-methanol with a single scan detection limit of 2 × 1020 spins (3 µl, 55M H2O) and detection limit of 2.9 × 1019 (200 µl, 617 mM 13C-methanol) spins with signal averaging. The system has a dynamic range of >3 orders of magnitude. Investigations of room-temperature relaxation dynamics of 13C-methanol show that sample dilution can be used in lieu of sample heating to acquire spectra with linewidths comparable to high-temperature spectra. These results indicate that the T1 and T2 mechanisms are governed by both the proton exchange rate and the dissolved oxygen in the sample. Finally, a 2D correlation spectroscopy experiment is reported, performed in the strong coupling regime that resolves the multiple resonances associated with the heteronuclear J-coupling. The spectrum was collected using 10 times less sample and in less than half the time from previous reports in the strong coupling limit.

YAP/TAZ Related BioMechano Signal Transduction and Cancer Metastasis.

Mechanoreciprocity refers to a cell's ability to maintain tensional homeostasis in response to various types of forces. Physical forces are continually being exerted upon cells of various tissue types, even those considered static, such as the brain. Through mechanoreceptors, cells sense and subsequently respond to these stimuli. These forces and their respective cellular responses are prevalent in regulating everything from embryogenic tissue-specific differentiation, programmed cell death, and disease progression, the last of which being the subject of extensive attention. Abnormal mechanical remodeling of cells can provide clues as to the pathological status of tissues. This becomes particularly important in cancer cells, where cellular stiffness has been recently accepted as a novel biomarker for cancer metastasis. Several studies have also elucidated the importance of cell stiffness in cancer metastasis, with data highlighting that a reversal of tumor stiffness has the capacity to revert the metastatic properties of cancer. In this review, we summarize our current understanding of extracellular matrix (ECM) homeostasis, which plays a prominent role in tissue mechanics. We also describe pathological disruption of the ECM, and the subsequent implications toward cancer and cancer metastasis. In addition, we highlight the most novel approaches toward understanding the mechanisms which generate pathogenic cell stiffness and provide potential new strategies which have the capacity to advance our understanding of one of human-kinds' most clinically significant medical pathologies. These new strategies include video-based techniques for structural dynamics, which have shown great potential for identifying full-field, high-resolution modal properties, in this case, as a novel application.

Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of mice bearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation.

Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.

Aerosol delivery into small anatomical airway model through spontaneous engineered breathing.

Pulmonary administration is a noninvasive drug delivery method that, in contrast to systemic administration, reduces drug dosage and possible side effects. Numerous testing models, such as impingers and impactors, have previously been developed to evaluate the fate of inhaled drugs. However, such models are limited by the lack of information regarding several factors, such as pulmonary morphology and breathing motion, which are required to fully interpret actual inhaled-drug deposition profiles within the human respiratory tract. In this study, a spontaneous breathing-lung model that integrates branched morphology and deformable alveolar features was constructed using a multilayered fabrication technology to mimic the complex environment of the human lower respiratory tract. The developed model could emulate cyclic and spontaneous breathing motions to inhale and exhale aerosols generated by a nebulizer under diseaselike conditions. Results of this research demonstrate that aerosols (4.2 µm) could reach up to the deeper lung regions (generation 19 of the branched lung structure) within the obstructivelike model, whereas lesser penetration (generation 17) was observed when using the restrictivelike model. The proposed breathing-lung model can serve as a testing platform to provide a comprehensive understanding of the pharmacokinetics of pulmonary drugs within the lower lungs.

Natural Killer Cell Recruitment and Activation Are Regulated by CD47 Expression in the Tumor Microenvironment.

Elevated CD47 expression in some cancers is associated with decreased survival and limited clearance by phagocytes expressing the CD47 counterreceptor SIRPα. In contrast, elevated CD47 mRNA expression in human melanomas was associated with improved survival. Gene-expression data were analyzed to determine a potential mechanism for this apparent protective function and suggested that high CD47 expression increases recruitment of natural killer (NK) cells into the tumor microenvironment. The CD47 ligand thrombospondin-1 inhibited NK cell proliferation and CD69 expression in vitro Cd47 -/- NK cells correspondingly displayed augmented effector phenotypes, indicating an inhibitory function of CD47 on NK cells. Treating human NK cells with a CD47 antibody that blocks thrombospondin-1 binding abrogated its inhibitory effect on NK cell proliferation. Similarly, treating wild-type mice with a CD47 antibody that blocks thrombospondin-1 binding delayed B16 melanoma growth, associating with increased NK cell recruitment and increased granzyme B and interferon-γ levels in intratumoral NK but not CD8+ T cells. However, B16 melanomas grew faster in Cd47 -/- than in wild-type mice. Melanoma-bearing Cd47 -/- mice exhibited decreased splenic NK cell numbers, with impaired effector protein expression and elevated exhaustion markers. Proapoptotic gene expression in Cd47-/- NK cells was associated with stress-mediated increases in mitochondrial proton leak, reactive oxygen species, and apoptosis. Global gene-expression profiling in NK cells from tumor-bearing mice identified CD47-dependent transcriptional responses that regulate systemic NK activation and exhaustion. Therefore, CD47 positively and negatively regulates NK cell function, and therapeutic antibodies that block inhibitory CD47 signaling can enhance NK immune surveillance of melanomas.

High-throughput phenotyping of cell-to-cell interactions in gel microdroplet pico-cultures.

Microbiomes exert significant influence on our planet's ecology. Elucidating the identities of individual microbes within these communities and how they interact is a vital research imperative. Using traditional plating and culturing methods, it is impractical to assess even a small fraction of the interactions that exist within microbial communities. To address this technology gap, we integrated gel microdroplet technology with microfluidics to generate millions of microdroplet cultures (MDs) that sequester individual cells for phenotyping MDs, facilitating rapid analysis and viable recovery using flow cytometry. Herein, we describe a validated high-throughput phenotyping pipeline that elucidates cell-to-cell interactions for millions of combinations of microorganisms. Through iterative co-culturing of an algae and a pool of environmentally sourced microbes, we successfully isolated bacteria that improved algal growth.

Multistep Fluidic Control Network toward the Automated Generation of Organ-on-a-Chip.

Organ-on-a-chip, which mimics physiological functions of organs, is a potential tool for drug development and precision medicine. This chip, accompanied by a suitable culture environment and appropriate culture procedure, allows cells to form functional tissues that can be used in drug tests. Due to difficulties in the maintenance of cells and the complex nature of the tissue development process, it is essential to develop an automated culture platform to avoid contamination and reduce operational errors during long-term tissue culture. In this study, we developed a semiautomatic culture platform that integrates with a multistep fluidic control network, which allows multiple culture steps to be controlled and meets the requirement of the air-liquid interface (ALI), while maintaining a dynamic flow onto the cells. The culture platform was assembled with a culture chip, a reservoir, a miniaturized peristaltic pump, and a fluidic control base to connect each component and to operate the multiple culture steps. To demonstrate the capability of the culture platform, we have successfully controlled the multiple cell culture steps by switching the operation modes, allowing (1) cell proliferation under a liquid-liquid interface, (2) medium change from proliferation medium to differentiation medium, (3) cell differentiation under ALI conditions, and (4) repeated mucus washing. The dynamics and ALI culture conditions can simulate a physiological environment that is capable of maintaining and enabling cell differentiation for tissue-specific functions. The results demonstrate that bronchial tissue develops in the culture chip after 4 weeks of tissue culture. A versatile combination of culture steps makes the tissue culture platform suitable as an in vitro organ-on-a-chip culture model, especially for the tissues that involve the ALI culture, such as lung and skin. This platform, with multilogic control procedures, holds promise for enabling the long-term cultivation of differentiated tissues for advanced pharmacological and toxicological applications.

Microfluidic channel optimization to improve hydrodynamic dissociation of cell aggregates and tissue.

Maximizing the speed and efficiency at which single cells can be liberated from tissues would dramatically advance cell-based diagnostics and therapies. Conventional methods involve numerous manual processing steps and long enzymatic digestion times, yet are still inefficient. In previous work, we developed a microfluidic device with a network of branching channels to improve the dissociation of cell aggregates into single cells. However, this device was not tested on tissue specimens, and further development was limited by high cost and low feature resolution. In this work, we utilized a single layer, laser micro-machined polyimide film as a rapid prototyping tool to optimize the design of our microfluidic channels to maximize dissociation efficiency. This resulted in a new design with smaller dimensions and a shark fin geometry, which increased recovery of single cells from cancer cell aggregates. We then tested device performance on mouse kidney tissue, and found that optimal results were obtained using two microfluidic devices in series, the larger original design followed by the new shark fin design as a final polishing step. We envision our microfluidic dissociation devices being used in research and clinical settings to generate single cells from various tissue specimens for diagnostic and therapeutic applications.

Micromachining of Polyurethane Membranes for Tissue Engineering Applications.

Engineered tissue barrier models offer in vitro alternatives in toxicology and disease research. To mimic barrier-tissue microenvironment, a porous membrane that can approach the stiffness of physiological basement membranes is required. While several biocompatible membranes with micrometer range thickness (10 µm) and a stiffness less than polystyrene (3 GPa) or polyethylene (PET, 2 GPa), have been developed, there has been little effort to optimize the process to enable rapid and reproducible pore production in these membranes. Here, we investigate the use of laser irradiation with femtosecond (fs) pulses because the combination of high-precision and cold-ablation causes minimal damage to polymeric membranes. This process enables automated, high-throughput and reproducible fabrication of thin, microporous membranes that can be utilized to culture cells at air-liquid interface (ALI), a unique culture technique that simulates the tissue-barrier microenvironment. We show the optimization of laser parameters on a thin polyurethane membrane and patterned pores with an average diameter of 5 µm. Tissue was cultured at ALI for 28 days to demonstrate the membrane's utility in constructing a tissue barrier model. These results confirm the utilization of fs laser machining as a viable method for creating a porous barrier substrate in tissue engineering platforms.

A microfluidic method to measure bulging heights for bulge testing of polydimethylsiloxane (PDMS) and polyurethane (PU) elastomeric membranes.

Thin and flexible elastomeric membranes are frequently used in many microfluidic applications including microfluidic valves and organs-on-a-chip. The elastic properties of these membranes play an important role in the design of such microfluidic devices. Bulge testing, which is a common method to characterize the elastic behavior of these membranes, involves direct observation of the changes in the bulge height in response to a range of applied pressures. Here, we report a microfluidic approach to measure the bulging height of elastic membranes to replace direct observation of the bulge height under a microscope. Bulging height is measured by tracking the displacement of a fluid inside a microfluidic channel, where the fluid in the channel was designed to be directly in contact with the elastomeric membrane. Polydimethylsiloxane (PDMS) and polyurethane (PU) membranes with thickness 12-35 µm were fabricated by spin coating for bulge testing using both direct optical observation and the microfluidic method. Bulging height determined from the optical method was subject to interpretation by the user, whereas the microfluidic approach provided a simple but sensitive method for determining the bulging height of membranes down to a few micrometers. This work validates the proof of principle that uses microfluidics to accurately measure bulging height in conventional bulge testing for polydimethylsiloxane (PDMS) and polyurethane (PU)eElastomeric membranes.

CD47 Expression in Natural Killer Cells Regulates Homeostasis and Modulates Immune Response to Lymphocytic Choriomeningitis Virus.

CD47 is a ubiquitous cell surface receptor that directly regulates T cell immunity by interacting with its inhibitory ligand thrombospondin-1 and limits clearance of cells by phagocytes that express its counter-receptor signal-regulatory protein-α. Murine natural killer (NK) cells express higher levels of CD47 than other lymphocytes, but the role of CD47 in regulating NK cell homeostasis and immune function remains unclear. Cd47-/- mice exhibited depletion of NK precursors in bone marrow, consistent with the antiphagocytic function of CD47. In contrast, antisense CD47 knockdown or gene disruption resulted in a dose dependent accumulation of immature and mature NK cells in spleen. Mature Cd47-/- NK cells exhibited increased expression of NK effector and interferon gene signatures and an increased proliferative response to interleukin-15 in vitro. Cd47-/- mice showed no defect in their early response to acute Armstrong lymphocytic choriomeningitis virus (LCMV) infection but were moderately impaired in controlling chronic Clone-13 LCMV infection, which was associated with depletion of splenic NK cells and loss of effector cytokine and interferon response gene expression in Cd47-/- NK cells. Broad CD47-dependent differences in NK activation, survival, and exhaustion pathways were observed in NK cell transcriptional signatures in LCMV infected mice. These data identify CD47 as a cell-intrinsic and systemic regulator of NK cell homeostasis and NK cell function in responding to a viral infection.

Crk adaptor proteins regulate CD3ζ chain phosphorylation and TCR/CD3 down-modulation in activated T cells.

T cell receptor (TCR) recognition of a peptide antigen in the context of MHC molecules initiates positive and negative cascades that regulate T cell activation, proliferation and differentiation, and culminate in the acquisition of effector T cell functions. These processes are a prerequisite for the induction of specific T cell-mediated adaptive immune responses. A key event in the activation of TCR-coupled signaling pathways is the phosphorylation of tyrosine residues within the cytoplasmic tails of the CD3 subunits, predominantly CD3ζ. These transiently formed phosphotyrosyl epitopes serve as docking sites for SH2-domain containing effector molecules, predominantly the ZAP70 protein tyrosine kinase, which is critical for signal propagation. We found that CrkI and CrkII adaptor proteins also interact with CD3ζ in TCR activated-, but not in resting-, T cells. Crk binding to CD3ζ was independent of ZAP70 and also occurred in ZAP70-deficient T cells. Binding was mediated by Crk-SH2 domain interaction with phosphotyrosine-containing motifs on CD3ζ, via a direct physical interaction, as demonstrated by Far-Western blot. CrkII binding to CD3ζ could also be demonstrated in a heterologous system, where coexpression of a catalytically active Lck was used to phosphorylate the CD3ζ chain. TCR activation-induced Crk binding to CD3ζ resulted in increased and prolonged phosphorylation of CD3ζ, as well as ZAP70 and LAT, suggesting a positive role for CrkI/II binding to CD3ζ in regulation of TCR-coupled signaling pathways. Furthermore, Crk-dependent increased phosphorylation of CD3ζ coincided with inhibition of TCR downmodulation, supporting a positive role for Crk adaptor proteins in TCR-mediated signal amplification.

TCR crosslinking promotes Crk adaptor protein binding to tyrosine-phosphorylated CD3ζ chain.

T cell antigen receptor (TCR) binding of a peptide antigen presented by antigen-presenting cells (APCs) in the context of surface MHC molecules initiates signaling events that regulate T cell activation, proliferation and differentiation. A key event in the activation process is the phosphorylation of the conserved tyrosine residues within the CD3 chain immunoreceptor tyrosine-based activation motifs (ITAMs), which operate as docking sites for SH2 domain-containing effector proteins. Phosphorylation of the CD3ζ ITAMs renders the CD3 chain capable of binding the ζ-chain associated protein 70 kDa (ZAP70), a protein tyrosine kinase that is essential for T cell activation. We found that TCR/CD3 crosslinking in Jurkat T cells promotes the association of Crk adaptor proteins with the transiently phosphorylated CD3ζ chain. Pull down assays using bead-immobilized GST fusion proteins revealed that the Crk-SH2 domain mediates binding of phospho-CD3ζ. Phospho-CD3ζ binding is selective and is mediated by the three types of Crk, including CrkI, CrkII, and CrkL, but not by other SH2 domain-containing adaptor proteins, such as Grb2, GRAP and Nck. Crk interaction with phospho-CD3ζ is rapid and transient, peaking 1 min post TCR/CD3 crosslinking. The results suggest the involvement of Crk adaptor proteins in the early stages of T cell activation in which Crk might help recruiting effector proteins to the vicinity of the phospho-CD3ζ and contribute to the fine-tuning of the TCR/CD3-coupled signal transduction pathways.

Hollow fiber integrated microfluidic platforms for in vitro Co-culture of multiple cell types.

This study demonstrates a rapid prototyping approach for fabricating and integrating porous hollow fibers (HFs) into microfluidic device. Integration of HF can enhance mass transfer and recapitulate tubular shapes for tissue-engineered environments. We demonstrate the integration of single or multiple HFs, which can give the users the flexibility to control the total surface area for tissue development. We also present three microfluidic designs to enable different co-culture conditions such as the ability to co-culture multiple cell types simultaneously on a flat and tubular surface, or inside the lumen of multiple HFs. Additionally, we introduce a pressurized cell seeding process that can allow the cells to uniformly adhere on the inner surface of HFs without losing their viabilities. Co-cultures of lung epithelial cells and microvascular endothelial cells were demonstrated on the different platforms for at least five days. Overall, these platforms provide new opportunities for co-culturing of multiple cell types in a single device to reconstruct native tissue micro-environment for biomedical and tissue engineering research.

In vivo regulation of human CrkII by cyclophilin A and FK506-binding protein.

Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼ 5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼ 5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly(219)-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.