Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage

T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In “frustrated” conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.     

End-binding protein 1 controls signal propagation from the T cell receptor

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome.     

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co‐localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site‐specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild‐type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild‐type Arabidopsis. Thus, we conclude that the PM‐associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity‐dependent manner, rather than its subcellular translocation.     

EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and fusion

During muscle differentiation, myoblasts elongate and fuse into syncytial myotubes [1]. An early event during this process is the remodeling of the microtubule cytoskeleton, involving disassembly of the centrosome and, crucially, the alignment of microtubules into a parallel array along the long axis of the cell [2-5]. To further our understanding on how microtubules support myogenic differentiation, we analyzed the role of EB1-related microtubule-plus-end-binding proteins. We demonstrate that EB3 [6] is specifically upregulated upon myogenic differentiation and that knockdown of EB3, but not that of EB1, prevents myoblast elongation and fusion into myotubes. EB3-depleted cells show disorganized microtubules and fail to stabilize polarized membrane protrusions. Using live-cell imaging, we show that EB3 is necessary for the regulation of microtubule dynamics and microtubule capture at the cell cortex. Expression of EB1/EB3 chimeras on an EB3-depletion background revealed that myoblast fusion depends on two specific amino acids in the calponin-like domain of EB3, whereas the interaction sites with Clip-170 and CLASPs are dispensable. Our results suggest that EB3-mediated microtubule regulation at the cell cortex is a crucial step during myogenic differentiation and might be a general mechanism in polarized cell elongation.     

Flanking V and J sequences of complementary determining region 3 of T cell receptor (TCR) δ1 (CDR3δ1) determine the structure and function of TCRγ4δ1

The γδ T cell receptor (TCR) differs from immunoglobulin and αβ TCR in its overall binding mode. In human, genes δ1, δ2, and δ3 are used for TCRδ chains. Previously, we have studied antigen binding determinants of TCRδ2 derived from dominant γδ T cells residing in peripheral blood. In this study we have investigated the critical determinants for antigen recognition and TCR function in TCRδ1 originated from gastric tumor-infiltrating γδ T lymphocytes using three independent experimental strategies including complementary determining region 3 (CDR3) of TCRδ1 (CDR3δ1)-peptide mediated binding, CDR3δ1-grafted TCR fusion protein-mediated binding, and TCRγ4δ1- and mutant-expressing cell-mediated binding. All three approaches consistently showed that the conserved flanking V and J sequences but not the diverse D segment in CDR3δ1 determine the antigen binding. Most importantly, we found that mutations in the V and J regions of CDR3δ1 also abolish the assembly of TCR and TCR-CD3 complexes in TCRγ4δ1-transduced J.RT3-T3.5 cells. Together with our previous studies on CDR3δ2 binding, our finding suggests that both human TCRδ1 and TCRδ2 recognize antigen predominately via flanking V and J regions. These results indicate that TCRγδ recognizes antigens using conserved parts in their CDR3, which provides an explanation for a diverse repertoire of γδTCRs only recognizing a limited number of antigens.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

Protein kinase C-δ negatively regulates T cell receptor-induced NF-κB activation by inhibiting the assembly of CARMA1 signalosome

T-cell receptor (TCR)-induced T-cell activation is a critical event in adaptive immune responses. The engagement of TCR complex by antigen along with the activation of the costimulatory receptors trigger a cascade of intracellular signaling, in which caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) is a crucial scaffold protein. Upon stimulation, CARMA1 recruits downstream molecules including B-cell CLL/lymphoma 10 (Bcl10), mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1), and TRAF6 to assemble a specific TCR-induced signalosome that triggers NF-κB and JNK activation. In this report, we identified protein kinase Cδ (PKCδ) as a CARMA1-associated protein by a biochemical affinity purification approach. PKCδ interacted with CARMA1 in TCR stimulation-dependent manner in Jurkat T cells. Overexpression of PKCδ inhibited CARMA1-mediated NF-κB activation, whereas knockdown of PKCδ potentiated TCR-triggered NF-κB activation and IL-2 secretion in Jurkat T cells. Reconstitution experiments with PKCδ kinase-dead mutant indicated that the kinase activity of PKCδ was dispensable for its ability to inhibit TCR-triggered NF-κB activation. Furthermore, we found that PKCδ inhibited the interaction between MALT1 and TRAF6, but not the association of CARMA1 with PKCθ, Bcl10, or MALT1. These observations suggest that PKCδ is a negative regulator in T cell activation through inhibiting the assembly of CARMA1 signalosome.     

A Model for the Evolution of the Mammalian T-cell Receptor α/δ and μ Loci Based on Evidence from the Duckbill Platypus

The specific recognition of antigen by T cells is critical to the generation of adaptive immune responses in vertebrates. T cells recognize antigen using a somatically diversified T-cell receptor (TCR). All jawed vertebrates use four TCR chains called α, β, γ, and δ, which are expressed as either a αβ or γδ heterodimer. Nonplacental mammals (monotremes and marsupials) are unusual in that their genomes encode a fifth TCR chain, called TCRμ, whose function is not known but is also somatically diversified like the conventional chains. The origins of TCRμ are also unclear, although it appears distantly related to TCRδ. Recent analysis of avian and amphibian genomes has provided insight into a model for understanding the evolution of the TCRδ genes in tetrapods that was not evident from humans, mice, or other commonly studied placental (eutherian) mammals. An analysis of the genes encoding the TCRδ chains in the duckbill platypus revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy (IgH) chain V genes (VH) and related to V genes used in TCRμ. They are expressed as part of TCRδ repertoire (VHδ) and similar to what has been found in frogs and birds. This, however, is the first time a VHδ has been found in a mammal and provides a critical link in reconstructing the evolutionary history of TCRμ. The current structure of TCRδ and TCRμ genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the IgH and TCRδ genes, as well as translocations of TCRδ genes out of the TCRα/δ locus early in mammals, creating the TCRμ locus.     

Chimeric IgH-TCRα/δ translocations in T lymphocytes mediated by RAG

Translocations involving the T cell receptor alpha/delta (TCRα/δ) chain locus, which bring oncogenes in the proximity of the TCRα enhancer, are one of the hallmark features of human T cell malignancies from ataxia telangiectasia (AT) and non-AT patients. These lesions are frequently generated by the fusion of DNA breaks at the TCRα/δ locus to a disperse region centromeric of the immunoglobulin heavy chain (IgH) locus. Aberrant VDJ joining accounts for TCRα/δ associated DNA cleavage, but the molecular mechanism that leads to generation of the "oncogene partner" DNA break is unclear. Here we show that in ATM deficient primary mouse T cells, IgH/TCRα/δ fusions arise at a remarkably similar frequency as in human AT lymphocytes. Recombinase-activating gene (RAG) is responsible for both TCRα/δ as well as IgH associated breaks on chromosome 12 (Chr12), which are subject to varying degrees of chromosomal degradation. We suggest a new model for how oncogenic translocations can arise from two non-concerted physiological DSBs.     

A multicomponent assembly pathway contributes to the formation of acentrosomal microtubule arrays in interphase Drosophila cells

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both gamma-tubulins. However, gamma-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted gamma-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.     

Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: possible consequences for microtubule filament stability

The COP9 signalosome (CSN) is a regulatory particle of the ubiquitin (Ub) proteasome system (UPS) consisting of eight subunits (CSN1-CSN8). We show that the CSN stabilizes the microtubule end-binding protein 1 (EB1) towards degradation by the UPS. EB1, the master regulator of microtubule plus ends, controls microtubule growth and dynamics. Therefore, regulation of EB1 stability by the CSN has consequences for microtubule function. EB1 binds the CSN via subunit CSN5. The C terminus of EB1 is sufficient for interaction with the CSN. Dimerization of EB1 is a prerequisite for complex association and subsequent CSN-mediated phosphorylation, as revealed by studies with the EB1I224A mutant, which is unable to dimerize. In cells, EB1 and CSN co-localize to the centrosome, as demonstrated by confocal fluorescence microscopy. EB1 is ubiquitinated and its proteolysis can be inhibited by MG132, demonstrating that it is a substrate of the UPS. Its degradation is accelerated by inhibition of CSN-associated kinases. HeLa cells permanently expressing siRNAs against CSN1 (siCSN1) or CSN3 (siCSN3) exhibit reduced levels of the CSN complex accompanied by lower steady-state concentrations of EB1. In siCSN1 cells, EB1 is less phosphorylated as compared with control cells, demonstrating that the protein is most likely protected towards the UPS by CSN-mediated phosphorylation. The CSN-dependent EB1 stabilization is not due to the CSN-associated deubiquitinating enzyme USP15. Treatment with nocodazole revealed a significantly increased sensitivity of siCSN1 and siCSN3 cells towards the microtubule depolymerizing drug accompanied by a collapse of microtubule filaments. A nocodazole-induced cell-cycle arrest was partially rescued by CSN1 or EB1. These data demonstrate that the CSN-dependent protection of EB1 is important for microtubule function.     

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.     

β‐arrestin‐1 mediates the TCR‐triggered re‐routing of distal receptors to the immunological synapse by a PKC‐mediated mechanism

T‐cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen‐presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β‐arrestin‐1 as a ligand of non‐phosphorylated resting TCRs. Using dominant‐negative and knockdown approaches we demonstrate that β‐arrestin‐1 is required for the internalization and downregulation of non‐engaged bystander TCRs. Furthermore, TCR triggering provokes the β‐arrestin‐1‐mediated downregulation of the G‐protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β‐arrestin‐1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR‐triggered PKC‐mediated phosphorylation of β‐arrestin‐1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.     

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

T cell antigen receptor–proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor–induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cells.     

A novel localization pattern for an EB1-like protein links microtubule dynamics to endomembrane organization

A group of microtubule-associated proteins called +TIPs (plus end tracking proteins), including EB1 family proteins, label growing microtubule ends specifically in diverse organisms and are implicated in spindle dynamics, chromosome segregation, and directing microtubules toward cortical sites. Here, we report three new EB1-like proteins from Arabidopsis and provide the intracellular localization for AtEB1, which differs from all known EB1 proteins in having a very long acidic C-terminal tail. In marked contrast to other EB1 proteins, the GFP-AtEB1 fusion protein localizes not only to microtubule plus ends but also to motile, pleiomorphic tubulovesicular membrane networks that surround other organelles and frequently merge with the endoplasmic reticulum. AtEB1 behavior thus resembles that of +TIPs, such as the cytoplasmic linker protein CLIP-170, that are known to associate with and pull along membrane tubules in animal systems but for which homologs have not been identified in plants. In addition, though EB1 proteins are believed to stabilize microtubules, a different behavior is observed for AtEB1 where instead of stabilizing a microtubule it localizes to already stabilized regions on a microtubule. The dual localization pattern of AtEB1 suggests links between microtubule plus end dynamics and endomembrane organization during polarized growth of plant cells.     

Spatial organization of centrosome-attached and free microtubules in 3T3 fibroblasts

The spatial organization of microtubules is crucial for different cellular processes. It is traditionally supposed that fibroblasts have radial microtubule arrays consisting of long microtubules that run from the centrosome. However, a detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study, we used laser photobleaching to analyze the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, after which the growth of microtubules in the centrosome region and peripheral parts of cytoplasm was assayed in the bleached zone. In most cases, microtubule growth in the bleached zone occurred rectilinearly; at distances of up to 5 μm, microtubules seldom bend more than 10°–15°. We considered a growing fragment of the microtubule as a vector with the beginning at the point of occurrence and the end at the point where the growth terminated (or the end point after 30 s if microtubule persistent growth proceeded for longer). We defined the direction of microtubule growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome, we directed a comparison vector inside the bleached zone from the centrosome to the beginning of the growing microtubule segment; in the lamella and trailing part of the fibroblast, we used the vector of comparison directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing straight away from the centrosome grew along the cell radius. However, at a distance of 10 μm from the centrosome, radially growing microtubules comprised 40% of the overall number, while at a distance of 20 μm, they made up only 25%. The rest of the microtubules grew in different directions, with the preferred angle between their growth direction and cell radius equaling around 90 °. In the lamella and trailing part of the fibroblast, 80% of all microtubules grew along the long axis of the cell or at an angle of no more than 20 °; 10–15% of microtubules grew along axis of the cell but towards the centrosome. Thus, in 3T3 fibroblasts, the radial system of microtubules is perturbed starting at a distance of several microns from the centrosome. In the internal cytoplasm, the microtubule system is completely disordered and, in the stretched parts of the polarized cell (lamella, trailing edge), the microtubule system again becomes well organized; microtubules are preferentially oriented along the long axis of the cell. From the results obtained, we conclude that the orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome; rather, their orientation is preset by local factors.     

Rab35 and its GAP EPI64C in T cells regulate receptor recycling and immunological synapse formation

Upon antigen recognition, T-cell receptor (TCR/CD3) and other signaling molecules become enriched in a specialized contact site between the T cell and antigen-presenting cell, i.e. the immunological synapse (IS). Enrichment occurs via mechanisms that include polarized secretion from recycling endosomes, but the Rabs and RabGAPs that regulate this are unknown. EPI64C (TBC1D10C) is an uncharacterized candidate RabGAP we identified by mass spectrometry as abundant in human peripheral blood T cells that is preferentially expressed in hematopoietic cells. EPI64C is a Rab35-GAP based both on in vitro Rab35-specific GAP activity and findings in transfection assays. EPI64C and Rab35 dominant negative (DN) constructs each impaired transferrin export from a recycling pathway in Jurkat T-cells and induced large vacuoles marked by transferrin receptor, TCR, and SNAREs implicated in TCR-polarized secretion. Rab35 localized to the plasma membrane and to intracellular vesicles where it substantially colocalized with TfR and with TCR. Rab35 was strongly recruited to the IS. Conjugate formation was impaired by transfection with Rab35-DN or EPI64C and by EPI64C knock down. TCR enrichment at the IS was impaired by Rab35-DN. Thus, EPI64C and Rab35 regulate a recycling pathway in T cells and contribute to IS formation, most likely by participating in TCR transport to the IS.     

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.     

Th1 and Th2 cells form morphologically distinct immunological synapses

The arrangement of molecules at the interface between T cells and APCs is known as the immunological synapse (IS). We conducted experiments with supported planar bilayers and transfected fibroblast APC to examine the IS formed by polarized Th1 and Th2 cells. Th1 cells formed typical "bull's-eye" IS with a ring of adhesion molecules surrounding MHC/TCR interactions at all Ag concentrations tested, while Th2 cells formed multifocal IS at high concentrations of Ag. At low Ag concentrations, the majority of Th2 cells formed IS with a compact, central accumulation of MHC/TCR, but ICAM-1 was not excluded from the center of the IS. Additionally, CD45 was excluded from the center of the interface between Th1 cells and APC, while CD45 was found at the center of the multifocal IS formed by Th2 cells. Finally, phosphorylated signaling molecules colocalized with MHC/TCR to a greater extent in Th2 IS. Together, our results indicate that the IS formed by Th1 and Th2 cells are distinct in structure, with Th2 cells failing to form bull's-eye IS.     

Arl4D-EB1 interaction promotes centrosomal recruitment of EB1 and microtubule growth

ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. End-binding 1 (EB1) is a microtubule (MT) plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing MTs and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates MT nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EB homology region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal MT nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased MT nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and the EB1, which is important for MT stabilization. Together, our results indicate that Arl4D modulates MT nucleation through regulation of the EB1–p150 association at the centrosome.