Martin Poenie

Dynamic Polarization of the Microtubule Cytoskeleton during CTL-Mediated Killing

Efficient unidirectional killing by cytotoxic T lymphocytes (CTL) requires translocation of the microtubule organizing center (MTOC) to the target cell contact site. Here we utilize modulated polarization microscopy and computerized 3D reconstruction of tubulin and LFA-1 immunofluorescence images to investigate how this is accomplished. The results show that the MTOC is drawn vectorially to the contact site by a microtubule sliding mechanism. Once the MTOC arrives at the contact site, it oscillates laterally. Microtubules loop through and anchor to a ring-shaped zone (pSMAC) defined by the dense clustering of LFA-1 at the target contact site. Microtubules that run straight between the MTOC and pSMAC and then turn sharply may indicate the action of a microtubule motor such as dynein.

Recruitment of dynein to the Jurkat immunological synapse

Binding of T cells to antigen-presenting cells leads to the formation of the immunological synapse, translocation of the microtubule-organizing center (MTOC) to the synapse, and focused secretion of effector molecules. Here, we show that upon activation of Jurkat cells microtubules project from the MTOC to a ring of the scaffolding protein ADAP, localized at the synapse. Loss of ADAP, but not lymphocyte function-associated antigen 1, leads to a severe defect in MTOC polarization at the immunological synapse. The microtubule motor protein cytoplasmic dynein clusters into a ring at the synapse, colocalizing with the ADAP ring. ADAP coprecipitates with dynein from activated Jurkat cells, and loss of ADAP prevents MTOC translocation and the specific recruitment of dynein to the synapse. These results suggest a mechanism that links signaling through the T cell receptor to translocation of the MTOC, in which the minus end-directed motor cytoplasmic dynein, localized at the synapse through an interaction with ADAP, reels in the MTOC, allowing for directed secretion along the polarized microtubule cytoskeleton.

NEW FLUORESCENT CALCIUM INDICATORS DESIGNED FOR CYTOSOLIC RETENTION OR MEASURING CALCIUM NEAR MEMBRANES

A new family of fluorescent calcium indicators has been developed based on a new analog of BAPTA called FF6. This new BAPTA analog serves as a versatile synthetic intermediate for developing Ca2+ indicators targeted to specific intracellular environments. Two of these new Ca2+ indicators, fura-PE3 and fura-FFP18, are described in this report. Fura-PE3 is a zwitterionic indicator that resists the rapid leakage and compartmentalization seen with fura-2 and other polycarboxylate calcium indicators. In contrast to results obtained with fura-2, cells loaded with PE3 remain brightly loaded and responsive to changes in concentration of cytosolic free calcium for hours. Fura-FFP18 is an amphipathic indicator that to binds to liposomes and to cell membranes. Studies to be detailed later indicate that FFP18 functions as a near-membrane Ca2+ indicator and that calcium levels near the plasma membrane rise faster and higher than in the cytosol.

Modulated polarization microscopy: a promising new approach to visualizing cytoskeletal dynamics in living cells.

In an effort to visualize cytoskeletal filaments in living cells, we have developed modulated polarization microscopy. Modulated polarization microscopy visualizes cytoskeletal filaments based on their birefringence but differs from the standard polarization microscopy by exploiting the angle dependence of birefringence. A prototype instrument has been developed using two Faraday rotators under computer control to change the angle of plane polarized light at a known rate. By placing one Faraday rotator before and one after the specimen, rotation produced by the first Faraday rotator is cancelled by the second. This allows the use of fixed polarizer and analyzer in a crossed configuration and continuous imaging of the specimen between crossed polarizers. The variation in polarization angle of light illuminating the specimen causes birefringent elements to oscillate in brightness. Images acquired as polarization angle is varied are then processed by a Fourier filter image-processing algorithm. The Fourier filtering algorithm isolates those signals that vary at the proper rate, whereas static or random signals are removed. Here we show that the modulated polarization microscope can reveal cytoskeletal elements including stress fibers and microtubules in living cells.

Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright

Regulation of BCR signalling strength is crucial for B‐cell development and function. Bright is a B‐cell‐restricted factor that complexes with Brutons tyrosine kinase (Btk) and its substrate, transcription initiation factor‐I (TFII‐I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII‐I and is then discharged from lipid rafts as a Sumo‐I‐modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant‐negative titration of rafts‐specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post‐translational modification of a transcription factor with implications for B‐cell tolerance and autoimmunity.

Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150Glued; however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10–44, is sufficient for binding p150Glued. Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150Glued form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150Glued fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150Glued. Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion

Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150Glued) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1–enhanced GFP fusion) were activated by Staphylococcus enterotoxin E–coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150Glued, which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B–containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150Glued in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell–focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively.

Murine T-lymphomas corresponding to the immature CD4-8+ thymocyte subset.

N-methyl-N-nitrosourea induces murine CD4-8+ T-lymphomas that express high levels of J11d and low levels of CD5 antigens, a phenotype characteristic of immature CD4-8+ thymocytes. This assignment is supported by the fact that CD4-8+ lymphoma cell lines acquire CD4 expression after intrathymic (i.t.) transfer, a finding consistent with the established precursor potential of the normal immature CD4-8+ subset. CD4+8+ lymphomas recovered after i.t. transfer maintain a CD4+8+ phenotype in long-term culture. Northern blot analyses reveal that CD4 expression is regulated at the transcriptional level in immature CD4-8+ and CD4+8+ cell lines. CD4-8+ lymphomas express low levels of functional CD3/TCR complexes that mediate intracellular Ca2+ mobilization in response to CD3 or α/β-TCR monoclonal antibody. These data suggest that the immature CD4-8+ subset contains cells capable of undergoing TCR-mediated signaling and selection events. In contrast to normal immature CD4-8+ cells, which comprise a heterogeneous and transient subset, the CD4-8+ lymphoma lines provide stable, monoclonal models of the immature CD4-8+ stage of thymocyte development.

Role of the MTOC in T Cell Effector Functions

T cells play important roles in defending the host against infections, in allergic responses, and in the destruction of tumor cells. The directed or focused delivery of effector molecules to another cell is minimally achieved by a two-step process that involves focusing of secretory vesicles around the microtubule-organizing center (MTOC) and movement of the MTOC up to the site of contact with the target cell. This chapter is focused on mechanisms involved in the movement of the MTOC to the target contact site in T cells. Modulated polarization microscopy (MPM) and several other imaging methods were employed to visualize the cytoskeleton in general and in particular, the dynamics of MTOC movement. Understanding the processes of MTOC translocation has important medical ramifications that are addressed in this chapter.

Translocation of the Microtubule Organizing Center: How Localized Signaling Leads to Localized Secretion in T Cells

When T cells make contact with cells displaying antigen, signaling through the T cell receptor leads to rapid formation of a specialized contact site known as the immunological synapse. The synapse represents the localized assembly of signaling, cytoskeletal, and adhesion complexes that orchestrate the T cell effector response, which at its heart, entails secretion. This secretory response depends on translocation of the microtubule organizing center (MTOC) up to the synapse bringing with it attached secretory vesicles[1, 2] and spatially directing subsequent vesicle movements[3]. In order to better understand how the MTOC translocated to the synapse we developed a new type of microscopy called modulated polarization microscopy that allowed us to monitor individual microtubules and the MTOC in living cytotoxic T cells[4]. Modulated polarization microscopy works by modulating the plane of polarized light presented to the specimen and then demodulating the light after passing through the specimen. This causes birefringent elements to exhibit a sinusoidal optical signal against a dark background that can be isolated and quantified using a single frequency Fourier filtering algorithm. Using this instrument, we were able to follow movements of the MTOC and showed that these movements correlated with tensioning of microtubules anchored to the cortex. Real-time studies of microtubule and MTOC movements suggested that motor proteins (presumably dynein) formed a circular array at the synapse[5]. Using computerized 3D reconstructions of antibody-labeled microtubules together with enhancement using 3D identification and filtering algorithms we obtained clear pictures of the microtubule array that showed that that there was a circular zone where microtubules came in contact with the cortex and this zone corresponded to regions where the integrin LFA-1 was clustered at the synapse, a region known as the pSMAC. Subsequent studies using Jurkat cells showed that certain antibodies against the dynein intermediate chain (DIC) labeled a circular ring-like structure at the synapse whereas other antiDIC antibodies labeled only microtubules and the MTOC. The dynein ring colocalized with ADAP, a scaffold protein that is part of the T cell signaling cascade as well as some known dynein binding proteins including β-catenin and PLAC-24[6]. Interestingly, it did not correlate with the localization of dynactin, which was more clearly associated with microtubules and the MTOC. When ADAP expression was reduced, dynein failed to accumulate at the synapse and MTOC translocation was blocked. Although ADAP seems to be essential for MTOC translocation it is not required in mouse T cells making it likely that other conserved molecules are involved. One of these appears to be LIS-1 which also part of the dynein complex at the synapse. 990 doi:10.1017/S1431927610062859 Microsc. Microanal. 16 (Suppl 2), 2010