It is becoming increasingly clear that cell polarity also plays a central role in lymphocyte function,^1,2 a fact that belies the textbook depiction of lymphocytes as featureless and spherically symmetric. While patrolling secondary lymphoid organs and peripheral tissues, lymphocytes adopt a “hand-mirror” configuration consisting of a lamellipodial leading edge followed by a stalk-like uropod (Fig. 1). In this manner, they survey the surfaces of other cells for molecular indicators of pathology. T cells and B cells bind to antigenic peptides and proteins, respectively, while NK cells sense surface markers of cellular distress. Recognition of any of these components leads to the formation of a specialized cell-cell contact between the lymphocyte and the target cell called an immunological synapse (IS),³ which is accompanied by a dramatic change in cellular morphology. First, the lymphocyte gloms onto the side of the target cell, forming a radially symmetric contact that is sealed by a dense ring of actin and integrins. Then, the MTOC or centrosome, of the lymphocyte moves to a position just beneath the interface. MTOC reorientation effectively aligns the lymphocytes' secretory apparatus with the IS, thereby enabling the release of soluble factors directionally toward the target cell.² This is crucial from maintaining the specificity of secretory responses. For example, MTOC reorientation is the reason cytotoxic T cells and NK cells can specifically kill target cells without damaging the surrounding tissue. In addition, several recent studies have suggested that T cells undergo asymmetric cell division in response to antigenic stimulation by dendritic cells.^4,5 In this context, polarization of the MTOC would presumably be important for establishing a division plane parallel to the IS.

MTOC reorientation to the IS was first characterized in T cells close to 30 years ago.^6,7 It has been difficult to study, however because the process occurs so quickly (<5 minutes) and because lymphocytes are so small. In the intervening years, however, considerable progress has been made toward understanding cell polarity in more tractable systems such as fibroblasts, astrocytes and epithelial cells.^8,9 Studies in these cell types have indentified a number of distinct protein complexes that accumulate in a polarized manner within defined regions of the plasma membrane (Fig. 1). The mutual inhibition of some complexes by others acts to establish and stabilize the polarized state. Interestingly, migrating fibroblasts, astrocytes and neurons reorient their MTOC toward the leading edge of the cell, and it has been tempting to speculate that the machinery used for MTOC polarization in these systems is shared by lymphocytes. It is important to note, however, that in adherent cell types polarity is established slowly, over a period of hours, and that it can persist for days or even longer. Lymphocyte polarity, by contrast, is highly dynamic and often transient. Hence, it is not unreasonable to expect that distinct molecular mechanisms are at work during MTOC polarization to the IS.

Our lab uses a combination of photochemistry and single cell imaging to examine lymphocyte signaling and cytoskeletal dynamics with high spatial and temporal resolution. Below, I will discuss recent progress we have made toward understanding the molecular mechanisms that drive MTOC reorientation to the IS in T cells. I will then attempt to place this work in the context of what is known about polarity in other cell types, and speculate about the extent to which molecular pathways and design concepts derived from other systems can be used to guide future studies in lymphocytes.

One of the most important effector enzymes recruited to the LAT-SLP76 complex by TCR signaling is phospholipase C-γ (PLC-γ), which hydrolyzes phosphatidyl-inositol bis-phosphate (PIP2) to yield two second messengers, inositol tris-phosphate (IP₃) and diacylglycerol (DAG). IP₃ stimulates the influx of calcium (Ca²⁺) into the cytoplasm, while DAG recruits proteins to the plasma membrane that contain “typical” C1 domains. Because DAG accumulates specifically in the IS after TCR stimulation, we investigated whether it might play an instructive role in guiding the polarization of the MTOC.¹² Using the C1 domains of protein kinase C-δ (PKCδ) as a biosensor for DAG, we were able to show in TCR photoactivation experiments that DAG accumulates at the site of TCR stimulation ′10 s prior to MTOC reorientation. A small molecule inhibitor of PLC-γ blocked the polarization response, consistent with a role for localized DAG in this process. Stimulation of unpolarized DAG-dependent signaling with phorbol myristate acetate (PMA) completely disrupted MTOC reorientation. Furthermore, inhibition of DAG kinases (DGKs), which convert DAG into phosphatidic acid, destabilized synaptic DAG accumulation and impaired MTOC recruitment to the IS. In contrast, blocking Ca²⁺ signaling with extracellular and intracellular chelators had no effect on polarization. Hence, it is DAG signaling, and not Ca²⁺, that plays the operative role in this pathway downstream of PLC-γ.

To further explore the mechanisms by which DAG influences the MTOC, we focused next on the PKC family of enzymes, which have been implicated in polarity induction in multiple cell types. PKCs can be divided into three subfamilies based on their regulatory properties.¹⁷ Classical PKCs (cPKCs) require both DAG and Ca²⁺ for activation, novel PKCs (nPKCs) require DAG but not Ca²⁺, and atypical PKCs (aPKCs) require neither DAG nor Ca²⁺. Because DAG, but not Ca²⁺, is necessary for MTOC reorientation,¹² we chose to investigate the nPKC isoforms, of which there are four: PKCδ, PKCε, PKCη and PKCθ. Of these, PKCθ was known to be involved in TCR signaling, having been implicated previously in transcriptional activation and the upregulation of integrin-mediated adhesion.^18,19 Less was known about the other three proteins. Indeed, some reports suggested that PKCε and PKCη played no part in the TCR signaling network.^20,21

Using our photoactivation and imaging approach, we demonstrated that PKCε, PKCη, and PKCθ, but not PKCδ, are recruited to the IS in an ordered cascade (Fig. 3).²² Approximately 15 seconds before MTOC reorientation, PKCε and PKCη accumulate in a broad region of membrane centered at the site of TCR stimulation. PKCθ is recruited ~5 seconds later, and it occupies a more restricted zone that is fully contained within the region of PKCε and PKCη accumulation. To explore the functional relevance of these three enzymes, we employed siRNA knockdown and also made use of available knockout mice. In this manner, we showed that PKCθ is required for optimal MTOC reorientation, and that PKCε and PKCη function redundantly with each other to promote PKCí recruitment and subsequent cytoskeletal polarization. Redundancy between PKCε and PKCη is consistent with their observed similarities in recruitment pattern, and possibly explains why PKCε knockout mice display no obvious defect in T cell activation.²⁰

The observation that synaptically polarized lymphocytes, like epithelial cells, partition their membranes into distinct domains has led a number of labs to investigate the roles of polarity complexes during IS formation in T cells. Immunocytochemical studies have demonstrated that Par3 and phosphorylated PKCζ accumulate at the IS while Scribble and Dlg localize to the back of the cell.^26,27 Synaptic recruitment of Par3 is consistent with other work showing that the kinase Par1b, which inhibits Par3 function, dissociates from the plasma membrane in response to TCR stimulation.²⁸ Interestingly, polarized accumulation of the Par and Scrib complexes was only observed after 30 minutes of conjugation, well beyond the time required for MTOC reorientation to the IS (Fig. 4). This temporal discordance suggests that Par and Scrib components may not be involved in the initial polarization event. Nevertheless, functional experiments have indicated that they are required for T cell polarity at some level. Pharmacological inhibition or siRNA knockdown of PKCζ impaired MTOC reorientation,²⁶ as did expression of dominant negative forms of Par1b.²⁸ Knockdown of Scribble also disrupted MTOC localization to the IS, although this result may have been secondary to a profound adhesion defect observed in these T cells.²⁷ Importantly, the position of the MTOC was scored at relatively late timepoints (>20 minutes after TCR stimulation) in all of these studies, leaving open the possibility that initial polarization did occur.

When taken together with the delayed recruitment behavior of Par and Scrib components, these results suggest that polarity complexes may be important for long-term maintenance of the polarized state. Hence, MTOC reorientation to the IS can be divided into two stages: a direction-sensing phase driven by DAG and nPKCs, followed by a stabilization phase that requires the Par and Scrib complexes (Fig. 4). It will be interesting to determine how and when activated T cells transition from the first to the second phase of polarization. It is conceivable that sustained DAG and PKC signaling at the IS could induce the recruitment of polarity complexes. It is also possible that the MTOC, after moving to the IS, could itself trigger the requisite signaling events. The centrosome contains a large number of unique signaling proteins, and the close apposition of these proteins with plasma membrane components at the IS could profoundly affect local signaling dynamics.

Separating MTOC polarization into two distinct steps would presumably allow the transition between these steps to be regulated. In this manner, synapse stability could be tailored to serve specific biological functions. One might imagine that highly stable synapses would be required for targeted cytokine-mediated communication over a period of hours, or to prepare cells for asymmetric division. In contrast, serial killing by cytotoxic lymphocytes, which combines directional secretion of cytolytic factors with rapid movement between target cells, would perhaps be best served by transient direction sensing without subsequent stabilization. Further studies will be required to test these ideas. Clearly, however, close analysis of synaptic polarity has reinforced the concept of the IS as a structure that evolves in time to suite the needs of the lymphocyte and its partner.

We must also guard against focusing on one analogy to the exclusion of others. Our work has demonstrated that MTOC reorientation in T cells is guided by a gradient of a lipid second messenger, DAG, and that perturbing the enzymes responsible for maintaining this gradient, PLCγ and the DGKs, disrupts polarization. This mechanism is remarkably similar to lipid-based direction sensing during leukocyte migration,³⁰ which is based on a polarized phosphatidyl-inositol tris-phosphate (PIP3) gradient that is generated by the coordinated activity of PI-3 kinases and lipid phosphatases (Fig. 1). In retrospect, the similarities between DAG-dependent MTOC polarization and PIP3-dependent direction sensing represents an excellent example of biological analogy, but it was one that we missed while pursuing other hypotheses that were, more often than not, based on different analogies.