Microtubule-organizing Center Reorientation Research Articles

The Rap1-cofilin-1 pathway coordinates actin reorganization and MTOC polarization at the B cell immune synapse.

B cells that bind antigens displayed on antigen-presenting cells (APCs) form an immune synapse, a polarized cellular structure that optimizes the dual functions of the B cell receptor (BCR), signal transduction and antigen internalization. Immune synapse formation involves polarization of the microtubule-organizing center (MTOC) towards the APC. We now show that BCR-induced MTOC polarization requires the Rap1 GTPase (which has two isoforms, Rap1a and Rap1b), an evolutionarily conserved regulator of cell polarity, as well as cofilin-1, an actin-severing protein that is regulated by Rap1. MTOC reorientation towards the antigen contact site correlated strongly with cofilin-1-dependent actin reorganization and cell spreading. We also show that BCR-induced MTOC polarization requires the dynein motor protein as well as IQGAP1, a scaffolding protein that can link the actin and microtubule cytoskeletons. At the periphery of the immune synapse, IQGAP1 associates closely with F-actin structures and with the microtubule plus-end-binding protein CLIP-170 (also known as CLIP1). Moreover, the accumulation of IQGAP1 at the antigen contact site depends on F-actin reorganization that is controlled by Rap1 and cofilin-1. Thus the Rap1-cofilin-1 pathway coordinates actin and microtubule organization at the immune synapse.

Polarized granzyme release is required for antigen-driven transendothelial migration of human effector memory CD4 T cells.

Human effector memory CD4 T cells may transmigrate across endothelial cell (EC) monolayers either in response to inflammatory chemokines or in response to TCR recognition of Ag presented on the surface of the EC. The kinetics, morphologic manifestations, and molecular requirements of chemokine- and TCR-driven transendothelial migration (TEM) differ significantly. In this study, we report that, whereas the microtubule organizing center (MTOC) and cytosolic granules follow the nucleus across the endothelium in a uropod during chemokine-driven TEM, MTOC reorientation to the contact region between the T cell and the EC, accompanied by dynein-driven transport of granzyme-containing granules to and exocytosis at the contact region, are early events in TCR-driven, but not chemokine-driven TEM. Inhibitors of either granule function or granzyme proteolytic activity can arrest TCR-driven TEM, implying a requirement for granule discharge in the process. In the final stages of TCR-driven TEM, the MTOC precedes, rather than follows, the nucleus across the endothelium. Thus, TCR-driven TEM of effector memory CD4 T cells appears to be a novel process that more closely resembles immune synapse formation than it does conventional chemotaxis.

Polarized actin structural dynamics in response to cyclic uniaxial stretch.

Endothelial cell (EC) alignment to directional flow or stretch supports anti-inflammatory functions, but mechanisms controlling polarized structural adaptation in response to physical cues remain unclear. This study aimed to determine whether factors associated with early actin edge ruffling implicated in cell polarization are prerequisite for stress fiber (SF) reorientation in response to cyclic uniaxial stretch. Time-lapse analysis of EGFP-actin in confluent ECs showed that onset of either cyclic uniaxial or equibiaxial stretch caused a non-directional increase in edge ruffling. Edge activity was concentrated in a direction perpendicular to the stretch axis after 60 min, consistent with the direction of SF alignment. Rho-kinase inhibition caused reorientation of both stretch-induced edge ruffling and SF alignment parallel to the stretch axis. Arp2/3 inhibition attenuated stretch-induced cell elongation and disrupted polarized edge dynamics and microtubule organizing center reorientation, but it had no effect on the extent of SF reorientation. Disrupting localization of p21-activated kinase (PAK) did not prevent stretch-induced SF reorientation, suggesting that this Rac effector is not critical in regulating stretch-induced cytoskeletal remodeling. Overall, these results suggest that directional edge ruffling is not a primary mechanism that guides SF reorientation in response to stretch; the two events are coincident but not causal.

From lipid second messengers to molecular motors: microtubule-organizing center reorientation in T cells.

In T lymphocytes, polarization of the microtubule-organizing center (MTOC) to the immunological synapse enables the directional secretion of cytokines, cytolytic factors, and other soluble molecules toward the antigen-presenting cell. This is likely to be crucial for maintaining the specificity of T-cell effector responses. Here, we review recent advances in our understanding of MTOC reorientation in T cells, focusing first on the importance of diacylglycerol and protein kinase C isozymes and then on the molecular motor proteins that function downstream to drive MTOC movement.

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

Paxillin Associates with the Microtubule Cytoskeleton and the Immunological Synapse of CTL through Its Leucine-Aspartic Acid Domains and Contributes to Microtubule Organizing Center Reorientation

The cytoskeletal adaptor protein paxillin localizes to the microtubule organizing center (MTOC) in T cells and, upon target cell binding, is recruited to the supramolecular activation complex (SMAC). We mapped the region of paxillin that associates with both the MTOC and SMAC to the leucine-aspartic acid (LD) domains and showed that a protein segment containing LD2-4 was sufficient for MTOC and SMAC recruitment. Examination of the localization of paxillin at the SMAC revealed that paxillin localizes to the peripheral area of the SMAC along with LFA-1, suggesting that LFA-1 may contribute to its recruitment. LFA-1 or CD3 engagement alone was insufficient for paxillin recruitment because there was no paxillin accumulation at the site of CTL contact with anti-LFA-1- or anti-CD3-coated beads. In contrast, paxillin accumulation was detected when beads coated with both anti-CD3 and anti-LFA-1 were bound to CTL, suggesting that signals from both the TCR and LFA-1 are required for paxillin accumulation. Paxillin was shown to be phosphorylated downstream of ERK, but when we generated a mutation (S83A/S130A) that abolished the mobility shift as a result of phosphorylation, we found that paxillin still bound to the MTOC and was recruited to the SMAC. Furthermore, ERK was not absolutely required for MTOC reorientation in CTL that require ERK for killing. Finally, expression of the LD2-4 region of paxillin substantially reduced MTOC reorientation. These studies demonstrated that paxillin is recruited, through its LD domains, to sites of integrin engagement and may contribute to MTOC reorientation required for directional degranulation.

Lymphocyte polarity, the immunological synapse and the scope of biological analogy

Lymphocytes such as T cells, B cells and natural killer (NK) cells form specialized contacts, called immunological synapses, with other cells in order to engage in specific intercellular communication and killing. Synapse formation is associated with the polarization of the microtubule-organizing center (MTOC) toward the contact site, which enables the directional secretion of cytokines and lytic factors. Although MTOC reorientation to the synapse is crucial for lymphocyte function, it has been difficult to study because of technical constraints. We have developed a photoactivation and imaging strategy that enables high-resolution analysis of cytoskeletal dynamics in individual T cells. Using this approach, we have demonstrated that the lipid second messenger diacylglycerol plays a crucial role in promoting MTOC reorientation by recruiting three members of the protein kinase C family to the synapse. Here, I will discuss these results along with studies from other labs, which have explored the role of polarity-inducing protein complexes after synapse formation. I will also propose a two-step model for MTOC reorientation in lymphocytes that reflects what we now know about the subject. Finally, I will consider the extent to which lymphocyte polarity resembles analogous cell polarity systems in other cell types.

SH3BP1, an Exocyst-Associated RhoGAP, Inactivates Rac1 at the Front to Drive Cell Motility

The coordination of the several pathways involved in cell motility is poorly understood. Here, we identify SH3BP1, belonging to the RhoGAP family, as a partner of the exocyst complex and establish a physical and functional link between two motility-driving pathways, the Ral/exocyst and Rac signaling pathways. We show that SH3BP1 localizes together with the exocyst to the leading edge of motile cells and that SH3BP1 regulates cell migration via its GAP activity upon Rac1. SH3BP1 loss of function induces abnormally high Rac1 activity at the front, as visualized by in vivo biosensors, and disorganized and instable protrusions, as revealed by cell morphodynamics analysis. Consistently, constitutively active Rac1 mimics the phenotype of SH3BP1 depletion: slow migration and aberrant cell morphodynamics. Our finding that SH3BP1 downregulates Rac1 at the motile-cell front indicates that Rac1 inactivation in this location, as well as its activation by GEF proteins, is a fundamental requirement for cell motility.

A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell is driven by the accumulation of diacylglycerol at the immunological synapse (IS). The mechanisms that couple diacylglycerol to the MTOC are not known. Using single-cell photoactivation of the T cell receptor, we demonstrated that three distinct protein kinase C (PKC) isoforms are recruited by diacylglycerol to the IS in two steps. PKC-ε and PKC-η accumulated first in a broad region of membrane, while PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation, and that PKC-ε and PKC-η operated redundantly to promote PKC-θ recruitment and subsequent polarization responses. These results establish a previously uncharacterized role for PKCs in T cell polarity.

Paxillin associates with the microtubule cytoskeleton and the immunological synapse of CTL through its LD domains and contributes to microtubule organizing center reorientation (112.24)

Abstract The cytoskeletal adaptor protein paxillin localizes to the microtubule organizing center (MTOC) in T cells and upon target cell binding is recruited to the supramolecular activation complex (SMAC). We mapped the region of paxillin that associates with both the MTOC and SMAC to the leucine-aspartic acid (LD) domains and showed that a protein segment containing LD2-4 was sufficient for MTOC and SMAC recruitment. Paxillin localizes to the peripheral area of the SMAC along with LFA-1 suggesting that LFA-1 may contribute to its recruitment. To test this possibility, beads coated with anti-CD3 were used alongside non-antigen bearing target cells, thus physically separating adhesion and TCR signals. Paxillin preferentially localized to sites of integrin engagement rather than to sites of TCR engagement, further suggesting that LFA-1 contributes to paxillin recruitment. These sites of antigen-independent integrin binding were not sufficient to induce MTOC reorientation. Paxillin has been shown to be phosphorylated downstream of ERK, but when we generated a mutation that abolished detectable phosphorylation we found that paxillin still bound to the MTOC and was recruited to the SMAC. Finally, expression of the LD2-4 region of paxillin reduced MTOC reorientation. These studies demonstrate that paxillin is recruited, through its LD domains, to sites of integrin engagement and may contribute to CTL adhesion and subsequent MTOC reorientation required for directional degranulation.

Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells

The reorientation of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell enables the directional secretion of cytokines and lytic factors. By single-cell photoactivation of the T cell antigen receptor, we show that MTOC polarization is driven by localized accumulation of diacylglycerol (DAG). MTOC reorientation was closely preceded first by production of DAG and then by recruitment of the microtubule motor protein dynein. Blocking DAG production or disrupting the localization of DAG impaired MTOC recruitment. Localized DAG accumulation was also required for cytotoxic T cell-mediated killing. Furthermore, photoactivation of DAG itself was sufficient to induce transient polarization. Our data identify a DAG-dependent pathway that signals through dynein to control microtubule polarity in T cells.

Receptor Tyrosine Kinase Ror2 Mediates Wnt5a-induced Polarized Cell Migration by Activating c-Jun N-terminal Kinase via Actin-binding Protein Filamin A

The receptor tyrosine kinase Ror2 has recently been shown to act as an alternative receptor or coreceptor for Wnt5a and to mediate Wnt5a-induced migration of cultured cells. However, little is known about the molecular mechanism underlying this migratory process. Here we show by wound-healing assays that Ror2 plays critical roles in Wnt5a-induced cell migration by regulating formation of lamellipodia and reorientation of microtubule-organizing center (MTOC). Wnt5a stimulation induces activation of the c-Jun N-terminal kinase JNK at the wound edge in a Ror2-dependent manner, and inhibiting JNK activity abrogates Wnt5a-induced lamellipodia formation and MTOC reorientation. Additionally, the association of Ror2 with the actin-binding protein filamin A is required for Wnt5a-induced JNK activation and polarized cell migration. We further show that Wnt5a-induced JNK activation and MTOC reorientation can be suppressed by inhibiting PKCzeta. Taken together, our findings indicate that Wnt5a/Ror2 activates JNK, through a process involving filamin A and PKCzeta, to regulate polarized cell migration.

CAMP Inhibits Cell Migration by Interfering with Rac-induced Lamellipodium Formation

Cell migration is critical for animal development and physiological as well as pathological responses. One important step during cell migration is the formation of lamellipodia at the leading edge of migrating cells. Here we report that the second messenger cAMP inhibits the migration of mouse embryonic fibroblast cells and mouse breast tumor cells. cAMP acts downstream of the small GTPase Rac and interferes with the formation of lamellipodia. Moreover, cAMP decreases the phosphorylation of the myosin light chain at the leading edge of cells and increases the phosphorylation of the vasodilator-stimulated phosphoprotein. Together with our previous report of a positive role of another second messenger, cGMP, in lamellipodium formation, our data indicate that cAMP and cGMP play opposite roles in modulating lamellipodium formation.

The Formin mDia Regulates GSK3β through Novel PKCs to Promote Microtubule Stabilization but Not MTOC Reorientation in Migrating Fibroblasts

In migrating cells, external signals polarize the microtubule (MT) cytoskeleton by stimulating the formation of oriented, stabilized MTs and inducing the reorientation of the MT organizing center (MTOC). Glycogen synthase kinase 3beta (GSK3beta) has been implicated in each of these processes, although whether it regulates both processes in a single system and how its activity is regulated are unclear. We examined these issues in wound-edge, serum-starved NIH 3T3 fibroblasts where MT stabilization and MTOC reorientation are triggered by lysophosphatidic acid (LPA), but are regulated independently by distinct Rho GTPase-signaling pathways. In the absence of other treatments, the GSK3beta inhibitors, LiCl or SB216763, induced the formation of stable MTs, but not MTOC reorientation, in starved fibroblasts. Overexpression of GSK3beta in starved fibroblasts inhibited LPA-induced stable MTs without inhibiting MTOC reorientation. Analysis of factors involved in stable MT formation (Rho, mDia, and EB1) showed that GSK3beta functioned upstream of EB1, but downstream of Rho-mDia. mDia was both necessary and sufficient for inducing stable MTs and for up-regulating GSK3beta phosphorylation on Ser9, an inhibitory site. mDia appears to regulate GSK3beta through novel class PKCs because PKC inhibitors and dominant negative constructs of novel PKC isoforms prevented phosphorylation of GSK3beta Ser9 and stable MT formation. Novel PKCs also interacted with mDia in vivo and in vitro. These results identify a new activity for the formin mDia in regulating GSK3beta through novel PKCs and implicate novel PKCs as new factors in the MT stabilization pathway.

Role of Fyn in the Rearrangement of Tubulin Cytoskeleton Induced through TCR

The translocation of the microtubule-organizing center (MTOC), its associated signaling complex, and the secretory apparatus is the most characteristic early event that involves the tubulin cytoskeleton of T or NK cells after their interaction with APC or target cells. Our results show that Fyn kinase activity is essential for MTOC reorientation in an Ag-dependent system. Moreover, T cells from Fyn-deficient mice are unable to rearrange their tubulin cytoskeleton in response to anti-CD3-coated beads. Analysis of conjugates of T cells from transgenic OT-I mice with dendritic cells revealed that an antagonist peptide induces translocation of the MTOC, and that this process is impaired in T cells from Fyn(-/-) OT-I mice. In addition, Fyn deficiency significantly affects the MTOC relocation mediated by agonist peptide stimulation. These results reveal Fyn to be a key regulator of tubulin cytoskeleton reorganization in T cells.

Nuclear Movement Regulated by Cdc42, MRCK, Myosin, and Actin Flow Establishes MTOC Polarization in Migrating Cells

The microtubule-organizing center (MTOC) is reoriented between the nucleus and the leading edge in many migrating cells and contributes to directional migration. Models suggest that the MTOC is moved to its position during reorientation. By direct imaging of wound-edge fibroblasts after triggering MTOC reorientation with soluble factors, we found instead that the nucleus moved away from the leading edge to reorient the MTOC, while the MTOC remained stationary. Rearward nuclear movement was coupled with actin retrograde flow and was regulated by a pathway involving Cdc42, MRCK, myosin, and actin. Nuclear movement was unaffected by the inhibition of dynein, Par6, or PKCzeta, yet these components were essential for MTOC reorientation, as they maintained the MTOC at the cell centroid. These results show that nuclear repositioning is an initial polarizing event in migrating cells and that the positions of the nucleus and the MTOC are established by separate regulatory pathways.

Regulation of microtubule‐organizing center orientation and actomyosin cytoskeleton rearrangement during immune interactions

The reorganization of membrane, cytoskeletal and signaling molecules during immune interactions is critical for the generation of immune response. At the initiation of the T cell-antigen presenting cell (APC) interaction, antigen-independent weak adhesion forces allow the scanning of the APC surface by the T cell receptor for specific antigens. The stabilization of T cell-APC conjugates involves the segregation of membrane and intracellular signaling proteins, driven by reorganization of membrane microdomains and cytoskeletal changes. In early T cell-APC cognate interactions, the microtubular cytoskeleton undergoes drastic changes that lead to microtubule-organizing center (MTOC) reorientation to the vicinity of the cell-cell contact area. Recent data on the dynamics of MTOC redistribution and its influence in T cell-APC conjugate stabilization, together with the description of an increasing number of signaling molecules associated to this complex, underscore the key role of MTOC translocation in the T cell response. We focus on the mechanisms that control the early MTOC reorientation during T cell-APC interaction and the relevance of this process to T cell activation.

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1–3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.

CD8+ T cell cytolytic activity independent of mitogen-activated protein kinase / extracellular regulatory kinase signaling (MAP kinase / ERK)

Cytotoxicity is a major effector function of CD8(+) T cells. Although mitogen-activated protein kinase (MAP kinase) / extracellular regulatory kinase (ERK) activity is indispensable for cytotoxic activity of most CD8(+) T cells a portion of CD8(+) T cells appears resistant to MEK inhibition as cytotoxicity of bulk cultures was partially preserved in the presence of a MEK inhibitor. We have also identified a long-term CD8(+) T cell line with unaltered cytolytic activity after prevention of ERK activation. Antigen-induced microtubule organizing center (MTOC) reorientation was not prevented in this CD8(+) cell line by MEK inhibition, in sharp contrast to the MTOC reorientation prevention in a CD8(+) T cell clone with MEK inhibition-sensitive cytolytic activity. These findings suggest that resistance of lysis to MEK inhibition may be due to a lack of ERK control over MTOC reorientation in some CD8(+) T cells. Thus, there appears to be a heterogeneity of ERK-regulated cytolytic activity in CD8(+) T cells, most likely resulting from a differential control of ERK over MTOC motility.

TCR, LFA-1, and CD28 Play Unique and Complementary Roles in Signaling T Cell Cytoskeletal Reorganization

T cells interacting with APCs undergo rearrangement of surface receptors and cytoskeletal elements to face the zone of contact with the APC. This polarization process is thought to affect T cell signaling by organizing a specialized domain on the T cell surface and to direct T cell effector function toward the appropriate APC. We have investigated the contribution of TCR, CD28, and LFA-1 signaling to T cell cytoskeletal polarization by assaying the response of an Ag-specific Th1 clone toward a panel of transfected APCs expressing MHC class II alone or in combination with ICAM-1 or B7-1. We show that polarization of talin, an actin-binding protein, occurs in response to integrin engagement. In contrast, reorientation of the T cell microtubule-organizing center (MTOC) is dependent on and directed toward the site of TCR signaling, regardless of whether integrins or costimulatory molecules are engaged. MTOC reorientation in response to peptide-MHC complexes is sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. CD28 coengagement overcomes this sensitivity, as does activation via Ab cross-linking of the TCR or via covalent peptide-MHC complexes, suggesting that phosphatidylinositol 3-kinase is not required per se but rather plays a role in signal amplification. Engagement of TCR in trans with LFA-1 results in separation of MTOC reorientation and cortical cytoskeletal polarization events, indicating that the two processes are not directly mechanistically linked. These studies show that T cells mobilize individual cytoskeletal components in response to distinct and specific cell surface interactions.