Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1

Epac1 is a Rap-specific guanine-nucleotide exchange factor (GEF) which is activated by the binding of cAMP to a cyclic nucleotide monophosphate (cNMP)-binding domain. We investigated the equilibrium and dynamics of the interaction of cAMP and Epac1 using a newly designed fluorescence analogue of cAMP, 8-MABA-cAMP. We observed that the interaction of cAMP, measured by competition with 8-MABA-cAMP, with an isolated cNMP binding domain of Epac1 has an overall equilibrium constant (Kd) of 4 microM and that the kinetics of the interaction are highly dynamic. The binding properties of cAMP are apparently not affected when the catalytic domain is present, despite the fact that binding of cAMP results in activation of Epac1. This indicates that for the activation process, no appreciable binding energy is required. However, when bound to Rap1b, the apparent Kd of Epac to cAMP was about fivefold lower, suggesting that substrate interaction stabilizes cAMP binding. Since the fluorescent analogues used here were either less able or unable to induce activation of Epac1, we concluded that the binding of nucleotide to Epac and the activation of GEF activity are uncoupled processes and that thus appropriate cAMP analogues can be used as inhibitors of the Epac1-mediated signal transduction pathway of Rap.     

Role of microtubule cytoskeleton in regulation of endothelial barrier function

Cytoplasmic microtubules are an obligatory component of the cytoskeleton of all types of cells. Microtubules are involved in many cellular processes including directed transport of vesicles and signaling molecules and changes in cell shape during its spreading, polarization, and movement. The intracellular organization of the system of microtubules and their dynamic properties are different in different types of cells because they play a key role in the implementation of a variety of cell and tissue functions, including the regulation of the endothelial barrier function. This review presents an overview of current studies on the properties of endothelial microtubules, their interaction with other components of the cytoskeleton and cell adhesion structures, and the role of microtubules in the regulation of the endothelial barrier function.     

Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control

Epac1 and its effector Rap1 are important mediators of cAMP induced tightening of endothelial junctions and consequential increased barrier function. We have investigated the involvement of Rap1 signalling in basal, unstimulated, barrier function of a confluent monolayer of HUVEC using real time Electric Cell-substrate Impedance Sensing. Depletion of Rap1, but not Epac1, results in a strong decrease in barrier function. This decrease is also observed when cells are depleted of the cAMP independent Rap exchange factors PDZ-GEF1 and 2, showing that PDZ-GEFs are responsible for Rap1 activity in control of basal barrier function. Monolayers of cells depleted of PDZ-GEF or Rap1 show an irregular, zipper-like organization of VE-cadherin and live imaging of VE-cadherin-GFP reveals enhanced junction motility upon depletion of PDZ-GEF or Rap1. Importantly, activation of Epac1 increases the formation of cortical actin bundles at the cell–cell junctions, inhibits junction motility and restores barrier function of PDZ-GEFs depleted, but not Rap1 depleted cells. We conclude that PDZ-GEF activates Rap1 under resting conditions to stabilize cell–cell junctions and maintain basal integrity. Activation of Rap1 by cAMP/Epac1 induces junctional actin to further tighten cell–cell contacts.     

Rap1 signaling in endothelial barrier control

The small G-protein Rap1 plays an important role in the regulation of endothelial barrier function, a process controlled largely by cell–cell adhesions and their connection to the actin cytoskeleton. During the various stages of barrier dynamics, different guanine nucleotide exchange factors (GEFs) control Rap1 activity, indicating that Rap1 integrates multiple input signals. Once activated, Rap1 induces numerous signaling cascades, together responsible for the increased endothelial barrier function. Most notably, Rap1 activation results in the inhibition of Rho to decrease radial stress fibers and the activation of Cdc42 to increase junctional actin. This implies that Rap regulates endothelial barrier function by dual control of cytoskeletal tension. The molecular details of the signaling pathways are becoming to be elucidated.     

Epac/Rap and PKA are novel mechanisms of ANP-induced Rac-mediated pulmonary endothelial barrier protection

Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP-and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.     

Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function

The passage of leukocytes out of the blood circulation and into tissues is necessary for the normal inflammatory response, but it also occurs inappropriately in many pathological situations. This process is limited by the barrier presented by the junctions between adjacent endothelial cells that line blood vessels. Here we show that activation of the Rap1 GTPase in endothelial cells accelerated de novo assembly of endothelial cell-cell junctions and increased the barrier function of endothelial monolayers. In contrast, depressing Rap1 activity by expressing Rap1GAP led to disassembly of these junctions and increased their permeability. We also demonstrate that endogenous Rap1 was rapidly activated at early stages of junctional assembly, confirming the involvement of Rap1 during junctional assembly. Intriguingly, elevating Rap1 activity selectively within endothelial cells decreased leukocyte transendothelial migration, whereas inhibiting Rap1 activity by expression of Rap1GAP increased leukocyte transendothelial migration, providing physiological relevance to our hypothesis that Rap1 augments barrier function of inter-endothelial cell junctions. Furthermore, these results suggest that Rap1 may be a novel therapeutic target for clinical conditions in which an inappropriate inflammatory response leads to disease.     

PDZ-GEF1, a guanine nucleotide exchange factor specific for Rap1 and Rap2

The small GTPase Rap1 has been implicated in a variety of cellular processes including the control of cell morphology, proliferation, and differentiation. Stimulation of a large variety of cell surface receptors results in the rapid activation of Rap1, i.e. an increase in the GTP-bound form. This activation is mediated by second messengers like calcium, cAMP, and diacylglycerol, but additional pathways may exist as well. Here we describe a ubiquitously expressed guanine nucleotide exchange factor of 200 kDa that activates Rap1 both in vivo and in vitro. This exchange factor has two putative regulatory domains: a domain with an amino acid sequence related to cAMP-binding domains and a PDZ domain. Therefore, we named it PDZ-GEF1. PDZ-GEFs are closely related to Epacs, Rap-specific exchange factors with a genuine cAMP binding site, that are directly regulated by cAMP. The domain related to cAMP-binding domains, like the cAMP binding site in Epac, serves as a negative regulatory domain. However, PDZ-GEF1 does not interact with cAMP or cGMP. Interestingly, PDZ-GEF1 also activates Rap2, a close relative of Rap1. This is the first example of an exchange factor acting on Rap2. We conclude that PDZ-GEF1 is a guanine nucleotide exchange factor, specific for Rap1 and Rap2, that is controlled by a negative regulatory domain.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

Rap1 GTPases: an emerging role in the cardiovasculature

The Ras related GTPase Rap has been implicated in multiple cellular functions. A vital role for Rap GTPase in the cardiovasculature is emerging from recent studies. These small monomeric G proteins act as molecular switches, coupling extracellular stimulation to intracellular signaling through second messengers. This member of the Ras superfamily was once described as the transformation suppressor with the ability to ameliorate the Ras transformed phenotype; however, further studies uncovered a unique set of guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and effector proteins for Rap suggesting a more sophisticated role for this small GTPase. At least three different second messengers can activate Rap, namely cyclic AMP (cAMP), calcium and diacylglycerol. More recently, an investigation of Rap in the cardiovasculature has revealed multiple pathways of regulation involving Rap in this system. Two closely related isoforms of Rap1 exist, 1a and 1b. Murine genetic models exist for both and have been described. Although thought at first to be functionally redundant, these isoforms have differing roles in the cardiovasculature. The activation of Rap1a and 1b in various cell types of the cardiovasculature leads to alterations in cell attachment, migration and cell junction formation. This review will focus on the role of these Rap1 GTPases in hematopoietic, endothelial, smooth muscle, and cardiac myocyte function, and conclude with their potential role in human disease.     

Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

Prostaglandin E(2) (PGE(2)) and prostacyclin are lipid mediators produced by cyclooxygenase and implicated in the regulation of vascular function, wound repair, inflammatory processes, and acute lung injury. Although protective effects of these prostaglandins (PGs) are associated with stimulation of intracellular cAMP production, the crosstalk between cAMP-activated signal pathways in the regulation of endothelial cell (EC) permeability is not well understood. We studied involvement of cAMP-dependent kinase (PKA), cAMP-Epac-Rap1 pathway, and small GTPase Rac in the PGs-induced EC barrier protective effects and cytoskeletal remodeling. PGE(2) and PGI(2) synthetic analog beraprost increased transendothelial electrical resistance and decreased dextran permeability, enhanced peripheral F-actin rim and increased intercellular adherens junction areas reflecting EC barrier-protective response. Furthermore, beraprost dramatically attenuated thrombin-induced Rho activation, MLC phosphorylation and EC barrier dysfunction. In vivo, beraprost attenuated lung barrier dysfunction induced by high tidal volume mechanical ventilation. Both PGs caused cAMP-mediated activation of PKA-, Epac/Rap1- and Tiam1/Vav2-dependent pathways of Rac1 activation and EC barrier regulation. Knockdown of Epac, Rap1, Rac-specific exchange factors Tiam1 and Vav2 using siRNA approach, or inhibition of PKA activity decreased Rac1 activation and PG-induced EC barrier enhancement. Thus, our results show that barrier-protective effects of PGE(2) and prostacyclin on pulmonary EC are mediated by PKA and Epac/Rap pathways, which converge on Rac activation and lead to enhancement of peripheral actin cytoskeleton and adherens junctions. These mechanisms may mediate protective effects of PGs against agonist-induced lung vascular barrier dysfunction in vitro and against mechanical stress-induced lung injury in vivo.     

Rap1 promotes endothelial mechanosensing complex formation,NO release and normal endothelial function

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin‐ and cadherin‐mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO‐dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex—comprised of PECAM‐1, VE‐cadherin and VEGFR2‐ and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.     

Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis

Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2^−/− and wild-type mice. However, the testes of RA-GEF-2^−/− male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2^−/− males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2^−/− mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2^−/− testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice.     

Ras and Rap1: two highly related small GTPases with distinct function

The Ras-like family of small GTPases includes, among others, Ras, Rap1, R-ras, and Ral. The family is characterized by similarities in the effector domain. While the function of Ras is, at least in part, elucidated, little is known about other members of the family. Currently, much attention is focused on the small GTPase Rap1. Initially, this member was identified as a transformation suppressor protein able to revert the morphological phenotype of Ras-transformed fibroblasts. This has led to the hypothesis that Rap1 antagonizes Ras by interfering in Ras effector function. Recent analysis revealed that Rap1 is activated rapidly in response to activation of a variety of receptors. Rap1 activation is mediated by several second messengers, including calcium, diacylglycerol, and cAMP. Guanine nucleotide exchange factors (GEFs) have been identified that mediate these effects. The most interesting GEF is Epac, an exchange protein directly activated by cAMP, thus representing a novel cAMP-induced, protein kinase A-independent pathway. Furthermore, Rap1 is inactivated by specific GTPase-activating proteins (GAPs), one of which is regulated through an interaction with Galphai. While Ras and Rap1 may share some effector pathways, evidence is accumulating that Ras and Rap1 each regulate unique cellular processes in response to various extracellular ligands. For Rap1 these functions may include the control of cell morphology.     

RA-GEF-1, a guanine nucleotide exchange factor for Rap1, is activated by translocation induced by association with Rap1*GTP and enhances Rap1-dependent B-Raf activation

We previously identified RA-GEF-1, a novel guanine nucleotide exchange factor (GEF) for Rap1 with the ability to associate with Rap1.GTP at its Ras/Rap1-associating (RA) domain. Because it possesses a PSD-95/DlgA/ZO-1 (PDZ) domain, it was also named PDZ-GEF. In this report, we have examined the role of the RA domain of this protein in Rap1-mediated cellular responses. A mutant of RA-GEF-1 (RA-GEF-1DeltaRA) carrying a 21-residue deletion at its RA domain fully retains the in vitro GEF activity toward Rap1 but completely loses the Rap1 binding activity. In contrast, RA-GEF-1DeltaRA, expressed in COS-7 cells, exhibits a 3-fold reduction in its in vivo GEF activity toward Rap1 compared with wild-type RA-GEF-1 as examined by the Rap1 pull-down assay. Correspondingly, when coexpressed with wild-type Rap1, RA-GEF-1DeltaRA is unable to further activate B-Raf, whereas RA-GEF-1 stimulates B-Raf as efficiently as activated Rap1. Consistent with these observations, coexpression of activated Rap1 induces translocation of RA-GEF-1, which is otherwise located in the cytoplasm, to the perinuclear compartment, where Rap1 is also predominantly localized. This localization almost coincides with that of the Golgi apparatus, which was detected by anti-trans-Golgi-network 38 antibody. RA-GEF-1DeltaRA fails to show the translocation. These results indicate that RA-GEF-1 defines a novel category of GEF that is translocated to a particular subcellular compartment by association with the GTP-bound form of a small GTPase and catalyzes activation of the GDP-bound form present in the compartment, thereby causing an amplification of cellular responses induced by the small GTPase.     

Ras is required for the cyclic AMP-dependent activation of Rap1 via Epac2

Exchange proteins activated by cAMP (cyclic AMP) 2 (Epac2) is a guanine nucleotide exchange factor for Rap1, a small G protein involved in many cellular functions, including cell adhesion, differentiation, and exocytosis. Epac2 interacts with Ras-GTP via a Ras association (RA) domain. Previous studies have suggested that the RA domain was dispensable for Epac2 function. Here we show for the first time that Ras and cAMP regulate Epac2 function in a parallel fashion and the Ras-Epac2 interaction is required for the cAMP-dependent activation of endogenous Rap1 by Epac2. The mechanism for this requirement is not allosteric activation of Epac2 by Ras but the compartmentalization of Epac2 on the Ras-containing membranes. A computational modeling is consistent with this compartmentalization being a function of both the level of Ras activation and the affinity between Ras and Epac2. In PC12 cells, a well-established model for sympathetic neurons, the Epac2 signaling is coupled to activation of mitogen-activated protein kinases and contributes to neurite outgrowth. Taken together, the evidence shows that Epac2 is not only a cAMP sensor but also a bona fide Ras effector. Coincident detection of both cAMP and Ras signals is essential for Epac2 to activate Rap1 in a temporally and spatially controlled manner.     

Role of adhesion and contraction in Rac 1-regulated endothelial barrier function in vivo and in vitro

We demonstrated previously that inhibition of the small GTPase Rac-1 by Clostridium sordellii lethal toxin (LT) increased the hydraulic conductivity (L(p)) of rat venular microvessels and induced gap formation in cultured myocardial endothelial cells (MyEnd). In MyEnd cells, we also demonstrated that both LT and cytochalasin D reduced cellular adhesion of vascular endothelial (VE)-cadherin-coated beads. Here we further evaluate the contribution of actin depolymerization, myosin-based contraction, and VE-cadherin linkage to the actin cytoskeleton to LT-induced permeability. The actin-depolymerizing agent cytochalasin D increased L(p) in single rat mesenteric microvessels to the same extent as LT over 80 min. However, whereas the actin-stabilizing agent jasplakinolide blunted the L(p) increase due to cytochalasin D by 78%, it had no effect on the LT response. This conforms to the hypothesis that the predominant mechanism whereby Rac-1 stabilizes the endothelial barrier in intact microvessels is separate from actin polymerization and likely at the level of the VE-cadherin linkage to the actin cytoskeleton. In intact vessels, neither inhibition of contraction (butanedione monoxime, an inhibitor of myosin ATPase) nor inhibition of Rho kinase (Y-27632) modified the response to LT, even though both inhibitors lowered resting L(p). In contrast butanedione monoxime and inhibition of myosin light chain kinase completely inhibited LT-induced intercellular gap formation and largely reduced the LT-induced permeability increase in MyEnd monolayers. These results support the hypothesis that the contractile mechanisms that contribute to the formation of large gaps between cultured endothelial cells exposed to inflammatory conditions do not significantly contribute to increased permeability in intact microvessels.     

Discriminatory residues in Ras and Rap for guanine nucleotide exchange factor recognition

The inability of the S17N mutant of Rap1A to sequester the catalytic domain of the Rap guanine nucleotide exchange factor C3G (van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845-850) prompted us to study possible fundamental differences in the way Rap1 interacts with C3G compared with the interaction of Ras with the catalytic domain of the mouse Ras guanine nucleotide exchange factor Cdc25(Mm). A variety of mutants in both Ras and Rap1A were designed, and both the C3G and Cdc25(Mm) catalyzed release of guanine nucleotide from these mutants was studied. In addition, we could identify regions in Rap2A that are responsible for the lack of recognition by C3G and induce high C3G activity by replacement of these residues with the corresponding Rap1A residues. The different Ras and Rap mutants showed that many residues were equally important for both C3G and Cdc25(Mm), suggesting that they interact similarly with their substrates. However, several residues were also identified to be important for the exchange reaction with only C3G (Leu70) or only Cdc25(Mm) (Gln61 and Tyr40). These results are discussed in the light of the structure of the Ras-Sos complex and suggest that some important differences in the interaction of Rap1 with C3G and Ras with Cdc25(Mm) indeed exist and that marker residues have been identified for the different structural requirements.     

Characterisation of PDZ-GEFs,a family of guanine nucleotide exchange factors specific for Rap1 and Rap2

PDZ-GEF1 (RA-GEF/nRapGEP/CNrasGEF) is a guanine nucleotide exchange factor (GEF) characterised by the presence of a PSD-95/DlgA/ZO-1 (PDZ) domain, a Ras-association (RA) domain and a region related to a cyclic nucleotide binding domain (RCBD). These domains are in addition to a Ras exchange motif (REM) and GEF domain characteristic for GEFs for Ras-like small GTPases. PDZ-GEF1 efficiently exchanges nucleotides of both Rap1 and Rap2, but has also been implicated in mediating cAMP-induced Ras activation through binding of cAMP to the RCBD. Here we describe a new family member, PDZ-GEF2, of which we isolated two splice variants (PDZ-GEF2A and 2B). PDZ-GEF2 contains, in addition to the domains characteristic for PDZ-GEF1, a second, less conserved RCBD at the N-terminus. PDZ-GEF2 is also specific for both Rap1 and Rap2. We further investigated the possibility that PDZ-GEF2, like PDZ-GEF1, is a cAMP-responsive GEF for Ras. However, in contrast to previous results, we did not find any effect of either PDZ-GEF1 or PDZ-GEF2 on Ras in the absence or presence of cAMP. Moreover, affinity measurements by isothermic calorimetry showed that the RCBD of PDZ-GEF1 does not bind cAMP with a physiologically relevant affinity. We conclude that both PDZ-GEF1 and 2 are specific for Rap1 and Rap2 and unresponsive to cAMP and various other nucleotides.     

Septin GTPases spatially guide microtubule organization and plus end dynamics in polarizing epithelia

Establishment of epithelial polarity requires the reorganization of the microtubule (MT) cytoskeleton from a radial array into a network positioned along the apicobasal axis of the cell. Little is known about the mechanisms that spatially guide the remodeling of MTs during epithelial polarization. Septins are filamentous guanine triphosphatases (GTPases) that associate with MTs, but the function of septins in MT organization and dynamics is poorly understood. In this paper, we show that in polarizing epithelia, septins guide the directionality of MT plus end movement by suppressing MT catastrophe. By enabling persistent MT growth, two spatially distinct populations of septins, perinuclear and peripheral filaments, steer the growth and capture of MT plus ends. This navigation mechanism is essential for the maintenance of perinuclear MT bundles and for the orientation of peripheral MTs as well as for the apicobasal positioning of MTs. Our results suggest that septins provide the directional guidance cues necessary for polarizing the epithelial MT network.