Lateral Border Recycling Compartment Research Articles

Human CD99L2 Regulates a Step in TEM Independent of PECAM and CD99 Regulated Steps

CD99‐like 2 (CD99L2; L2) is a highly glycosylated 52 kDa type‐1 membrane protein that is important for leukocyte transendothelial migration (TEM) in mice. Inhibiting L2 using function blocking antibody or genetic ablation significantly reduces the recruitment of leukocytes to sites of inflammation in vivo. Similarly, endothelial L2 knockout mice have an inherent defect in leukocyte transmigration into sites of inflammation, but not the preceding steps in inflammation. The position of neutrophil arrest suggests L2 regulates a step in TEM independent of platelet endothelial cell adhesion molecule (PECAM). However, the role of L2 in inflammation has only been studied in mice and the mechanism by which it regulates TEM is not known.In order to study the relevance to human inflammation, we studied the role of L2 on primary human cells in vitro. Using immunofluorescence and flow cytometry, our data show that human L2 is constitutively expressed at the borders of endothelial cells and on the surfaces of leukocytes. Inhibiting L2 using antibody blockade, L2‐Fc chimera decoy molecules, or genetic knockdown in endothelial cells significantly reduced transmigration of human neutrophils and monocytes across endothelial cells. Furthermore, our data also show that L2 regulates a specific, sequential step of TEM between PECAM and CD99, rather than operating in parallel or redundantly with these molecules. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment (LBRC) to sites of TEM, specifically downstream of PECAM initiation. Collectively, our data identify a novel functional role for human L2 in TEM and elucidate a mechanism that is distinct from PECAM and CD99.

Spatiotemporal restriction of endothelial cell calcium signaling is required during leukocyte transmigration

Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.

Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis.

Transendothelial migration (TEM) of leukocytes across the endothelium is critical for inflammation. In the endothelium, TEM requires the coordination of membrane movements and cytoskeletal interactions, including, prominently, recruitment of the lateral border recycling compartment (LBRC). The scaffold protein IQGAP1 was recently identified in a screen for LBRC-interacting proteins. Knockdown of endothelial IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. Expression of truncated IQGAP1 constructs demonstrated that the calponin homology domain is required for IQGAP1 localization to endothelial borders and that the IQ domain, on the same IQGAP1 polypeptide, is required for its function in TEM. This is the first reported function of IQGAP1 requiring two domains to be present on the same polypeptide. Additionally, we show for the first time that IQGAP1 in the endothelium is required for efficient TEM in vivo. These findings reveal a novel function for IQGAP1 and demonstrate that IQGAP1 in endothelial cells facilitates TEM by directing the LBRC to the site of TEM.

Endothelial Cell IQGAP1 is Required to Support Efficient Leukocyte Transmigration both In Vitro and In Vivo

Inflammation is the body's fundamental response to tissue insult and damage. When operating correctly, it allows the organism to fight infection, repair injuries, and return the body to homeostasis. When the response is incorrectly directed, whether insufficient, excessive, or against an improper target, it contributes to and even directly causes numerous human pathologies. One of the critical components of inflammation is the recruitment of leukocytes to the target site. This process is a carefully choreographed series of sequential steps that leads to the net efflux of leukocytes out of the blood stream and into tissue. In the culminating and putative committed stage, the leukocyte squeezes between adjacent endothelial cells in a process commonly referred to as transendothelial migration (TEM). TEM is a critical step that requires several intercellular interactions and thus represents an attractive therapeutic target for regulating aberrant inflammation. During TEM, endothelial cells direct the lateral border recycling compartment (LBRC), a sub‐junctional membrane network, to the site of leukocyte migration to facilitate its passage. All manipulations that affect the function and delivery of the LBRC substantially reduce leukocyte efflux.To better understand the molecular components and function of the LBRC, we recently conducted a screen for LBRC‐interacting proteins and identified IQGAP1. IQGAP1 is a large multi‐domain cytoplasmic protein that is reported to bind several signaling proteins in other systems. Knockdown of endothelial cell IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. To determine which domains of IQGAP1 are required for its function in TEM, we expressed a series of truncated constructs and examined their ability to rescue the knockdown of endogenous IQGAP1. We found that the actin‐binding domain of IQGAP1 is required for its localization to endothelial borders. Furthermore, both the actin‐binding domain and the IQ‐motifs are required for its function in TEM. We extended these studies in vivo by generating bone marrow chimeras, in which IQGAP1 knockout animals were reconstituted with wild‐type leukocytes. These studies show that IQGAP1 in endothelium is required for efficient TEM in vivo in two different inflammatory models. Together these findings detail a novel function for IQGAP1 in TEM that involves both its actin‐binding domain and IQ motifs which together coordinate the directed movement of the LBRC to the site of TEM.Support or Funding InformationFunding Support: R01HL046849, R01HL064774This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of IQGAP1 in transendothelial migration: from in vitro identification to in vivo validation

Although inflammation is the body's stereotyped response to tissue damage, the clinical outcomes of this complex process, whether beneficial or detrimental, are rooted in the recruitment and subsequent function of the leukocytes at site of insult. It therefore follows that the regulation of leukocyte recruitment represents an acutely tractable target for therapeutic intervention. In order to reach the target tissue, leukocytes must undergo a carefully choreographed series of sequential intercellular interactions with the endothelium which culminate in transendothelial migration (TEM). As the leukocyte prepares to traverse the endothelial cell layer requisite protein‐protein interactions between the two cells directs the recruitment of the endothelial cell sub‐junctional Lateral Border Recycling Compartment (LBRC). Several of the endothelial proteins crucial for this process reside in the LBRC and its function in TEM is required for efficient leukocyte efflux.To identify additional proteins that are involved in LBRC function in an unbiased approach, we adapted a biochemical purification method known as the diaminobenzidine‐induced density shift to specifically label and recover membrane fractions enriched the LBRC proteins. Through this process we identified IQ domain containing GTPase Activating Protein 1 (IQGAP1) as a previously unknown LBRC associated proteins. IQGAP1 is a cytoplasmic multidomain scaffolding protein that has been shown in other systems to integrate and propagate complex signaling pathways. Genetic ablation of IQGAP1 in endothelial cell cultures using shRNA transfection substantially reduced their ability to support efficient monocyte TEM. Re‐expression of full‐length IQGAP1 rescued this defect, confirming its essential function in this process. Interestingly, re‐expression of IQGAP1 constructs that lack the actin‐binding or IQ‐motif domains were not able to rescue the TEM defect shRNA mediated reduction of IQGAP1 expression did not affect the physical amount of LBRC membrane that could be isolated from endothelial cells suggesting instead that IQGAP1 plays a role in some signaling aspect or other function of the LBRC. To extend these studies in vivo we created bone marrow chimeras (i.e. wild type leukocytes in IQGAP1 knockout animals) and investigated the requirement of endothelial cell IQGAP1 in inflammation. We used two separate mouse models; croton oil dermatitis visualized with conventional confocal microscopy and IL‐1β induced inflammation in the cremaster muscle visualized by 4D intravital microscopy. In both models the ability of neutrophils to extravasate across the endothelium into inflamed tissues was significantly inhibited in animals that lacked IQGAP1. Together with our in vitro studies, these findings provide important insight into the critical role of endothelial cell IQGAP1 in leukocyte diapedesis.Support or Funding InformationThis research was supported by F32 AI084454 to DPS and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.

Identifying Key Domains in Endothelial IQGAP1 Critical for Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. The majority of leukocytes undergo transendothelial migration (TEM) by using a unique endothelial cell membrane system referred to as the lateral border recycling compartment (LBRC). IQ‐domain GTPase‐activating protein 1 (IQGAP1) was identified as an LBRC‐associated protein and subsequently found to be essential for TEM. IQGAP1 is a scaffolding protein comprised of six major interaction domains. It has been reported to interact productively with over 135 cellular proteins. In endothelial cells IQGAP1 partially localizes to cell borders and knocking down its expression prevents TEM. However, it is not known which of the many proteins it is known to interact with carries out its role in TEM. Since these proteins bind to specific IQGAP1 domains, as a first step toward understanding the mechanism by which IQGAP1 regulates TEM, we explored which of its six protein interactions domains were critical for its function. We knocked down IQGAP1 in endothelial cells and re‐expressed truncated forms of IQGAP1 with specific regions deleted to identify the regions (and thereby the potential binding partners) that are critical for TEM. We show that the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM.Human endothelial cells in which >80% of endogenous IQGAP1 was depleted by shRNA did not support TEM; re‐expression of wild‐type IQGAP1 restored TEM to control levels. Re‐expression of IQGAP1 with a GFP‐tagged construct after knockdown also showed IQGAP1 enrichment around transmigrating leukocytes. Re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM. Constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.After identifying the IQ domain as having a critical role in TEM, we generated specific point mutations in this domain within the full‐length construct. In particular, the isoleucine (I)‐glutamine (Q) motif after which the IQ domain is named has been identified in the literature as having an essential role in IQGAP1 function and we are currently testing whether they are also important in TEM. Using these constructs in future studies will help identify the role of the IQ domain in TEM and its interacting partner(s). Identifying the binding partners of IQGAP1 that are critical for inflammation will provide clues to its mechanism of action and potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationFunded by R01 HL046849, R01 HL064774, Alpha Omega Alpha Carolyn Kuckein Student Research Fellowship

Transendothelial migration: unifying principles from the endothelial perspective.

Transendothelial migration (TEM) of polymorphonuclear leukocytes (PMN) involves a carefully orchestrated dialog of adhesion and signaling events between leukocyte and endothelial cell. This article focuses on the contribution of endothelial cells to transmigration. The initiation of TEM itself generally requires interaction of PECAM on the leukocyte with PECAM at the endothelial cell border. This is responsible for the transient elevation of cytosolic-free calcium ions in endothelium that is required for TEM and for recruitment of membrane from the lateral border recycling compartment (LBRC). TEM requires LBRC to move to the site at which TEM will take place and for VE-cadherin to move away. Targeting of the LBRC to this site likely precedes movement of VE-cadherin and may play a role in clearing VE-cadherin from the site of TEM. The process of TEM can be dissected into steps mediated by distinct pairs of PMN/endothelial interacting molecules. CD99 regulates a step at or close to the end of TEM. CD99 signals through soluble adenylyl cyclase to activate PKA to trigger ongoing targeted recycling of the LBRC. Paracellular transmigration predominates (≥90% of events) in the cremaster muscle circulation, but transcellular migration may be more important at sites such as the blood-brain barrier. Both processes involve many of the same molecules and recruitment of the LBRC.

A Unique Role for Endothelial Cell Kinesin Light Chain 1, Variant 1 in Leukocyte Transendothelial Migration

A reservoir of parajunctional membrane in endothelial cells, the lateral border recycling compartment (LBRC), is critical for transendothelial migration (TEM). We have previously shown that targeted recycling of the LBRC to the site of TEM requires microtubules and a kinesin molecular motor. However, the identity of the kinesin and mechanism of cargo binding were not known. We show that microinjection of endothelial cells with a monoclonal antibody specific for kinesin-1 significantly blocked LBRC-targeted recycling and TEM. In complementary experiments, knocking down KIF5B, a ubiquitous kinesin-1 isoform, in endothelial cells significantly decreased targeted recycling of the LBRC and leukocyte TEM. Kinesin heavy chains move cargo along microtubules by one of many kinesin light chains (KLCs), which directly bind the cargo. Knocking down KLC 1 isoform variant 1 (KLC1C) significantly decreased LBRC-targeted recycling and TEM, whereas knocking down other isoforms of KLC1 had no effect. Re-expression of KLC1C resistant to the knockdown shRNA restored targeted recycling and TEM. Thus kinesin-1 and KLC1C are specifically required for targeted recycling and TEM. These data suggest that of the many potential combinations of the 45 kinesin family members and multiple associated light chains, KLC1C links the LBRC to kinesin-1 (KIF5B) during targeted recycling and TEM. Thus, KLC1C can potentially be used as a target for anti-inflammatory therapy.

Targeted Recycling of the Lateral Border Recycling Compartment Precedes Adherens Junction Dissociation during Transendothelial Migration

Leukocyte transendothelial migration (TEM) requires two major events: local dissociation of adherens junctions manifested as gaps in vascular endothelial (VE)-cadherin staining at the site of TEM and targeted trafficking of the lateral border recycling compartment (LBRC) to the site of TEM. However, the association between LBRC recycling and VE-cadherin gaps remains unknown. We found that when targeting of the LBRC is selectively inhibited using established methods, such as a function blocking anti-platelet endothelial cell adhesion molecule 1 antibody, depolymerizing microtubules, or microinjection of an antibody that inhibits kinesin, VE-cadherin gaps do not form around the blocked leukocyte. This is the first time that the LBRC has been implicated in this process. We obtained similar results for neutrophils and monocytes and in studies using live cell imaging microscopy conducted under fluid shear conditions. Depolymerizing microtubules did not affect the ability of leukocytes to induce tyrosine phosphorylation of VE-cadherin. A VE-cadherin double mutant (Y658F, Y731F) expressed in endothelial cells acted as a dominant negative and inhibited VE-cadherin gap formation and TEM, yet targeting of the LBRC still occurred. These data suggest that targeting of the LBRC to the site of TEM precedes VE-cadherin clearance. Recruitment of the LBRC may play a role in clearing VE-cadherin from the site of TEM.

The regulation of transendothelial migration: new knowledge and new questions.

Leucocyte transendothelial migration (TEM) involves a co-operative series of interactions between surface molecules on the leucocyte and cognate counter-ligands on the endothelial cell. These interactions set up a cascade of signalling events inside the endothelial cell that both allow for the junctions to loosen and for membrane to be recruited from the lateral border recycling compartment (LBRC). The LBRC is thought to provide an increased surface area and unligated receptors to the leucocyte to continue the process. The relative importance of the individual adhesion/signalling molecules that promote transmigration may vary depending on the type of leucocyte, the vascular bed, the inflammatory stimulus, and the stage of the inflammatory response. However, the molecular interactions between leucocyte and endothelial cell activate signalling pathways that disengage the adherens and tight junctions and recruit the LBRC to the site of transmigration. With the exception of disengaging the junctions, similar molecules and mechanisms promote transcellular migration as paracellular migration of leucocytes. This review will discuss the molecular interactions and signalling pathways that regulate transmigration, and the common themes that emerge from studying TEM of different leucocyte subsets under different inflammatory conditions. We will also raise some unanswered questions in need of future research.

Critical Roles for the IQ‐Motif and Actin‐Bnding Domain of Endothelial Cell IQGAP1 During Leukocyte Transendothelial Migration

In order to mount an appropriate inflammatory response, leukocytes must cross the endothelium to gain access to the target tissue and carry out their essential roles. This process is referred to as transendothelial migration (TEM) and requires a novel endothelial cell membrane compartment, the Lateral Border Recycling Compartment (LBRC), which is delivered to the migrating leukocyte to supply it with several requisite membrane proteins. IQGAP1, a large soluble scaffolding protein, is associated with the LBRC and is required for its function during TEM. In a variety of other processes, IQGAP1 integrates signals from diverse pathways to regulate cytoskeletal remodeling, Rho family GTPase activity, and other downstream readouts, however its role in TEM remains unclear. Here we show that genetic ablation of endothelial IQGAP1 expression arrests leukocytes during TEM by preventing the targeted delivery of the LBRC. Expression of a full length GFP‐tagged IQGAP1 construct completely restores this defect and also shows that endothelial IQGAP1 is enriched around the leukocyte during TEM. Furthermore, using fluorescently tagged IQGAP1 truncation constructs, we show that the actin‐binding domain of IQGAP1 is required for its localization to the junction in resting endothelial cells and its recruitment and function during TEM. Interestingly, the IQ motif of IQGAP1, which facilitates its interaction with calmodulin, is not required for localization to the junction or recruitment during TEM, but is necessary for IQGAP1′s function during TEM. Together these data suggest that IQGAP1 integrates signals from various sources to facilitate the function and recruitment of the LBRC during TEM. Supported by R01 HL046849 and R37 HL064774.

Endothelial CD99 Signals Through Soluble Adenylyl Cyclase and PKA to Regulate Leukocyte Transendothelial Migration

CD99 is a critical regulator of leukocyte transendothelial migration (TEM, diapedesis); however, it remains entirely unknown how CD99 signals during this process. In this study, we find that engagement of endothelial cell (EC) CD99 during TEM activates protein kinase A (PKA) via soluble adenylyl cyclase (sAC). Active PKA stimulates membrane trafficking from the lateral border recycling compartment (LBRC) to sites of leukocyte diapedesis, facilitating the passage of leukocytes across the EC junction. Pharmacologic or genetic inhibition of sAC, or PKA, recapitulates the phenotype of CD99 blockade on TEM, arresting leukocytes partway through EC junctions, both in vitro and in vivo. In contrast, inhibition of transmembrane adenylyl cyclases did not produce such effects. Furthermore, we show that a positively charged juxtamembrane region in the cytoplasmic tail of CD99 is critical for interacting with sAC and the A‐kinase anchoring protein, ezrin. Together, this signaling complex is responsible for the ability of CD99 to govern leukocyte extravasation. This is the first description of the CD99 signaling pathway in TEM as well as the first implication of sAC in leukocyte extravasation.

Isolation of the lateral border recycling compartment using a diaminobenzidine-induced density shift.

The migration of leukocytes across the endothelium and into tissue is critical to mounting an inflammatory response. The lateral border recycling compartment (LBRC), a complex vesicular-tubule invagination of the plasma membrane found at endothelial cell borders, plays an important role in this process. Although a few proteins have been shown to be present in the LBRC, no unique marker is known. Here, we detail methods that can be used to characterize a subcellular compartment that lacks an identifying marker. Initial characterization of the LBRC was performed using standard subcellular fractionation with sucrose gradients and took advantage of the observation that the compartment migrated at a lower density than other membrane compartments. To isolate larger quantities of the compartment, we modified a classic technique known as a diaminobenzidine (DAB)-induced density shift. The DAB-induced density shift allowed for specific isolation of membranes labeled with horseradish peroxidase-conjugated antibody. Because the LBRC could be differentially labeled at 4 °C and 37 °C, we were able to identify proteins that are enriched in the compartment, despite lacking a unique marker. These methods serve as a model to others studying poorly characterized compartments and organelles and are applicable to a wide variety of biological systems.

Endothelial cell IQGAP1 is an important regulator of the transendothelial migration of leukocytes (146.1)

In order to mount an appropriate inflammatory response, leukocytes must cross the endothelium to gain access to the target tissue and carry out their essential role. During this process (referred to as transendothelial migration, TEM) membrane from a novel endothelial cell compartment, the Lateral Border Recycling Compartment (LBRC), is delivered to the migrating leukocyte to supply it with several requisite proteins. In a proteomic screen, we discovered that IQGAP1 is associated with the LBRC and is required for its function during TEM. IQGAP1 is a large soluble scaffolding protein that integrates signals from diverse pathways to regulate cytoskeletal remodeling, Rho family GTPase activity, and other downstream readouts. Although it has been implicated in a variety of processes in other systems, its role in TEM remains unclear. In addition to being localized to the cytoplasm, in endothelial cells it is also enriched at lateral borders. Genetic ablation of IQGAP1 expression arrests leukocytes on the apical side of endothelial cells and prevents the targeted delivery of the LBRC similar to the phenotype of blocking PECAM. Expression of a full length GFP‐tagged IQGAP1 construct completely restores this defect and also shows that endothelial IQGAP1 is enriched around the leukocyte during TEM. Furthermore, using fluorescently tagged IQGAP1 truncation constructs, we have identified the domains of IQGAP that are required for its localization and function under resting conditions and during TEM. These data suggest that IQGAP1 is an important factor in the regulation of LBRC function. Preliminary studies in IQGAP1 knockout mice support a role for IQGAP in TEM in vivo.Grant Funding Source: Supported by F32 AI084454 to DPS and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM

CD99 signals through soluble adenylyl cyclase to regulate leukocyte transendothelial migration (146.6)

Transendothelial migration (TEM) is the process by which leukocytes extravasate out of the vasculature into the interstitium and is one the key steps of the inflammatory response. PECAM and CD99 are two critical proteins at the endothelial cell border that function sequentially to regulate TEM (PECAM upstream of CD99). These proteins partially reside in a novel, intracellular endothelial cell compartment, known as the Lateral Border Recycling Compartment (LBRC), which supplies the extravasating leukocyte with unligated proteins and membrane surface area to facilitate its movement across the endothelium, in a process called targeted recycling (TR). While much is known regarding the function of PECAM during TEM, very little is understood regarding how CD99 signals. The C‐terminal tail of CD99 is short (39AA) and contains no known signaling motifs. Blocking homophilic interaction of leukocyte CD99 with endothelial cell CD99 arrests leukocytes part way through the junctions; cross‐linking endothelial CD99 overcomes this block. Here we show that engagement of leukocyte CD99 with endothelial cell CD99 (or crosslinking CD99 on endothelial cells to mimic this) activates PKA via a soluble form of adenylyl cyclase, ADCY10 (sAC). Blocking either PKA or sAC, but not transmembrane adenylyl cyclase (tmAC), recapitulates the phenotype of CD99 arrest on TEM and also prevents crosslinking‐mediated restoration of CD99 blockade. Activation of PKA restores TR of LBRC membrane to the arrested leukocytes to overcome the CD99 blockade. Current research is focused on identifying the mediators downstream of PKA as well as the signaling events taking place between the cytoplasmic domain of CD99 and sAC.This research was supported by F30 HL116100‐01A1 to RLW and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.Grant Funding Source: NIH NRSA F30 HL116100‐01A1

How Endothelial Cells Regulate Transmigration of Leukocytes in the Inflammatory Response

Leukocytes attach to vascular endothelial cells at the site of inflammation via a series of intercellular adhesive interactions. In a separate step in leukocyte extravasation, transendothelial migration is regulated by molecules that play no role in the preceding steps of tethering, rolling, adhesion, and locomotion. Transendothelial migration itself can be dissected into a series of distinct interactions regulated sequentially by molecules concentrated at the endothelial cell border; these include platelet/endothelial cell adhesion molecule, poliovirus receptor (CD155), and CD99. These molecules are components of the lateral border recycling compartment (LBRC), a perijunctional network of interconnected tubulovesicular membrane that traffics to surround the leukocyte as it passes across the endothelial cell. This targeted recycling of LBRC requires kinesin to move the membrane along microtubules, and interfering with LBRC trafficking blocks transmigration of neutrophils, monocytes, and lymphocytes. The LBRC is also recruited to mediate transcellular migration when that occurs. Movement of the LBRC is coordinated with events on the luminal surface, such as clustering of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 under the migrating leukocyte, as well as movement of vascular endothelial cadherin and its associated catenins out of the junction at the site of transendothelial migration. How these events are coordinated is not known, but their regulation shares common signaling pathways that may serve to connect these steps.

Segregation of VE-cadherin from the LBRC depends on the ectodomain sequence required for homophilic adhesion.

The lateral border recycling compartment (LBRC) is a reticulum ofperijunctional tubulovesicular membrane that is continuous with the plasmalemma of endothelial cells and is essential for efficient transendothelial migration (TEM) of leukocytes. The LBRC contains molecules involved in TEM, such as PECAM, PVR and CD99, but not VE-cadherin. Despite its importance, how membrane proteins are included in or excluded from the LBRC is not known. Immunoelectronmicroscopy and biochemical approaches demonstrate that inclusion into the LBRC is the default pathway for transmembrane molecules present at endothelial cell borders. A chimeric molecule composed of the extracellular domain of VE-cadherin and cytoplasmic tail of PECAM (VE-CAD/PECAM) did not enter the LBRC, suggesting that VE-cadherin was excluded by a mechanism involving its extracellular domain. Deletion of the homophilic interaction domain EC1 or the homophilic interaction motif RVDAE allowed VE-CAD/PECAM and even native VE-cadherin to enter the LBRC. Similarly, treatment with RVDAE peptide to block homophilic VE-cadherin interactions allowed endogenous VE-cadherin to enter the LBRC. This suggests that homophilic interactions of VE-cadherin stabilize it at cell borders and prevent entry into the LBRC.

Neutrophil and monocyte recruitment by PECAM, CD99, and other molecules via the LBRC

The recruitment of specific leukocyte subtypes to the site of tissue injury is the cornerstone of inflammation and disease progression. This process has become an intense area of research because it presents several possible steps against which disease-specific therapies could be targeted. Leukocytes are recruited out of the blood stream by a series of events that include their capture, rolling, activation, and migration along the endothelium. In the last step, the leukocytes squeeze between adjacent endothelial cells to gain access to the inflamed tissue through a process referred to as transendothelial migration (TEM). Although many of the molecules, such as PECAM and CD99, that regulate these sequential steps have been identified, much less is understood regarding how they work together to coordinate the complex intercellular communications and dramatic shape changes that take place between the endothelial cells and leukocytes. Several of the endothelial cell proteins that function in TEM are localized to the lateral border recycling compartment (LBRC), an interconnected reticulum of membrane that recycles selectively to the endothelial borders. The recruitment of the LBRC to surround the migrating leukocyte is required for efficient TEM. This review will focus on the proteins and mechanisms that mediate TEM and specifically how the LBRC functions in the context of these molecular interactions and membrane movements.

Molecular characteristics determining entry into or exclusion from the endothelial cell (EC) lateral border recycling compartment (LBRC)

The LBRC is a recently discovered membrane compartment in EC. It contains molecules critical for leukocyte transmigration, PECAM, CD99, and JAM‐A, but excludes other junction components like VE‐cadherin. Since a mutation in the cytoplasmic tail (C‐tail) of PECAM prevented localization to the LBRC, we first determined whether the C‐tail of PECAM could bring a molecule not expressed on EC into the LBRC. A chimera with the extracellular domain (ECD) of Tac and C‐tail of PECAM localized to junctions, entered the LBRC, and recycled like PECAM. However, the control protein, Tac, entered the LBRC too, even though it was expressed diffusely. This suggested that entrance to the LBRC might be default and VE‐cadherin is selectively excluded. We constructed a VE‐cadherin/PECAM (CP) chimera with the ECD of VE‐cadherin and C‐tail of PECAM. We found CP went to junctions but was excluded from the LBRC, like VE‐cadherin. These findings suggested ECD excluded VE‐cadherin and CP from the LBRC. We then generated truncated VE‐cadherin and CP with deletion of the homophilic interaction domain EC1 or its specific motif RVDAE. Both VE‐cadherin and CP chimera lacking RVDAE motif or EC1 domain DID enter the LBRC. This suggests that homophilic interactions of VE‐cadherin with other VE‐cadherin molecules stabilize it at cell borders and prevent entry into the LBRC. Supported by AHA Postdoctoral Fellowship to GF and R01 HL046849 to WAM

Kinesin‐1 mediates targeted recycling of the lateral border recycling compartment

A critical step in the inflammatory response is transendothelial migration (TEM). Our lab has recently discovered a membrane compartment in endothelial cells (EC) that regulates TEM: The lateral border recycling compartment (LBRC). The LBRC contains a subset of membrane proteins, such as PECAM, CD99, and JAMA, which are required for TEM. During TEM, membrane from the LBRC is directed to the region of the EC where the leukocyte is transmigrating. This “targeted recycling” (TR) facilitates TEM by delivering LBRC membrane and proteins to the migrating leukocyte. Inhibiting targeted recycling of the LBRC blocks leukocyte TEM. Targeted recycling of the LBRC requires functional microtubules and kinesin motors. Microinjection of EC with an antibody against the conserved motor domain of kinesin inhibited TR and TEM to a similar magnitude as blocking PECAM or depolymerizing microtubules. We sought to identify the specific kinesin(s) that mediate targeted recycling. Preliminary experiments suggested the KIF5b gene product, kinesin‐1 as a likely candidate. Microinjection of EC with a kinesin‐1 mAb blocked TEM. Knocking down kinesin‐1 in EC decreased targeted recycling of LBRC and leukocyte TEM in preliminary experiments. This suggests that kinesin‐1 is a major molecular motor necessary for LBRC transport along microtubules during TR. Supported by an AHA Midwest Affiliate Predoctoral Fellowship and NIH R01 HL046849.