Parallels between single cell migration and barrier formation: The case of RhoB and Rac1 trafficking

ABSTRACT

The appearance of multicellularity implied the adaptation of signaling networks required for unicellular life to new functions arising in this remarkable evolutionary transition. A hallmark of multicellular organisms is the formation of cellular barriers that compartmentalize spaces and functions. Here we discuss recent findings concerning the role of RhoB in the negative control of Rac1 trafficking from endosomes to the cell border, in order to induce membrane extensions to restore endothelial barrier function after acute contraction. This role closely resembles that proposed for RhoB in controlling single cell migration through Rac1, which has also been observed in cancer cell invasion. We highlight these similarities as a signaling paradigm that shows that endothelial barrier integrity is controlled not only by the formation of cell-cell junctions, but also by a balance between ancestral mechanisms of cell spreading and contraction conserved from unicellular organisms and orchestrated by Rho GTPases.

Keywords:

• actin
• barrier function
• cell migration
• contraction
• endosomes
• inflammation
• lamellipodium
• permeability
• Rab5
• Rab7
• Rac1
• RhoB
• thrombin
• TNF

Introduction

The formation of cellular barriers that compartmentalize cavities is one of the first requirements for the acquisition of multi cellularity.¹ The organization of cells into single layers has facilitated morphogenetic processes in complex organisms, starting from gastrulation, which ensures the precise organization of the embryo into cell sheets that differentiate into tissues. Many of these tissues conserve an organization into cellular barriers, namely parenchymal epithelia and vascular endothelia.^2,3 It is thus probable that the molecular mechanisms regulating the life of single cells, for example, chemotaxis and migration, had to be remodeled in order to adapt cells to the mechanical constriction and new duties related to barrier function. Indeed, central regulators of cell barrier function, such as cell-cell adhesion molecules or the actin cytoskeleton, probably played barrier-independent roles in unicellular organisms before multicellularity arose.^4-6 This commentary highlights the similarities between the roles of the RhoGTPases RhoB and Rac1 during the extension of lamellipodia in single amoeboid-type cell migration, and those that regulate endothelial barrier reformation after acute contraction has been induced by inflammatory mediators.⁷

Rac1 endosomal trafficking mediates lamellipodial extension during single cell migration

The Rho GTPase family comprises 20 proteins, most of which cycle between GTP-loaded, active and a GDP-loaded, inactive state. Upon activation, Rho GTPases are translocated to membranes through the exposure of their C-terminus domain, which contains lipid modifications. Rho GTPases activate their effectors from the plasma membrane and also from internal cellular membranes such as those of the Golgi, or endosomes or even in the nucleus.^8,9 In addition, some Rho proteins, such as RhoB or Rac1, can localize in different compartments depending on the type and intensity of the stimulus.^7,9,10 In 2008, Palamidessi et al. showed that Rac1-mediated actin polymerization and membrane protrusion require spatially restricted endosomal trafficking, in which Rac1 is internalized in early endosomes, where it meets the Rho GEF Tiam1, becomes active and recycles back to the plasma membrane. Rac1 endosomal trafficking underlay the formation of actin structures such as dorsal ruffles in response to migration-promoting growth factors, namely HGF and VEGF.¹¹ Rac1 intracellular trafficking was detected not only in single motile mammalian tumor cells but also in primordial germinal cells from zebrafish, suggesting a general mechanism to locally restrict subcortical actin polymerization to regions where growth factor stimulus is detected.¹¹ In accordance, the endosomal accumulation of Rac and Rac GEFs has been observed in different eukaryotic organisms, from Dictyostelium¹² to humans.^11,13

Endosomal RhoB regulates actin polymerization and Rac1 activation in single cell migration

A few years before Palamidessi´s work, Margaret Frame´s laboratory reported that endosomal RhoB is required for translocation of the kinase Src from endosomes to the plasma membrane to mediate actin polymerization, which proved to be essential for lamellipodial extension in primary fibroblasts.¹⁴ RhoB-positive endosomes harbored actin and scar1, a protein that is involved in actin polymerization. This was the first indication that endosomes could locally regulate actin polymerization, which was later confirmed when Dia1 and mDia2 were found in RhoB-positive intracellular vesicles. In these endosomes, RhoB recruits diaphanous formins, which locally polymerize actin, thereby regulating actin-dependent endosomal mobility.^15,16 These studies did not address the potential role of other GTPases in endosomal trafficking, actin polymerization and cell motility. In a more pathological context, RhoB has been found to be involved not only in cell migration but also in cell transformation and invasion. In bronchial cell lines, knockdown of RhoB promotes migration and invasiveness. Moreover, RhoB absence favors the aggressiveness of lung adenocarcinoma.¹⁷ Interestingly, the role of RhoB constraining migration in bronchial cells is mediated through the negative regulation of Rac1 and Akt1 activity.^18,19 In smooth muscle cells and prostate cell lines, however, RhoB silencing has an opposing effect and decreases Rac1 activity and Rac-mediated lamellipodial protrusions, suggesting that RhoB activation positively regulates Rac1.²⁰ Together, these studies suggest that the effect of RhoB on Rac1 is cell and stimulus-specific.²¹ In vascular and lymphatic endothelial beds, these opposing roles of RhoB in migration-related events are striking. Loss of RhoB reduces angiogenesis in the vascular endothelium and promotes it in lymphatic endothelium.²²

RhoB regulates Rac1 trafficking and activity thereby regulating endothelial barrier function during inflammation

In a search for new proteins essential to the inflammatory response, we have found that RhoB is upregulated in response to inflammatory cytokines such as IL-1β and TNF. Such upregulation is quite specific and the expression of most of the Rho family members is unaltered upon cytokine exposure.⁷ RhoB can be considered as a stress protein that is upregulated in response to different stimuli. These include UV light,²³ hypoxia²⁴ TGFβ²⁵ and the aforementioned cytokines.^7,26 RhoB plays a role in cell survival and apoptosis, but its upregulation has an additional, often underestimated effect of increasing the total concentration and activity of the RhoA subfamily proteins on the stressed cell, which are the master promoters of actin polymerization. In human vascular endothelial cells exposed to TNF, quantification of the relative expression levels of upregulated RhoB with its closest relatives, RhoA and RhoC, revealed that RhoA, RhoB and RhoC are equally expressed and activated. Thus, upon RhoB upregulation, TNF increases the total protein expression in the RhoA subfamily, the main Rho kinase and actomyosin-regulators, between 40 and 50%.⁷ Indeed, these 3 Rho proteins play redundant roles in the control of filamentous (F-) actin levels. Specific Rho silencing with siRNA induces the upregulation of the remaining Rho proteins, compensating the total RhoA subfamily protein levels in a remarkably quantitative way. In addition, single and double Rho silencing have a minor effect on endothelial F-actin, whereas triple Rho knockdown, reduces F-actin levels to those obtained upon treatment with the Rho family inhibitor, C3 transferase.^7,27 Therefore, in endothelial barriers RhoA, RhoB and RhoC compensate each other to maintain the homeostasis of F-actin.

However, in addition to these cooperative roles, specific Rho silencing led us to identify a specific role for RhoB in endothelial cells pretreated with TNF, which strikingly resembled that observed in single cell migration. Thrombin is a protease that activates by proteolysis the protease activated receptors (PARs), which are G protein-coupled receptors that activate actomyosin-mediated acute contraction. Thrombin and PARs are crucial in inflammation-related vascular pathologies, such as sepsis and acute respiratory distress syndrome. They contribute to the vascular tone but also induce vascular barrier disruption, often with fatal consequences.^28,29 Triple Rho knockdown or inhibition of common downstream effectors, such as ROCKs, inhibits thrombin-induced acute contraction in endothelial barriers.^7,30 However, reduction of RhoB expression, but not of RhoA or RhoC, accelerates the recovery of barrier function after this acute contraction. Interestingly, this increase in the ability of the endothelium to reform the cellular barrier appears to be independent of cell-cell junction integrity and is more closely related to the greater capacity of endothelial cells to form plasma membrane extensions in the absence of RhoB expression, which favors and enhances the establishment of new cell-cell contacts. The intensity of this effect is correlated with the physiological levels of RhoB expression: in the absence of TNF exposure, RhoB levels are low and the effect of siRNA RhoB on barrier repair is weaker than in TNF-pretreated cells, which express high levels of this GTPase. In addition, the effect of RhoB depletion occurs mostly during the time of barrier reformation, which is associated with the increase in trafficking of RhoB-positive vesicles during this particular period.⁷

Taking into account the previous research on the role of RhoB in regulating Rac1 activity in tumor cell migration and the fact that Rac1 is the master regulator of lamellipodial extension, we investigated whether the effect of RhoB siRNA on barrier repair and plasma membrane extension after thrombin-induced contraction is mediated by Rac1 in endothelial cells. RhoB depletion increases Rac1 activation at the time period in which cells produce their lamellipodial-like extensions. Moreover, we observed that Rac1 is localized not only at the plasma membrane but also in intracellular vesicles (Fig. 1). During re-extension of the plasma membrane after acute contraction, Rac1-positive vesicle trafficking increases in a way similar to that previously observed for RhoB. In addition, pharmacological Rac1 inhibition during endothelial barrier reformation abrogates the increase in barrier recovery induced by RhoB depletion.⁷ Collectively, these results suggest that RhoB controls endothelial barrier reformation by increasing Rac1 activation thereby forming plasma membrane extensions. A relative quantification of Rac1 subcellular localization revealed that high RhoB activity or expression retains Rac1 in a late endosomal compartment, whereas RhoB silencing promotes Rac1 translocation to the cell border. Although Rac1 and RhoB were found in the same vesicles (Fig. 1), super-resolution confocal microscopy and proximity assays, suggest that both GTPases are in proximity, but in different membrane domains.⁷ Altogether, these findings indicate that RhoB regulates Rac1 dynamics between membrane early and late endosomal compartments. This Rac1 intracellular trafficking during endothelial barrier reformation closely resembles that found in single cell migration, although the effect of RhoB preventing Rac1 recycling to the cell border has not yet been addressed in migrating cells.

Figure 1. Rac1 co localizes with active RhoB in a vesicular compartment. The constitutively active mutant HA-RhoB-V14 and cherry-Rac1 were expressed in human microvascular endothelial cells. Cells were fixed, stained for HA and analyzed by confocal microscopy. Arrows point at vesicular structures containing active RhoB and Rac1. Top right and bottom images are enlargements of the squared area in the top left image. Bars, top left, 15 μm; top right, 4 μm.

A role for lamellipodia in endothelial barrier function has been suggested by other researchers.^31-33 Rac1 and the RacGEF Tiam1, a GEF that Rac1 finds in the endosomal compartment during its recycling toward the cell border during cell migration, are endothelial barrier enhancers.^34-37 In addition, soluble growth factors, namely HGF, that induce Rac1 endosomal trafficking in single cells, also preserve barrier function.^11,35,38 A close connection between lamellipodia and the maturation of cell-cell junctions has been described by Schnittler´s group.^33,39 At initial stages of endothelial barrier formation, cell-cell contact areas that are not yet occupied by adherens junctions emit circular lamellipodia that favor the formation of new VE-cadherin-mediated intercellular junctions. Membrane re-extension during endothelial barrier recovery may therefore concentrate the machinery required for adherens junction formation and maturation. Beyond intercellular contacts, but also in an inflammatory context, Rac1 promotes extension of ventral lamellipodia to repair microwounds caused by leukocytes undergoing paracellular and transcellular diapedesis.⁴⁰ However, the role of vesicular trafficking and Rac1 recycling has not yet been addressed for this membrane remodeling aimed to restore cell integrity.

Conclusions and future perspectives

Our findings about the role of RhoB and Rac1 trafficking have similarities with those reported in previous studies of its role on Rac1 activity and enhanced single cell migration in different cell types, including cancer cells (Fig. 2). Lamellipodial-like extensions in confluent endothelial cells are required not to displace a cell out of the monolayer, but to reform the endothelial barrier after its disruption. Hence, it is easy to envisage endothelial monolayers as a colony of single cells coordinating ancient mechanisms of contraction and spreading to form a barrier that compartmentalizes important functions in complex organisms, such as oxygen, nutrient and immune cell transport by the circulatory system. Exposure to inflammatory cytokines would diminish the ability of cells to form cellular protrusions and re-establish cell-cell contacts in an endothelial barrier, weakening the vascular wall, just as transformed cells activate mechanisms to prevent the extension of lamellipodia that would induce cell migration and cancer dissemination. It is of note that inflammatory signaling participates in cell migration-related events such as angiogenesis.^41-43 In particular, TNF, which induces the greatest upregulation of RhoB, plays a contextual role in promoting or inhibiting cell migration and angiogenesis.^44-47 It would be thus interesting to investigate whether the effects of TNF and other cytokines on cell migration and transformation are mediated by an alteration in the intracellular trafficking regulated by RhoB.

Figure 2. Rac endosomal trafficking during plasma membrane extension in migrating cells and in confluent endothelial cells. Left panel. In migrating cells, Rac1 internalization and recycling in Rab5-positive endosomes are necessary for its activation by the endosomal GEF Tiam1 and the subsequent actin-mediated membrane extension.¹¹ Central and right panels. In confluent endothelial cells RhoB and Rac1 co localize in Rab5 and Rab7-positive endosomes. RhoB-GTP increases Rac1 intracellular localization and prevents Rac1 activation and localization at the cell border, where it stabilizes cell-cell junctions, suggesting that RhoB activation affects GTP loading and recycling of endosomal Rac1.⁷ Active, GTP-loaded Rac is represented by a red star.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The expert technical advice of the confocal microscopy facility of CBMSO is gratefully acknowledged.

Additional information

Funding

This work was supported by grants SAF2014–57950-R (to J.M.), from the Ministerio de Economía y Competitividad; and Endocornea, convenio colaboración with Instituto de Investigación Sanitaria Fundación Jiménez Díaz. D.G.W. is recipient of a FPI fellowship from the Ministerio de Economía y Competitividad.