Arhgef6 (alpha‐PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence

Immunity is governed by successful T cell migration, optimized to enable a T cell to fully scan its environment without wasted movement by balancing speed and turning. Here we report that the Arhgef6 RhoGEF (aka alpha‐PIX; αPIX; Cool‐2), an activator of small GTPases, is required to restrain cell migration speed and cell turning during spontaneous migration on 2D surfaces. In Arhgef6−/− T cells, expression of Arhgef7 (beta‐PIX; βPIX; Cool‐1), a homolog of Arhgef6, was increased and correlated with defective activation and localization of Rac1 and CDC42 GTPases, respectively. Downstream of Arhgef6, PAK2 (p21‐activated kinase 2) and LIMK1 phosphorylation was reduced, leading to increased activation of Cofilin, the actin‐severing factor. Consistent with defects in these signaling pathways, Arhgef6−/− T cells displayed abnormal bilobed lamellipodia and migrated faster, turned more, and arrested less than wild‐type (WT) T cells. Using pharmacologic inhibition of LIMK1 (LIM domain kinase 1) to induce Cofilin activation in WT T cells, we observed increased migration speed but not increased cell turning. In contrast, inhibition of Cdc42 increased cell turning but not speed. These results suggested that the increased speed of the Arhgef6−/− T cells is due to hyperactive Cofilin while the increased turning may be due to abnormal GTPase activation and recruitment. Together, these findings reveal that Arhgef6 acts as a repressor of T cell speed and turning by limiting actin polymerization and lamellipodia formation.


INTRODUCTION
T cell migration is a complex, multistep process that enables the cell to scan its environment for antigenic signals. On 2D surfaces, these steps begin with a broad and thin protrusion called a lamellipodium, consisting of polymerized, branched actin. Actin polymerization at the cell membrane forms a lamellipodium that forces filaments back toward the cell center, a process known as retrograde actin flow. As the lamellipodium grows and attaches to the substrate using integrinbased adhesions, retrograde actin flow is transformed into a force that T cells can maximally scan their local environment while randomly migrating. 4 Cell turning during migration is controlled by proteins that regulate protrusion or lamellipodia formation. 2 The GTPase Cdc42 has long been known as a polarity factor in yeast, where it is required for placement of new buds after recruiting its activating GEF (Guanine nucleotide exchange factor). 5,6 Knockout of Cdc42 in dendritic cells results in multiple protrusions around the periphery of cells and highly twisting trajectories; thus Cdc42 suppresses protrusions in order to promote a single, dominant protrusion. 7 The GTPase Rac1 promotes lamellipodial extension by activating actin polymerization at the tips of lamellipodia. 8 Knockdown of Rac1 in fibroblasts inhibits peripheral protrusions resulting in an elongated cell that migrates persistently, whereas overexpression of Rac1 induces multiple lateral protrusions and twisting migration paths. 2,9 Rac1 activation of the WAVE (WASPfamily verprolin homologous protein 1) regulatory complex induces the Arp2/3 actin-branching complex required for stable lamellipodia, and inhibitors of Arp2/3 such as Arpin or Coronin affect protrusion formation, migration speed, and cell turning. [10][11][12] Rac1 and Cdc42 also promote polymerized actin in cell protrusions by inducing LIMK to inhibit Cofilin, which otherwise severs actin. 13 Thus, Cdc42 and Rac1 regulate cell protrusions, turning, and cell speed.
Although many actin-related proteins play roles in T cell migration, relatively few are negative regulators of T cell speed. One such protein is Arhgef6 (alpha-PIX), a RhoGEF exchange factor for Rho GTPases

Rac1 and Cdc42. Thymocytes and marginal zone B cells lacking
Arhgef6 migrate faster than their wild-type (WT) counterparts. 14,15 And Arhgef7 has been shown to increase cell speed by turning over nascent focal adhesions in fibroblasts. 16 Arhgef6 and its homolog Arhgef7 were first isolated as p21-activated kinase (PAK)-interacting proteins that activate Rac1 and Cdc42 by catalyzing a GDP-GTP nucleotide exchange. Arhgef6 and Arhgef7 bind constitutively to the GTPase-activating proteins GIT1 and GIT2, members of the Arf-GAP family of proteins that inactivate Arf (ADP-ribosylation factor) GTPases. 17 This complex is referred to as the PIX-GIT (p21-activated protein kinase exchange factor -GRK-interacting protein) complex and it is notable for connecting two major classes of GTPases: Rho GTPases, activators of actin dynamics, and the Arf GTPases, regulators of the WAVE complex, Arp2/3 and lamellipodia. 17 Together, the PIX-GIT complex relays signals from receptors to actin cytoskeletal rearrangements required for signaling.
Arhgef6 is an X-linked protein and has been identified as a mutation in patients with X-linked intellectual disabilities. 18 In neurons, Arhgef6 and Arhgef7 are required for formation of actin structures including neurites, spines, and growth cones, [18][19][20][21][22][23][24] and signal to Cofilin in these processes. [25][26][27] In Arhgef6 −/− mice, pyramidal neurons show increased dendritic length and spine density, while the mice display impairments in learning and behavior. 21 However, despite its importance to neuronal functions, the roles of Arhgef6 in T cells are less clear. Arhgef6 protein is highly expressed in immune cells compared to Arhgef7 17,28 and is required for formation of the immune synapse downstream of TCR signaling. 29 Additionally, Arhgef6 −/− mice have developmental defects at an early stage of T cell development in the thymus due to increased migration speed and reduced contact with antigenpresenting cells. 14 In order to investigate the mechanisms behind the increased speed of Arhgef6 −/− T cells, we employed immunocytochemistry and 2D integrin-based migration assays to assess lamellipodia, their regulators, and cell migration trajectories. We found that T cells lacking Arhgef6 have increased active Rac1, mislocalized Cdc42, and increased Cofilin activity, as well as larger and more frequent lamellipodia. The Arhgef6 −/− T cells migrated both faster and turned more than WT cells, revealing a role for Arhgef6 in coupling T cell speed and persistence in cell migration.

Migration assays
CD4 + T cells were resuspended in migration buffer (1× HBSS, 2% fatty acid-free BSA, 1 mM HEPES) and seeded at a density of 12,000 cells/well on slides coated with ICAM-1 as described earlier at 37 • C.
Loose and dead cells were carefully washed off with prewarmed migration buffer. Cells were imaged for 20 min at 20 s intervals using a 10× objective. Cells were manually tracked using the ImageJ manual tracking plugin. Tracks were analyzed using the Ibidi Chemotaxis tool v. 2.0 for velocity, displacement, and straightness (displacement over path length). Arrest coefficient is the percentage of time that a cell is not moving (speed between frames at <2 m/min) over the total time of the cell track. Autocorrelation, idling plots, cell tracks with idling marked, and plots of idling/active paths vs. angle changes were calculated using Excel VBA programs from Gorelik and Gautreau 36,37 and plotted using GraphPad Prism v.7.

Statistical analysis
All the data are reported as mean ± SD or SEM, as indicated, and means of different groups were compared using unpaired Student's t-test or 2-way ANOVA in GraphPad Prism v. 7. The difference between two data sets was considered significant for P-values <0.05.

Arhgef6 controls the recruitment and activation of GTPases and the Cofilin pathway
In previous work, we found that the loss of Arhgef6 alters the expression of its homolog and binding partner, Arhgef7, in thymocytes. 14  in localized Arhgef7 and of Cdc42 ( Fig. 2A, B). Cdc42-GTP was also increased in the TIRF contact plane but proportionally to Cdc42; thus Cdc42 activity was normal and only recruitment was increased ( Fig. 2B). We also assessed Arhgef7 and Cdc42 recruitment to the periphery of T cells where the leading edge of the lamellipodia would be found and observed slightly increased levels of recruitment of Arhgef7, Cdc42, and Cdc42-GTP to the periphery with respect to the total cell ( Fig. 2A, B). Furthermore, when we repeated the same analysis on beta-actin, the levels of actin at the TIRF contact zone were also increased (Fig. 2C), despite normal levels of -actin by Western blot in the Arhgef6 −/− T cells (Fig. 2D). Together, these data indicate that although overall levels of Cdc42 and -actin are normal, there is increased recruitment of both and of Arhgef7 to the integrin-ligand contact plane of the cell.
Arhgef6 and Arhgef7 bind to a Rac1 and Cdc42 effector, PAK2 kinase, which is the dominant PAK isoform in T cells. 38 Western blot analysis of total PAK2 and phospho-PAK2 in WT and Arhgef6 −/− T cells showed that expression of total PAK2 was higher in Arhgef6 knockout T cells but the level of phosphorylated PAK2 was lower (Fig. 3A). One target of active PAK2 kinase is the kinase LIMK1. LIMK1 is phosphorylated to become active and can then phosphorylate Cofilin on serine 3 to inactivate it. 13 We therefore examined phosphorylation of LIMK1 and Cofilin in Arhgef6 −/− T cells by Western blot and found that the phosphorylated forms of both LIMK1 and Cofilin were greatly reduced in Arhgef6 −/− T cells (Fig. 3B, C). As a loading control for pLIMK1, we used GAPDH (Fig. 3B) because the LIMK1 antibody did not work in Western blot, although it did work in immunocytochemistry. Thus, Arhgef6 is required for normal activation of a signaling pathway that includes PAK2, LIMK1, and Cofilin.

Arhgef6 restrains lamellipodia spread and inhibits local actin polymerization in T cells
The

Arhgef6 restrains speed and turning in T cells
Because  (Fig. 5C). To confirm this, we quantified the displacement, and straightness values for the faster cells (velocity > 10 m/min). The displacement was slightly reduced for Arhgef6 −/− T cells (P = 0.11, t-test), and the straightness was significantly reduced (Fig. 5D). To apply an additional test of straightness, we calculated the direction autocorrelation values, a statistical measure of correlation between turning angles in a cell's trajectory. 36 The higher position of the line for WT cells indicates greater correlation between turn-ing angles, consistent with straighter trajectories than for Arhgef6 −/− T cells (Fig. 5E). The increased turning of Arhgef6 −/− T cells suggested that Cdc42 was involved. To test the role of Cdc42 in T cell turning, we treated T cells with a Cdc42 inhibitor, Casin. Casin had no effect on T cell speed for either genotype and also no effect on overall track straightness (Fig. 5F). As expected, Arhgef6 −/− T cells were faster and twistier than WT cells (Fig. 5F). However, Casin significantly reduced straightness of fast-moving WT cells, showing that Cdc42 is required for maintaining a straighter cell migration path for fast-moving cells (Fig. 5F). Together, these data showed that Cdc42 may be involved in T cell turning and that Arhgef6 −/− T cells migrated faster than WT cells but turned more.
Increased cell migration speed is normally associated with reduced cell turning, and proteins that induce cell turning, such as Arpin, reduce cell speed. 3,37 In contrast, the Arhgef6 −/− T cells migrate faster and turn more than WT cells. To further investigate the increased turning, we used several programs from the Gautreau group to quan- been reported for other cell types 37 (Fig. 6C). However, these illustrations also revealed reduced pausing in Arhgef6 −/− T cells (Fig. 6C). To quantify the association between pausing and turning in Arhgef6 −/− T cells, we assessed turn angles in cells that either paused or migrated faster than 2 m/min for 1, 2, or 3 frames (Fig. 6D). Turning angles are expected to be lower for actively moving cells than for idling cells as idling is associated with turning, 37 and this was the case for both WT and Arhgef6 −/− T cells (Fig. 6D). In addition, there was no difference between WT and Arhgef6 −/− T cells in the turning angles of idling cells (Fig. 6D, "idling" cells, left panel). However, the actively moving Arhgef6 −/− T cells ("active" cells, right panel) displayed consistently higher turning angles than those of WT (Fig. 6D). Together, these data show that in Arhgef6 −/− T cells, cell turning is increased and is uncoupled from cell pausing.

LIMK inhibition increases T cell size and speed
To determine if the defective Cofilin pathway in Arhgef6 −/− T cells was responsible for one or both of the velocity and turning defects, we treated WT and mutant T cells with the LIMK1 inhibitor, LIMKi 3.

DISCUSSION
Random T cell migration enables the cell to thoroughly scan an environment for antigens without unnecessary energy expenditures. To achieve an efficient search pattern, T cells must tightly control both migration speeds and cell turn angles. 4 In this study, we have shown . Mean ± SD. *P < 0.05, ***P < 0.001, ****P < 0.0001, by Student's t-test F I G U R E 8 Schematic representation of Arhgef6-controlled signaling pathways. In wild-type (WT) cells, Arhgef6 and Arhgef7, RhoGEFs for Rac1 and Cdc42, repress signaling to actin reorganization and restrict lamellipodial formation to limit cell speed and maintain relative straightness. In T cells lacking Arhgef6, cells migrate faster and turn more. Cdc42 is mislocalized to the ICAM1-coated migration surface and Rac1 is overactivated. Moreover, PAK2, LIMK1, and Cofilin are all hypophosphorylated meaning that Cofilin, which promotes actin severing and polymerization, is overactivated. The mechanisms for Rac1 activation of lamellipodial extension are not characterized here but may include hyperativation of WAVE and Arp2/3, both required for lamellipodia extension. Arhgef7 expression is increased, likely due to its taking the place of Arhgef6 in the PIX-GIT complex, but it cannot compensate fully for the absence of Arhgef6 as the immune cell-specific Arhgef6 may be required for targeting the complex to T cell specific receptors of Cdc42 resulted in increased twistiness of fast cells but cell speed was unchanged. These data suggest that Arhgef6 controls two pathways critical to T cell migration: cell speed via Cofilin-mediated actin polymerization and cell turning, possibly via localization of Cdc42.
These observations highlight a role for Arhgef6 as a repressor of lamellipodia formation and of cell migration speed in T cells (Fig. 8).
We hypothesize that Arhgef6 signals through PAK2 and LIMK1 to inhibit Cofilin activation of actin severing and also restricts a newly formed lamellipodia to a site close to the old lamellipodia. Arhgef6 and Arhgef7 (aka PIX proteins) are normally found in a constitutive complex with GIT1 and GIT2 proteins. 17 In the absence of Arhgef6, Arhgef7 may be protected from degradation by taking the place of Arhgef6 in the complex, thus increasing Arhgef7 expression overall.
However, Arhgef7 may not be able to compensate properly for Arhgef6 in the complex, possibly because Arhgef6 possesses an actin-binding domain, called a CH domain, that is absent in Arhgef7 17 and which may target the PIX-GIT complex to actin at receptors in T cells. The increased Arhgef7 expression is accompanied by increased Rac1-GTP activity, which may activate WAVE and Arp2/3 complexes in the formation of lamellipodia. 39 And although activated Rac1 is increased, it fails to direct the PAK-LIMK signaling pathway to inhibit Cofilin, likely because the role of Arhgef6 is to localize the GTPases to a single, dom-inant lamellipodia. Thus, increased Arhgef7 at the periphery of T cells could mislocalize the complex of PIX-GIT proteins and Cdc42 and fail to repress Rac1.
Further support for the idea that Arhgef6 and Arhgef7 control placement of cellular projections by regulating expression and localization of downstream signaling proteins is found in other cell systems. In fibroblasts, Arhgef7 is initially recruited by Cdc42 to a nascent lamellipodium, where it then subsequently recruits Rac1 to polymerize actin and induce protrusion formation. 40 In neurons, GIT1 is enriched in synapses and acts with Arhgef7 to stabilize synaptic spines. 24 However, overexpression of Arhgef7 or of a truncated form of GIT1 mislocalizes these proteins, causing multiple dendritic protrusions. 24 Aberrant expression-either higher or lower, of Arhgef6-controlled proteins is often associated with multipolar protrusions. For example, overexpression of Arhgef7 in fibroblasts or its deletion in anterior visceral endoderm cells both cause multiple protrusions. 40 However, Arhgef6 −/− T cells were also 20-25% faster than WT cells.
Our results reveal a role for Arhgef6 in coupling T cell speeds with T cells turning. The Arp2/3 inhibitor, Arpin, is also involved in cell speed and turning but differs from Arhgef6 in that Arpin reduces cell speed because it increases cell turning. 10 In contrast, Arhgef6 represses both cell speed and cell turning, likely by signaling upstream of Rac1, Cofilin, WAVE complex activation and Arp2/3, and thus represents a nexus between the placement of a new lamellipodium and the extension of that lamellipodium. It has been demonstrated that actin flows mediate coupling between cell speed and turning, 45 and we show here that Arhgef6 is integral to this mechanism.
Immune cells in general, and T cells in particular, are the most motile cells in the body and can migrate at remarkable speeds through vessels as well as tissues and organs. 4 It is possible that T cells use Arhgef6 and Arhgef7 to act as the brakes on a migration motor that is always running, and then release the immune-specific Arhgef6 as needed at specific integrins or TCR to unleash actin reorganization. 28,46 We reported earlier that Arhgef6 −/− thymocytes displayed increased migration speeds and reduced arrest coefficients, leading to inefficient scanning of antigen-presenting cells and impaired T cell development. 14 We also observed decreased arrest of Arhgef6 −/− T cells here. Defects in scanning of antigen-presenting cells are also linked to cell turning in the case of T cells with a mutation in Myo1g. 47 Therefore, the loss of Arhgef6 control of lamellipodia formation is a molecular mechanism that would likely lead to defective T cell-based immunity in Arhgef6 knockout mice. In conclusion, Arhgef6 and Arhgef7 control Rac1, Cdc42, and Cofilin to restrain lamellipodia formation, cell turning, and cell migration speed.