Research Article

A composition-dependent molecular clutch between T cell signaling condensates and actin

Howard Hughes Medical Institute, Summer Institute, Marine Biological Laboratory, United States
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
University of Texas Southwestern Medical Center, United States
National Centre for Biological Sciences, Tata Institute for Fundamental Research, India
Howard Hughes Medical Institute, University of California, San Francisco, United States
Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, United States

Jul 3, 2019

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Abstract
Introduction
Results
Discussion
Materials and methods
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Abstract

During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments. We found that basic regions of Nck and N-WASP/WASP promote association and co-movement of LAT condensates with actin, indicating conversion of weak individual affinities to high collective affinity upon phase separation. Condensates lacking these components were propelled differently, without strong actin adhesion. In cells, LAT condensates lost Nck as radial actin transitioned to the concentric network, and engineered condensates constitutively binding actin moved aberrantly. Our data show that Nck and WASP form a clutch between LAT condensates and actin in vitro and suggest that compositional changes may enable condensate movement by distinct actin networks in different regions of the IS.

https://doi.org/10.7554/eLife.42695.001

Introduction

Biomolecular condensates are compartments in eukaryotic cells that concentrate macromolecules without an encapsulating membrane (Banani et al., 2017; Shin and Brangwynne, 2017). Numerous condensates are found in the cytoplasm and nucleoplasm, where they are involved in processes ranging from mRNA storage and degradation to DNA repair and ribosome biogenesis (Brangwynne et al., 2011; Feric et al., 2016; Luo et al., 2018; Protter and Parker, 2016). They are also found at membranes, where they control the organization, and likely the activity, of many signaling receptors (Banjade and Rosen, 2014; Case et al., 2019; Su et al., 2016; Zeng et al., 2018).

Condensates are thought to form through phase separation driven by multivalent interactions between molecules containing multiple binding elements (Banani et al., 2017; Banjade and Rosen, 2014; Li et al., 2012). Recent models suggest that a limited collection of proteins and/or RNA molecules forms the essential phase separating scaffold of particular condensates (Banani et al., 2016; Ditlev et al., 2018; Langdon et al., 2018). These molecules then recruit larger numbers of client proteins to complete the structure. Condensate composition is thus determined by the specificity of interactions among scaffolds and between scaffolds and clients. The concentrations, interactions, and dynamics of scaffolds and clients are believed to dictate the biochemical activities, and consequent cellular functions, of individual condensates (Banani et al., 2017; Holehouse and Pappu, 2018; Stroberg and Schnell, 2018). The composition of many condensates is known to change in response to signals, implying regulated changes in activity (Chen et al., 2008; Dellaire et al., 2006; Markmiller et al., 2018; Salsman et al., 2017; Youn et al., 2018). However, the relationships between composition and biochemical/cellular functions are not well understood in most cases.

During activation of a T cell by an antigen presenting cell (APC), condensates organized around the transmembrane adaptor protein LAT (Linker for Activation of T cells) play important roles in downstream signaling and resultant T cell activation (Balagopalan et al., 2013; Houtman et al., 2006; Kumari et al., 2015). These condensates are located at the interface between the T cell and APC, known as the immunological synapse (IS) (Bunnell et al., 2002). We recently provided evidence that these structures form through multivalency-driven phase separation of LAT and its intracellular binding partners Grb2, Sos1, SLP-76, Nck, and WASP (Su et al., 2016). In this system LAT, Grb2, and Sos1 appear to act as the key scaffolds, which recruit SLP-76, Nck, and WASP as clients. During T cell activation, LAT condensates that initially appear in the periphery of the IS are moved radially to the IS center by two different actin cytoskeletal networks, a peripheral dendritic meshwork and more central circular arcs (DeMond et al., 2008; Kaizuka et al., 2007; Mossman et al., 2005; Murugesan et al., 2016; Yu et al., 2010). This translocation is essential for proper T cell responses to antigen presenting cells (Babich et al., 2012; Ilani et al., 2009; Kumari et al., 2012; Yi et al., 2012; Yu et al., 2012).

It has remained unknown how LAT condensates engage actin to move across the IS and whether their composition plays a role in this process (Hammer et al., 2019). To address these questions, we analyzed interactions between LAT condensates and actin in reconstituted biochemical systems and in cells using quantitative fluorescence microscopy and statistical analysis. Our in vitro studies revealed that LAT condensates bind actin filaments in a composition-dependent fashion, primarily through interactions of basic regions in Nck and N-WASP/WASP. In cells, we observed that Nck dissipates from LAT condensates as they traverse the IS. This loss spatially parallels the change in actin architecture from dendritic network to circular arcs. LAT condensates containing a mutant Grb2, which caused them to constitutively bind actin filaments independently of Nck, exhibited aberrant movement across the IS. These data show that Nck and N-WASP/WASP can form a molecular clutch between LAT condensates and actin in vitro. The combined cellular and biochemical data suggest a model in which LAT condensates engage actin differently depending on the density of Nck and WASP proteins in them, such that switching between compositions and actin-binding modes enables them to move radially via the two actin networks at the IS.

Results

LAT condensates reconstituted in vitro within actin networks move in a composition-dependent manner

We previously reconstituted LAT condensates on supported lipid bilayers (SLBs) through addition of various T cell signaling proteins to membrane-attached phospho-LAT (pLAT) (Su et al., 2016). In separate work, we also reconstituted membrane-associated contractile actomyosin networks by attaching actin to SLBs via the membrane-anchored actin binding domain of ezrin (eABD) in the presence of myosin II and capping protein (Köster et al., 2016). To examine interactions of LAT condensates with actin networks, here we combined these two systems into a single assay (Figure 1A). We attached polyhistidine-tagged phospho-LAT (pLAT) and polyhistidine-tagged eABD to Ni-NTA functionalized lipids within the SLB. We induced LAT phase separation into condensates by adding an increasing subset of binding partners, in the order Grb2, Sos1, phospho-SLP-76 (pSLP-76), Nck, and, finally, WASP or N-WASP, as previously described (Su et al., 2016). Hereafter we use the nomenclature pLAT → X to indicate condensates containing pLAT and all binding partners up to X (e.g. if X is Nck, then the condensates would contain pLAT, Grb2, Sos1, pSLP-76, and Nck). In T cells, the main WASP family protein at the IS is WASP (Kumari et al., 2014), which acts as a constitutive complex with WASP Interacting Protein (WIP) (Antón et al., 2002; Ramesh et al., 1997). We performed experiments here with either WASP or N-WASP to examine how differences between the proteins, which are 48% identical in amino acid sequence, affect interactions of LAT condensates with dynamic actin networks. We fused the N-WASP/WASP binding fragment of WIP to the N-terminus of each protein in order to stabilize its EVH1 domain (Peterson et al., 2007; Volkman et al., 2002), and improve bacterial expression. Our use of full-length WASP proteins here represents a step closer to the natural signaling system than our previous reconstitution (Su et al., 2016), which used only a C-terminal fragment of N-WASP, enabling us to better capture essential features of cellular LAT condensates while still maintaining manageable complexity.

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Composition regulates condensate movement with actin networks.

(A) Schematic of biochemical reconstitution in solutions containing LAT and eABD attached to SLBs and LAT phase separation agents, actin filaments, and myosin filaments in solution. (B) TIRF microscopy images of LAT condensates of the indicated compositions (see text for nomenclature) without actin (left column), with actin-only networks (middle column), or with actomyosin networks (right column). pLAT-Alexa488 is green, rhodamine-actin is magenta. Scale bar = 5 μm. (C) Actin enrichment at condensates in reconstitution assays using actomyosin, calculated as the ratio of actin fluorescence intensity within condensates to actin intensity outside condensates (see Materials and methods). Shown are the individual data points and their mean ±s.d. from N = 15 fields of view from three independent experiments (with 5 FOV per experiment). P-values are for indicated distribution comparisons via Wilcoxon rank-sum test with Bonferroni correction to achieve a total type-I error of 0.05. (D) Example plots of condensate tracks. Scale bar = 5 μm. (E) Effective diffusion coefficients of the condensate tracks. Each measurement is shown as a violin plot of the distribution of values from analyzing N = 15 fields of view from three independent experiments (with 5 FOV per experiment). Green square shows median and magenta plus sign shows mean. Significance was determined by averaging results from 100 Wilcoxon rank-sum tests that compared pairs of 500 randomly-selected tracks.

https://doi.org/10.7554/eLife.42695.002

Figure 1—source data 1 Source data file for Figure 1C.: https://doi.org/10.7554/eLife.42695.004
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For experiments involving actin, we added polymerized actin filaments that bound to the SLB via anchored eABD. To induce actin filament movement we added muscle myosin II and ATP, as previously described (Köster et al., 2016). While T cells express only non-muscle myosin II, the muscle isoform is functionally similar, differing largely in making somewhat longer filaments (800 nm vs 300 nm average length under similar conditions [Vicente-Manzanares et al., 2009]), and is much easier to purify from tissues in biochemical quantities. Since most movement of T cell receptor condensates (which typically coincide with LAT condensates) is blocked by inhibition of myosin (Yi et al., 2012), more complex reconstitutions including actin filament assembly and disassembly dynamics were not warranted in our work here (Blanchoin et al., 2000; Didry et al., 1998; Shekhar and Carlier, 2017). Thus, our reconstituted system should retain key qualitative behaviors of T cell actomyosin, while remaining experimentally practical.

We induced LAT condensate formation without actin, with actin alone, or with active actomyosin networks, and imaged the system using total internal reflection fluorescence (TIRF) microscopy (Figure 1B, Videos 1, 2 and 3). We immediately observed that condensates containing Nck, Nck and WASP, or Nck and N-WASP associated with and wet actin filaments in both actin and actomyosin networks. In contrast, condensates lacking these proteins remained distributed across the SLB (Figure 1B). As a corollary, co-localization analysis (see Supplemental Methods) showed that actin enrichment in condensates increased significantly in the presence of Nck and N-WASP (Figure 1C).

Video 1

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posterframe for video — Reconstitution of LAT condensate formation and movement using different component mixtures.

TIRF microscopy revealed LAT condensate formation and movement on supported lipid bilayers. His₈-pLAT-Alexa488 was attached to Ni-functionalized SLBs at 500 molecules / μm². Condensate component mixtures are indicated at the top of each movie panel. All reconstitutions were performed in 75 mM KCl. Condensates were formed by adding 125 nM Grb2 and 125 nM Sos1 (pLAT → Sos1); 125 nM Grb2, 62.5 nM Sos1, and 62.5 nM pSLP-76 (pLAT → pSLP-76); 125 nM Grb2, 62.5 nM Sos1, 62.5 nM pSLP-76, a 125 nM Nck (pLAT → Nck); 125 nM Grb2, 62.5 nM Sos1, 62.5 nM pSLP-76, 125 nM Nck, and 125 nM N-WASP (pLAT → N-WASP); or 125 nM Grb2, 62.5 nM Sos1, 62.5 nM pSLP-76, 125 nM Nck, and 125 nM WASP (pLAT → WASP). After formation, most condensates were either immobile or displayed confined movement regardless of their composition. Movie shows a 32 μm x 32 μm field of view for each movie panel. The movie is played at nine fps with frame intervals of 15 s.

https://doi.org/10.7554/eLife.42695.005

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Video 3

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In all conditions, we automatically detected and tracked the condensates at the SLB for 15 min, and then measured their effective diffusion coefficients using Moment Scaling Spectrum analysis as a measure of mobility (Jaqaman et al., 2011; Jaqaman et al., 2008; Vega et al., 2018); see Materials and methods) (Figure 1—figure supplement 1). This analysis revealed that in the absence of actin, condensates exhibited a distribution of diffusion coefficients with medians in the range of 3 × 10⁻⁵ to 8 × 10⁻⁵ µm² / s (Figure 1D,E, Video 1). In the presence of actin alone (i.e. no myosin), condensate movement varied with composition. pLAT → Sos1 and pLAT → pSLP76 showed an increase in mobility (Figure 1D,E, Video 2), while pLAT → Nck, pLAT → WASP, and pLAT → N-WASP had a tendency to align with actin filaments and showed either a decrease or no change in mobility (Figure 1D,E, Video 2). Lastly, in the presence of active actomyosin networks, condensates of all compositions exhibited an overall increase in mobility (median effective diffusion coefficient range of 1 × 10⁻⁴ to 3 × 10⁻⁴ µm² / s). The increase for pLAT → Sos1 and pLAT → pSLP76 was subtle, larger for pLAT → Nck, and largest for pLAT → WASP and pLAT → N-WASP. These data suggest that condensates containing Nck or, Nck and WASP, or Nck and N-WASP, which wet filaments and show a large differential in behavior between actin alone and actomyosin conditions, can be viewed distinctly from condensates containing components only up to Sos1 or Sos1 and pSLP76, which do not wet filaments and show smaller differences between the two types of actin networks.

LAT condensates in vitro containing Nck and WASP or N-WASP move with contracting actomyosin networks with high fidelity

To delineate the effect of composition on the ability of LAT condensates to move with actin filaments, we devised an in vitro system where the actin filaments moved in a directional manner. This system enabled us to clearly distinguish between condensates that move with the actin filaments (because they would also exhibit directional movement) and those that do not. For this, we performed experiments where an SLB-associated actin network was induced to contract into asters by addition of myosin II filaments in the presence of low concentrations of salt and ATP (see Supplemental Methods). These experiments were especially susceptible to photo-damage caused by light exposure (Figure 2—figure supplement 1). Thus, imaging conditions were set to minimize light exposure and the actomyosin/condensate configuration at the end point of all time-lapses was compared to adjacent, non-imaged regions of the SLB. These actomyosin contraction experiments were performed with pLAT → Sos1, pLAT → WASP, and pLAT → N-WASP condensates. In the beginning of such experiments, pLAT → Sos1 condensates were randomly distributed on the membrane while pLAT → WASP or pLAT → N-WASP condensates were aligned along the actin filaments as observed above (Figure 2A). In all cases the filament network started to contract immediately upon myosin II addition and formed stable asters within 2 min (Video 4). As shown in Figure 2A, at the end of the contraction, most of the pLAT → Sos1 condensates remained scattered across the SLB, while virtually all of the pLAT → N-WASP condensates had moved with the actin into asters. pLAT → WASP condensates showed behavior intermediate between these extremes. To quantify these behaviors, we examined the speed and direction of both condensate movement and actin movement during actomyosin network contraction using Spatio-Temporal Image Correlation Spectroscopy (STICS) (Ashdown et al., 2014) (Figure 2B). We found that the speed of pLAT → N-WASP condensates correlated well with the speed of actin, while the speed of pLAT → Sos1 condensates did not (Figure 2C). pLAT → WASP condensates showed an intermediate degree of correlation. Additionally, the distribution of angles between the vectors of pLAT → N-WASP condensate movement and proximal actin movement showed clear preference for smaller angles, indicating a high degree of co-movement. In contrast, the angle distribution for pLAT → Sos1 showed only a slight preference for smaller angles, which was marginally significant (Figure 2D). Again pLAT → WASP condensates displayed an intermediate behavior. Together, the steady state (Figure 1) and contraction (Figure 2) analyses show that LAT condensates are influenced by actin network dynamics in a composition-dependent fashion. Condensates containing Nck or Nck and WASP/N WASP bind to and move with actomyosin filaments. In contrast, condensates lacking these proteins do not bind filaments appreciably, and move through a different mechanism, likely due to non-specific steric contacts. Thus, Nck and WASP/N WASP function as a molecular clutch between LAT condensates and actin in vitro.

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pLAT → N-WASP condensates bind to and move with moving actin filaments.

(A) TIRF microscopy images of pLAT → Sos1 condensates (left two columns), and pLAT → N-WASP condensates (middle two columns), and pLAT→ WASP (right two columns), formed in an actin network before (t = 0 min) and after (t = 2 min) addition of myosin II. pLAT condensates are green and actin is magenta in merge. Scale bar = 5 μm. (**B–D**) STICS analysis of actin and condensate movement. (B) Representative map of actin (magenta) and pLAT condensate (green) vector fields. Lower panels show magnification of box regions in upper panels. (C) Condensate speed vs. actin speed at same position. Condensate composition indicated above each heat map. Heat map indicates frequency in each bin, that is counts in each bin normalized by total number of counts. (D) Distribution of the angle between actin and condensate movement vectors for pLAT → Sos1 (blue), pLAT → N-WASP (gold), randomized pLAT → Sos1 (red) and randomized pLAT → N-WASP (purple) (see Materials and methods for randomization). P-values are for indicated distribution comparisons via Kolmogorov-Smirnov test. Data in (C) and (D) are pooled from 15 fields of view from three independent experiments (5 FOV per experiment).

https://doi.org/10.7554/eLife.42695.008

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Phase separation on membranes enhances interactions between Nck/N-WASP and actin filaments

The data above suggest that Nck, WASP, and N-WASP mediate binding of LAT condensates to actin filaments. To test this, we quantified the recruitment of preformed actin filaments (10% rhodamine-labeled) to SLBs by LAT condensates of different compositions in the absence of eABD. As shown in Figure 3A and Figure 3—figure supplement 1, only condensates containing Nck, Nck and WASP, or Nck and N-WASP recruited substantial amounts of actin filaments. In each case, condensates were elongated along filaments, as observed above.

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Basic regions of Nck and N-WASP mediate interaction of LAT condensates with actin filaments.

(A) (Left) TIRF microscopy images of rhodamine-actin (magenta) recruited to SLBs by the indicated LAT condensate compositions (green). Scale bar = 5 μm. All panels use the same intensity range. (Right) Normalized average rhodamine-actin fluorescence intensity on SLBs. Shown are the individual data points and their mean ±s.d. N = 15 fields of view from three independent experiments (5 FOV per experiment). P values were determined using a t-test. (B) (Left) TIRF microscopy images of rhodamine-actin recruited to SLBs by His-tagged Nck variants. Scale bar = 5 μm. All panels use the same intensity range. (Right) Normalized average rhodamine-actin fluorescence intensity on SLBs. Shown are the individual data points and their mean ±s.d. N = 9 fields of view from three independent experiments (3 FOV per experiment). P values were determined using a t-test. (C) (Left) TIRF microscopy images of rhodamine-actin recruited to SLBs by His-tagged N-WASP variants. All panels use the same intensity range. (Right) Normalized average rhodamine-actin fluorescence intensity on SLBs. Shown are the individual data points and their mean ±s.d. N = 15 fields of view from three independent experiments (5 FOV per experiment). P values were determined using a t-test. (D) TIRF microscopy images of pLAT → N-WASP_Basic condensates (left two columns) and pLAT → N-WASP_Neutral condensates (right two columns) formed in an actin network before (t = 0 min) and after (t = 2 min) addition of myosin II. LAT condensates are green and actin is magenta in merge. Scale bar = 5 μm. (E) Condensate speed vs. actin speed at same position from STICS analysis. Condensate composition indicated above each heat map. Heat map indicates frequency in each bin (as in Figure 2C). N = 15 fields of view from three independent experiments (5 FOV per experiments). (F) Distribution of the angle between actin and condensate movement vectors for pLAT → N-WASP_Basic (gold), randomized pLAT → N-WASP_Basic (purple), pLAT → N-WASP_WT (black, same data as in Figure 2), pLAT → N-WASP_Neutral (blue), and randomized pLAT → N-WASP_Neutral (red). N = 15 fields of view from three independent experiments (5 FOV per experiment). P-values are for indicated distribution comparisons via Kolmogorov-Smirnov test.

https://doi.org/10.7554/eLife.42695.011

Figure 3—source data 1 Source data file for Figure 3A.: https://doi.org/10.7554/eLife.42695.028
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Figure 3—source data 2 Source data file for Figure 3B.: https://doi.org/10.7554/eLife.42695.029
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Figure 3—source data 3 Source data file for Figure 3C.: https://doi.org/10.7554/eLife.42695.030
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To better understand these interactions, we next determined the effective dissociation constants for LAT → Nck and LAT → N-WASP condensates binding to actin filaments (Figure 3—figure supplement 2A). We measured the fluorescence intensity of recruited actin as a function of actin filaments in solution. Fluorescence intensity showed a sigmoidal dependence on actin filament concentration, indicating cooperative recruitment to the membrane, perhaps due to avidity effects based on multi-point interactions of longer filaments with the condensates (see below). Fitting the data to a Hill equation showed that LAT → N-WASP condensates bind actin more tightly than LAT → Nck condensates (K_D = 280 nM, 95% Confidence Interval (CI) [250 nM, 300 nM] versus 410 nM, 95% CI [380 nM, 450 nM]) and with similar Hill coefficients (3.6, 95% CI [3.0, 4.4] vs 3.2, 95% CI [2.4, 5.6]) (Figure 3—figure supplement 2B,C). LAT → N-WASP condensates also have a higher capacity for actin filaments than LAT → Nck condensates, based on a higher maximal recruited actin fluorescence intensity (Figure 3—figure supplement 2B,C). Thus, the condensates can recruit actin filaments to membranes with high effective affinity.

We next performed co-sedimentation assays to determine whether Nck or N-WASP could bind actin filaments in solution or whether efficient binding required the proteins to be arrayed on a two-dimensional surface (Figure 3—figure supplement 3). At 2 µM concentration, Nck did not appreciably co-sediment with actin filaments up to 7.9 µM actin filament concentration. While these data could not yield a K_D value for the interaction of Nck and actin in solution, they clearly show that binding affinity is much lower than when Nck is organized into LAT → Nck condensates on membranes. Consistent with previous reports, 2 µM N-WASP did bind filaments (Co et al., 2007), although only 24% of the protein co-sedimented with 7.9 µM F-actin. This contrasts with α-actinin, which has a reported K_D for actin filaments of 4.7 µM (Wachsstock et al., 1993) and shows an approximately equal distribution between supernatant and pellet (Figure 3—figure supplement 3). Estimating K_D of N-WASP:F-actin binding in solution from the 7.9 µM F-actin data point yields an approximate value of 25 µM. Thus, as for Nck, N-WASP has appreciably lower affinity for actin filaments in solution than when organized into LAT → N-WASP condensates on membranes.

This difference in N-WASP-actin affinity between the different formats could be due to A) effects of the other proteins in LAT → N-WASP condensates (e.g. inducing conformational changes in N-WASP that increase affinity) or B) effects of concentrating N-WASP into high-density condensates on the two-dimensional membrane surface. To test whether the collection of molecules within LAT condensates alone could increase affinity of N-WASP for actin, we performed co-sedimentation assays in which the composition of the solution was increased in complexity by adding Nck, pSLP-76, Grb2, and pLAT to N-WASP and actin filaments. In solution, addition of LAT condensate components did not enhance binding of N-WASP to actin filaments (Figure 3—figure supplement 4). These data suggest that it is the localization of N-WASP to LAT condensates on membranes, rather than conformation changes induced by ligands per se, that enables efficient actin filament binding. We do not know the exact source of these effects. They could arise from avidity when multiple N-WASP molecules in a high-density membrane condensate bind an actin filament. Alternatively, they could arise from actions of the membrane itself on the conformation of Nck-bound N-WASP.

Together, these data show that assembling Nck and N-WASP into phase separated condensates on membranes increases their apparent affinity for actin filaments, enabling efficient recruitment of actin filaments to membranes. This effect probably also accounts for the strong engagement of membrane-associated actin filament networks with LAT condensates containing Nck and N-WASP (Figures 1–3).

Basic regions of Nck and N-WASP couple LAT condensates to actin filaments

To identify the elements of Nck that mediate LAT condensate binding to actin filaments, we attached polyhistidine-tagged fragments of the proteins to SLBs and measured their ability to recruit actin filaments from solution. Nck is composed of three SH3 domains and an SH2 domain, connected by flexible linkers of 25–42 residues (Banjade et al., 2015). Of these seven elements, two contain dense basic patches, one contains dense acidic patches, and the remaining four are relatively free of dense charge patches. As detailed in Figure 3—figure supplement 5, Nck fragments containing an excess of basic elements recruited actin to the membrane, where greater excess resulted in more efficient recruitment, while neutral or acidic fragments did not. Similarly, mutating one of the basic elements (the linker between the first and second SH3 domains, L1, to neutralize it (Nck_Neutral) or to make it acidic (Nck_Acidic) greatly impaired actin recruitment, while making it more basic (Nck_Basic) enhanced actin recruitment (Figure 3B, Figure 3—figure supplement 6). These data indicate that basic regions of Nck likely contribute to binding of LAT condensates to actin filaments.

Like Nck, N-WASP also has a central basic region, amino acid residues 186–200. We thus asked whether this region and/or the two C-terminal WH2 motifs, which are known to bind actin monomers and filament barbed ends (Bieling et al., 2018; Co et al., 2007), contribute to coupling of LAT condensates to actin filaments. We generated pLAT → N-WASP condensates with N-WASP fragments consisting of the basic-proline elements (BP), basic-proline +VCA (BPVCA) elements, and a BPVCA protein with mutations in the WH2 motifs in the VCA region that impair filament binding (BPVCA_mut) (Co et al., 2007). All three types of condensates strongly recruited actin, indicating that WH2-actin interactions are not needed for actin filament recruitment by LAT condensates (Figure 3—figure supplement 7). To further examine the basic region, we generated three variants of His₆-tagged full length N-WASP (fused N-terminally to the WASP-binding region of WIP, as in the earlier experiments; Figure 3—figure supplement 7): one containing a doubled basic region (N-WASP_Basic), one wild-type (N-WASP_WT), and one containing a neutral linker instead of the basic region (N-WASP_Neutral). As shown in Figure 3C and Figure 3—figure supplement 8, these variants recruited actin in the order N-WASP_Basic >>N WASP_WT>N WASP_Neutral. Thus, for both Nck and N-WASP, the degree of positive charge in basic elements correlates with the ability of the proteins and their LAT condensates to recruit actin filaments to SLBs.

To test whether these basic regions are necessary to couple LAT condensate movement to actin movement (Figure 2), we performed actin contraction assays with condensates containing N-WASP_Basic or N-WASP_Neutral. Before myosin II addition, pLAT - > N WASP_Basic condensates aligned almost perfectly with actin filaments, while pLAT - > N WASP_Neutral condensates aligned only partially, consistent with the notion that the basic region mediates binding to actin filaments (Figure 3D). After actin network contraction, pLAT → N-WASP_Basic condensates localized with actin asters to a similar degree as pLAT → N-WASP_WT, while pLAT → N-WASP_Neutral localized less with actin (Figure 3D vs. Figure 2A, Video 5 vs. Video 4). STICS analysis revealed that the correlation of pLAT → N-WASP_Basic movement with local actin movement was slightly better than that of pLAT → N-WASP_WT, while the correlation of pLAT → N-WASP_Neutral was worse (Figure 3E,F). The remaining correlation for pLAT → N-WASP_Neutral is most likely due to the presence of Nck, which also contributes to actin binding.

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Together these data demonstrate that regions of Nck and N-WASP that contain dense basic patches can mediate the clutch-like behaviors of the proteins by directly interacting with actin filaments proportionally to the degree of positive charge, and that these interactions are necessary for LAT condensates to faithfully move with actin.

The composition of LAT condensates changes as they traverse the IS in cells

In Jurkat T cells activated by SLBs coated with ICAM-1 and OKT3, an antibody that recognizes the CD3ε subunit of the TCR, LAT condensates that form at the periphery of the IS move to its center over ~5 min as the cell-SLB contact matures. To investigate whether the composition-dependent interactions observed in our biochemical data might have consequences for the behavior of LAT condensates in cells, we examined the composition of LAT condensates as they moved in the plane of the plasma membrane during activation of live Jurkat T cells. We used Jurkat T cells because they retain many features of primary T cells relevant to movement and signaling from LAT condensates, but are easier to manipulate and analyze. In both cell types, LAT condensate formation is well-documented (Balagopalan et al., 2013; Lin et al., 1999; Su et al., 2016; Yokosuka et al., 2005), condensate movement across the IS is correlated with actin flow (DeMond et al., 2008; Kaizuka et al., 2007; Murugesan et al., 2016; Yi et al., 2012), and proximal biochemical signaling from LAT through SLP-76 is similar (Bartelt et al., 2009). However, the IS between Jurkat T cells and supported lipid bilayers is larger than that of primary T cells (Murugesan et al., 2016), and LAT condensates do not initiate actin polymerization to the same degree in Jurkat T cells as in primary T cells, which allows us to analyze the ability of condensates to couple to dynamic actin networks without accounting for their own self-generated polymerized actin (Kumari et al., 2015).

We co-expressed LAT-mCitrine with Grb2-mCherry or LAT-mCherry with Nck-sfGFP and LifeAct-BFP in Jurkat T cells. These cells bound to SLBs coated with mobile ICAM-1 and OKT3, producing an IS mimic with the SLB. In contrast to Jurkat T cells adhered to immobile substrates, which extend long lamellipodia (Babich et al., 2012), Jurkat T cells adhering to fluid SLBs here extend only short lamellipodia at the IS periphery. We used TIRF microscopy to capture images of activated cells every 5 s for up to 5 min. We then automatically detected and tracked LAT condensates from their formation at the periphery of the IS to their coalescence with the central supramolecular activation complex (cSMAC) at the synapse center (Jaqaman et al., 2008) (Figure 4—figure supplement 1A, Figure 4—figure supplement 2, Video 6), monitoring the fluorescence intensity of LAT and Grb2 or Nck. We tracked condensates based on the LAT channel, and then read out the Grb2 or Nck intensities at the condensate locations (‘master/slave’ channel analysis, as in Loerke et al., 2011). This scheme ensured accurate condensate detection and tracking because of the stronger signal in the LAT channel, especially when compared to the Nck channel, which tended to have high background due to soluble Nck molecules not associated with the membrane. To overcome the stochasticity and noise inherent to live-cell image data, we aligned tracks (in space or time, as described below) and averaged them to uncover underlying overall trends. For meaningful alignment and averaging, we filtered tracks by their duration, extent of directed movement, initial and final positions (to ensure that they traversed a sufficient radial distance across the IS), and initial Grb2 or Nck intensity to ensure that changes in intensity could be measured accurately (see Materials and methods for details).

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Initially we aligned tracks according to radial position in the synapse, defining distance from the center of the synapse to the synapse edge as ‘normalized radial position’ (equal to zero at the synapse center and one at the synapse edge; see Materials and methods). This analysis revealed that Grb2 colocalized with LAT condensates at the edge of the synapse (Figure 4A, Video 7) and its fluorescence intensity in the condensates was maintained throughout the trajectory to the center of the synapse (Figure 4B,C). In contrast, while Nck also colocalized with LAT condensates at the edge of the synapse (Figure 4D, Video 8), its fluorescence diminished relative to LAT during the trajectory. The rate of change was largest in the ~0.8–0.5 interval of normalized radial positions. The decrease in the Nck:LAT ratio within condensates compared to the peak value was statistically significant starting at the 0.7–0.6 normalized radial position interval, based on a Rank-Sum test of the medians (Figure 4C,E). The change in composition slowed down to an almost negligible rate after the 0.6–0.5 normalized radial position interval, that is in the central half of the IS (Figure 4C). The emergence of this pattern from averaging 125 spatially aligned tracks from 25 cells suggests that spatial position is a key determinant of Nck residence in LAT condensates. In contrast, when the condensates were aligned according to the time of their appearance (Figure 4—figure supplement 1B), the composition measurements per time point were less well defined (as reflected by larger 95% confidence intervals around the median), the pattern over time was noisier, and the median Nck intensity more or less steadily decreased over the course of the trajectory. The decrease in Nck intensity is not the result of photobleaching (Figure 4—figure supplement 3). This suggests that in this experimental setting, position plays a more instructive role than time in determining the residence of Nck in condensates (and presumably the residence of WASP, which is recruited to LAT condensates via Nck). However, since time and space are coupled, our data do not rule out a role for time in this process, as has previously been observed in experimental conditions where LAT condensates were immobilized (Barda-Saad et al., 2005; Yi et al., 2019).

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LAT condensates change composition as they move across the IS.

(A) TIRF microscopy image of Jurkat T cell expressing Grb2-mCherry (magenta in merge) and LAT-mCitrine (green in merge) activated on an SLB coated with OKT3 and ICAM-1. Scale Bar = 5 μm. (B) Magnification of boxed region in (A), with normalized radial position (one at synapse edge, 0 at cSMAC center) indicated above images and time indicated below. Scale bar = 2 μm. (C) Quantification of fluorescence intensity ratios of Grb2/LAT (gold) and Nck/LAT (blue) in LAT condensates in Jurkat T cells at different normalized radial positions. Measurements were made at identical relative locations but data points are slightly offset in the graph for visual clarity. Plot displays median and its 95% confidence interval from N = 125 condensates from 25 cells expressing Nck-sfGFP and LAT-mCherry from five independent experiments and 82 condensates from 11 cells expressing Grb2-mCherry and LAT-mCitrine from five independent experiments. Only tracks in which the mean Grb2 or Nck intensity was greater than one standard deviation above background during the first three measurements were used for this analysis. Asterisks indicate data points whose values differ significantly from the reference data point (radial position = 1.0–0.9 for Grb2 and radial position = 0.9–0.8 for Nck) as determined using a Wilcoxon rank-sum test with Bonferroni correction. The Bonferroni correction was used to achieve a total type-I error of 0.05. For eight comparisons, this leads to a significance threshold per pair = 0.006. (D) TIRF microscopy image of Jurkat T cell expressing Nck-sfGFP (green in merge) and LAT-mCherry (magenta in merge) activated on an SLB by OKT3 and ICAM-1. Scale Bar = 5 μm. (E) Magnification of boxed region in (D). Scale bars and details as in (B). (F) Fluorescence polarization microscopy image of a Jurkat T cell sparsely labeled with SiR-Actin and activated on an SLB coated with OKT3 and ICAM-1 (left, scale bar = 10 µm), magnification of boxed region in image (middle, scale bar = 2 µm), and fraction of filaments perpendicular (+ / - 45^o) to the synapse edge (right). Cyan lines in left and center images indicate the orientation of the fluorescence dipole of the SiR fluorophore which in turn is oriented orthogonal to the underlying actin filament (see Figure 4—figure supplement 5). Shown are the mean ±s.d. from N = 13,052 particles from 6 cells. Asterisks indicate data points whose values differ significantly from the reference data point (radial position = 1.0–0.9) as determined using a t-test with Bonferroni correction to achieve a total type-I error of 0.05.

https://doi.org/10.7554/eLife.42695.033

Figure 4—source data 1 Source data file for Figure 4F.: https://doi.org/10.7554/eLife.42695.040
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Nck dissipation spatially parallels the changes in actin architecture from dendritic network to concentric arcs

Translocation of LAT condensates from the synapse periphery to the center of the IS is driven by motion of the actin cytoskeleton (DeMond et al., 2008; Kaizuka et al., 2007; Mossman et al., 2005; Yu et al., 2010). Recent work has shown that two actin networks are generated at the IS in activated T cells (Murugesan et al., 2016; Yi et al., 2012). The outer ~1/3 of the synapse is enriched in a dendritic actin meshwork generated by the Arp2/3 complex, where the filaments are on average directed radially, perpendicular to the synapse edge. Progressing toward the cSMAC this meshwork is replaced by formin-generated concentric actin arcs that are directed parallel to the synapse edge (Figure 4—figure supplement 4) (Hammer and Burkhardt, 2013; Yi et al., 2012). The arcs dominate in the central ~1/2 of the IS. Both filament networks move through the action of myosin motors as the cell-cell conjugate matures; however, the nature of this movement is different in the two cases. The outer dendritic network moves in a direction perpendicular to the edge of the synapse in a process termed retrograde flow (DeMond et al., 2008; Kaizuka et al., 2007; Mossman et al., 2005; Yu et al., 2010), analogous to actin flow observed at the leading edge of migrating cells (Ponti et al., 2005; Ponti et al., 2004). In contrast, the inner concentric arcs sweep toward the center of the synapse in a telescoping manner and appear to have components of motion both perpendicular and parallel to the synapse edge (Murugesan et al., 2016).

The spatial pattern of Nck dissipation from LAT condensates is similar to the reported actin transition from dendritic architecture to arc architecture (Hammer and Burkhardt, 2013; Murugesan et al., 2016; Yi et al., 2012). To corroborate this in our cells, we incubated Jurkat T cells with the dye SiR-Actin, which binds to actin filaments with a defined orientation (perpendicular to the filament orientation; Figure 4—figure supplement 5) (Nordenfelt et al., 2017). This dye enabled us to use instantaneous polarization TIRF microscopy (Mehta et al., 2016) to evaluate the orientation of actin filaments at the IS. We found that SiR-actin at concentrations higher than 50 nM blocked actin flow at the IS, but speckle labeling with 10 nM SiR-actin allowed for actin flow from the edge to the center (data not shown). Since the actin networks were stained as sparsely distributed speckles of SiR-actin, our analysis procedure does not involve (or require) identification of individual filaments in the images to characterize their orientations. Rather, by observing the fluorescence polarization orientation of single speckles randomly bound to actin filaments, we can build a spatial map of filament orientations across the IS. We found that actin filaments in the outer 30% of the synapse were generally oriented perpendicular to the synapse edge, while those closer to the center of the synapse were parallel to the synapse edge (Figure 4F), in good agreement with earlier super-resolution work (Murugesan et al., 2016). The rate of change of orientation was largest in the ~0.8–0.6 interval of normalized radial positions. T-test statistical analysis, in which actin filament orientation at each spatial point was compared with filament orientation at the edge of the IS, demonstrated a significant change in actin filament orientation relative to the edge starting at a normalized radial position of 0.7–0.6 and was maintained to the center of the IS. This spatial pattern of actin filament orientation correlates well with the spatial pattern of Nck dissipation from LAT condensates (compare Figure 4C and F). The location of the changes in actin orientation is unrelated to the overall three-dimensional geometry of the cell, indicating that actin architecture at the IS, as observed via TIRF microscopy, is not determined by the position of the cell body above the membrane surface (Figure 4—figure supplement 6).

These data show that the changes in the actin network at the IS, from a peripheral dendritic organization to a more central collection of circular arcs, are spatially correlated with the changes in composition of the LAT condensates, from high levels of Nck enrichment at the periphery to low levels nearer the center.

Constitutive engagement of LAT condensates with actin leads to their aberrant movement across the IS

Our combined biochemical and cellular data thus far indicate that Nck and WASP mediate LAT condensate engagement with actin, and that LAT condensates lose these proteins as they move from the dendritic actin meshwork in the outer part of the synapse to the contractile arcs closer to the synapse center. The biochemical data suggest that this change in composition should allow LAT condensates to interact differently with the two actin networks, with stronger adhesion to the dendritic meshwork and weaker adhesion to the contractile arcs. We hypothesized that this change in interaction might be necessary for the proper radial movement of LAT condensates at the IS, given the different orientation (perpendicular vs. parallel to the synapse edge) and movement (retrograde flow vs. telescoping motion) of filaments in the two actin networks. To test this hypothesis, we altered the adhesion of LAT condensates to the actin filament network by fusing Grb2, which remains in the condensates throughout their trajectories (Figure 4A–C), with the doubled basic region of N-WASP (Grb2_Basic).

In biochemical assays, this fusion protein generated LAT condensates that bound actin filaments in the absence of Nck or WASP. The pLAT-Grb2_basic complex recruited actin filaments to SLBs, while pLAT alone or the pLAT-Grb2 complex did not (Figure 5A). In actomyosin contraction assays, condensates of pLAT/Grb2_Basic/Sos1 initially wet filaments and then localized to actin asters after myosin II-induced contraction to a greater degree than pLAT/Grb2/Sos1, although to a lesser degree than pLAT → N-WASP (Figure 5B, Video 9 vs. Video 4). Similarly, during actomyosin network contraction, the movement of pLAT/Grb2_Basic/Sos1 condensates was correlated more strongly with actin movement than condensates containing Grb2 (Figure 5—figure supplement 1), but less strongly than pLAT → N-WASP condensates (Figure 2C,D). Together, these data demonstrate that the double basic motif of N-WASP, when added to Grb2, can act as a molecular clutch, coupling LAT condensates to actin in vitro.

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Grb2 fused to a basic molecular clutch can couple LAT condensates to actin.

(A) TIRF microscopy images of rhodamine-actin recruited to SLBs by His-tagged pLAT or condensates of pLAT → Grb2 or pLAT → Grb2_Basic. Scale bar = 5 μm. (B) TIRF microscopy images of pLAT → Sos1 condensates containing Grb2_WT (top row; data from Figure 2) or Grb2_Basic (bottom row) formed in an actin network before (t = 0 min) and after (t = 2 min) addition of myosin II. Actin shown in magenta and LAT condensates in green. Scale bar = 5 μm. (C) TIRF microscopy image of Jurkat T cell expressing Grb2-mCherry (top, magenta in merge) or Grb2_Basic-mCherry (bottom, magenta in merge) and LAT-mCitrine (green in merge) activated on an SLB coated with OKT3 and ICAM-1. Scale Bar = 5 μm. (D) Fluorescence intensity ratios of Grb2/LAT (blue, data from Figure 4C) or Grb2_Basic / LAT in condensates (gold) at different normalized radial positions. Measurements were made at identical relative locations but data are slightly offset in the graph for visual clarity. Plot displays median and notches from boxplot for 95% confidence interval from N = 44 condensates from 12 cells expressing Grb2_Basic-mCherry and LAT-mCitrine from seven independent experiments and 82 condensates from 11 cells expressing Grb2-mCherry and LAT-mCitrine from five independent experiments (same cells as in Figure 4C). Only tracks in which the mean Grb2 or Grb2_Basic intensity was greater than one standard deviation above background during the first three measurements were used to generate this plot. Asterisk indicates data point whose value differs significantly from the reference data point (radial position = 1.0–0.9) as determined using a Wilcoxon rank-sum test with Bonferroni correction. The Bonferroni correction was used to achieve a total type-I error of 0.05. For eight comparisons, this leads to a significance threshold per pair = 0.006. (E) Trajectories of LAT condensates in Jurkat T cells expressing Grb2-mCherry (top) or Grb2_Basic-mCherry (bottom) recorded over 2 to 5 min of imaging. Trajectories are color-coded as indicated in the legend at right according to deviation from a straight line between the estimated starting point of actin engagement and just before entering the cSMAC (see Materials and methods). Scale bar = 5 μm. (F) Distribution of deviations from a straight line for condensates in Jurkat T cells expressing Grb2-mCherry (red) or Grb2_Basic-mCherry (blue). N = 44 condensates from 12 cells expressing Grb2_Basic-mCherry and LAT-mCitrine from seven independent experiments and 82 condensates from 11 cells expressing Grb2-mCherry and LAT-mCitrine from independent experiments (same cells as in Figure 4C). P-value is for comparing the two distributions via a Kolmogorov-Smirnov test. Only tracks in which the mean Grb2 or Grb2_Basic intensity was greater than one standard deviation above background during the first three measurements were used to generate this plot.

https://doi.org/10.7554/eLife.42695.043

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We next asked whether expression of Grb2_Basic in Jurkat T cells would perturb the radial movement of LAT condensates due to their constitutively engaged clutch, including in the medial region of the IS where they encounter actin arcs. We quantified the deviation from a straight path of condensate trajectories between the start of persistent inward radial movement and coalescence with the cSMAC (see Supplemental Methods for details). For these experiments, cells expressing Grb2_Basic-mCherry were activated on SLBs as above. Similar to cells expressing Grb2-mCherry, LAT condensates that formed at the periphery of the IS retained Grb2_Basic-mCherry throughout their trajectories to the cSMAC (Figure 5C,D). However, statistical analysis of these trajectories demonstrated a small, but significant, deviation from a straight path when compared with condensates in cells expressing Grb2-mCherry (Figure 5E,F, Video 10 vs. Video 7). This behavior is consistent with abnormally high adhesion of condensates containing Grb2_Basic to actin filaments, even after Nck has presumably dissipated, leading to trajectories that reflected more the telescoping, circular component of the contractile actin arc motion.

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Formin activity is necessary for LAT condensate composition change

Finally, we asked whether the transition from the dendritic actin architecture to the contractile arcs might play a role in changing the composition of LAT condensates. Previous data showed that the contractile arcs are generated by the formin mDia1 and could be eliminated by the formin inhibitor, SMIFH2 (Murugesan et al., 2016). We found that in contrast to control cells treated with DMSO, where Nck dissipated normally from LAT condensates (Figure 6A and B, Figure 6—figure supplement 1, Video 11), cells treated with SMIFH2 for five minutes prior to imaging displayed LAT condensates with virtually constant Nck intensities throughout their trajectories from the periphery to the cSMAC (Figure 6A and C, Figure 6—figure supplement 1, Video 12). Thus, the activity of formin proteins, and/or perhaps the actin arcs that they generate, act to alter the composition of LAT condensates, likely altering their downstream signaling activities in the central region of the IS. We note that the SMIFH2 data further support the notion that in unperturbed cells, space, rather than time, is the key determinant of Nck residence in condensates (assuming that formins do not also create a temporal signal). Our combined data suggest that the two actin networks in activated Jurkat T cells not only spatially organize the immunological synapse by moving LAT condensates, but may also contribute to creation of specific signaling zones.

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Formin activity is necessary for Nck dissipation from LAT condensates.

(A) Fluorescence intensity ratios of Nck/LAT in condensates in Jurkat T cells treated with DMSO (blue) or the formin inhibitor, SMIFH2, (gold) at different normalized radial positions. Measurements were made at identical relative locations but data are slightly offset in the graph for visual clarity. Plot displays median and notches from boxplot for 95% confidence interval from N = 43 condensates from 11 DMSO-treated cells from five individual experiments and 102 condensates from 14 SMIFH2-treated cells from five individual experiments. Only tracks in which the mean Nck intensity was greater than one standard deviation above background during the first three measurements were used to generate this plot. The first DMSO data point (radial position = 1.0–0.9) does not appear in the plot because the number of detected condensates was too small (<10) to generate a statistically meaningful measurement. Asterisks indicate data points whose values differ significantly from the reference data point (radial position = 0.9–0.8) as determined using a Wilcoxon rank-sum test with Bonferroni correction. The Bonferroni correction was used to achieve a total type-I error of 0.05. For eight comparisons, this leads to a significance threshold per pair = 0.006. (**B, C**) Magnification of boxed regions from Figure 6—figure supplement 1 of condensates containing LAT-mCherry (magenta in merge) and Nck-sfGFP (green in merge) during their trajectories across the IS in a cell treated with DMSO (B) or SMIFH2 (C). Normalized radial position indicated above image panels and time below panels. Scale bar = 2 μm.

https://doi.org/10.7554/eLife.42695.047

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Discussion

Compositional changes of biomolecular condensates in response to signals have been well documented (Chen et al., 2008; Dellaire et al., 2006; Markmiller et al., 2018; Salsman et al., 2017; Youn et al., 2018). However, the functional consequences of these compositional changes have generally not been elucidated. Here, we show that compositional changes alter the interactions of LAT condensates on membranes with active actomyosin networks. Our in vitro data demonstrate that Nck, WASP, and N-WASP act as a clutch that mediates strong binding of LAT condensate to actin filaments, thus coupling movement of the structures to movement of actomyosin filaments. LAT condensates lacking Nck and WASP or N-WASP can still be propelled by other, possibly steric, interactions with moving actomyosin filaments, but less efficiently than when the clutch is present. We also show that the composition of LAT condensates in activated Jurkat T cells changes as they move radially from the edge to the center of the IS. This change spatially parallels the transition from the peripheral dendritic actin network to the circular actin arcs, and inhibition of the formin mDia1 prevents loss of Nck from LAT condensates. Mutations that add a basic sequence to Grb2, thus constitutively engaging LAT condensates with actin filaments, cause aberrant movement of the condensates with a higher tendency to deviate from a straight line.

These data suggest a potential model for movement of LAT condensates across the IS by navigating the distinct cortical actin networks. Condensates form in the outer region of the IS with the full complement of signaling molecules, including Grb2, Sos1, SLP-76, Nck, and WASP. Within this collection, the basic regions on Nck and WASP could act as a molecular clutch, enabling the condensates to adhere tightly to the outer dendritic actin network, and to travel radially with the network as it moves by retrograde flow. In the transition to the formin-generated actin arcs, a formin-dependent signal causes loss of Nck, and likely WASP, which is present in condensates largely through its interactions with Nck (Cannon et al., 2001) (although interactions between TCR, DOCK-8, WIP, and WASP could also contribute [Janssen et al., 2016]). We do not yet know the nature of this signal, but one possibility would be dephosphorylation of SLP-76, as Nck is known to join condensates primarily through binding SLP-76 phosphotyrosines (Barda-Saad et al., 2010; Pauker et al., 2012). Other mechanisms involving weakening of different interactions, appearance of competing Nck binding partners, or mechanical disruptions are also possible. Our biochemical data suggest that loss of Nck/WASP should decrease adhesion of the LAT condensates to actin. In this state, the condensates should still be movable by actin, based on our in vitro data on LAT → Sos1 condensates, but likely through more transient, weaker contacts. This is consistent with previous observations that in the actin arcs region of the IS, LAT condensates are repeatedly hit by arcs that move them briefly but then release (Murugesan et al., 2016). The circular movement of the telescoping actin arcs is randomly directed clockwise and counterclockwise, and repeated hits by arcs moving oppositely should produce no net circular motion on the condensates. But the radial component is consistently directed toward the center of the IS, and thus repeated hits could constructively produce a net movement in a radial direction. Thus, this model predicts that LAT condensates would continue to move linearly toward the center of the IS in the arc region. If the condensates were to adhere tightly to actin in the arcs region (i.e. if they contained Nck and WASP there), they would no longer undergo repeated hits by arcs moving in opposite directions, and the circumferential force would not average to zero. In the simplest case, condensates would attach to the first arc they encounter and move circumferentially with it. Such effects could account for the aberrant movement of LAT condensates containing the Grb2 mutant artificially equipped with a basic clutch, which should produce inappropriately strong adhesion to the telescoping actin arcs. Future cellular studies will directly test our proposed molecular clutch, in part by analysis of condensates lacking basic elements of Nck and WASP, and their movement in the periphery of the IS.

While we have examined Jurkat T cells here, existing data suggest that the behaviors we have described and the model we have proposed are likely relevant to primary T cells as well. The IS formed by both Jurkat T cells and primary T cells is composed of a dendritically-branched actin network at the edge of the IS, followed immediately by a concentric actomyosin cable network near the center of the IS (Murugesan et al., 2016). Thus, condensates must be moved across two distinct actin networks in Jurkat and primary T cells. In primary T cells, condensate-associated actin polymerization localizes mostly (although not entirely) in the outer region of the IS and dissipates in the region adjacent to the branched actin network, where ICAM-1 localizes (Kumari et al., 2015), which would be consistent with loss of Nck and WASP toward the center of the IS. Similarly, PLC-γ1 activation by WASP-promoted actin polymerization appears to localize mostly in the dSMAC where we observe strong Nck co-localization with LAT (Kumari et al., 2015). One difference between the two cell types is that WASP-promoted actin polymerization is much weaker in Jurkat T cells than in primary T cells (Kumari et al., 2015). In primary cells, this actin assembly may also play a role in the movement of condensates from the cell edge to the center, in addition to myosin-driven movement of the cortical actin. Future work addressing the modes of movement, and the precise signals that dictate compositional change, will elucidate the mechanisms by which LAT condensates move across the IS in primary T cells.

LAT condensates represent one particular type of biomolecular condensate. It is generally thought that the functions of condensates are intimately connected to their compositions, and that changes in composition could cause changes in function (Banani et al., 2016). Our data here demonstrate that when Nck and N-WASP are arrayed on membranes they can bind actin filaments efficiently, even though both bind filament sides only weakly in solution. This adhesion enables condensates containing the proteins to be moved over long distances in response to actomyosin contraction. Adhesion is lost when Nck and WASP or N-WASP depart. Thus, the composition of LAT condensates plays an important role in their coupling to actin and their mode of movement at the IS. These behaviors of the LAT system are produced by generalizable features of membrane-associated condensates - their high density and composition based on regulatable interactions. Analogous behaviors are likely to be widely observed as the biochemical and cellular activities of other condensates are explored.

Construct	Sequence	Notes
Ezrin	ISHMHHHHHHHHHHKCKVYEPVSYHVQESLQDEGAEPTGYSAELSSEGIRDDRNEEKRITEAEKNERVQRQLLTLSSELSQARDENKRTHNDIIHNENMRQGRDKYKTLRQIRQGNTKQRIDEFEAL	Human, Ezrin actin binding domain, residues 477–586, with N-terminal His₁₀ - KCK fusion.
Grb2	GPLGSMEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNV	Human, residues 1–217.
Grb2_Basic	GPLGSMEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNVKEKKKGKAKKKRLTKGKEKKKGKAKKKRITKA	Human, residues 1–217 with the basic region of N-WASP (x2) fused to the C-terminal.
LAT	GGSLEHHHHHHHHGIQFKRPHTVAPWPPAFPPVTSFPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASFENEEPACEDADEDEDDFHNPGYLVVLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYENLQELN	Human, residues 48–233 (short isoform) with His₈N-terminal fusion. This construct only contains the four C-terminal Tyr residues (Y132, Y171, Y191, and Y226) that are sufficient for TCR signaling. Y171, Y191, and Y226 are the three tyrosines that are recognized by Grb2 when phosphorylated. All other tyrosine residues were mutated to Phe residues.
Nck	GHMCMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 1–377, with mutations C139S, C232A, C266S, C340S. A single Cys was added to the N-terminus.
His-Nck (FL)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 1–377, with N-terminal His₈-GGSC(GGS)₄ fusion and C139S, C232A, C266S, and C340S mutations.
His-Nck (FLΔL1)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSGGSAGGSAGGSATLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 2–377, with N-terminal His₈-GGSC(GGS)₄fusion and C139S, C232A, C266S, and C340S mutations. ARKASIVKNLKD in the N-terminal portion of L1 has been replaced with GGSAGGSAGGSA.
His-Nck (L1-S2-L2-S3-L3-SH2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 59–377 (No S1), with N-terminal His₈-GGSC(GGS)₄ fusion and C139S, C232A, C266S, and C340S mutations.
His-Nck (L1(KtoE)-S2-L2-S3-L3-SH2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEKNSAREASIVENLEDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 59–377 (No S1), with N-terminal His₈-GGSC(GGS)₄fusion and C139S, C232A, C266S, and C340S mutations. The basic N-terminal portion of L1 has the following basic to acidic mutations: K64E, K69E, and K72E.
His-Nck (L1(HM)-S2-L2-S3-L3-SH2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEKNSARKASNSKNSKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 59–377, with N-terminal His₈-GGSC(GGS)₄fusion and C139S, C232A, C266S, and C340S mutations. Additional Hydrophobic Mutations in L1: I67N, V68S, L71S.
His-Nck (S1-L1-S3-L3-SH2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 2–108 and 191–377, with N-terminal His₈-GGSC(GGS)₄ fusion and C232A, C266S, and C340S mutations.
His-Nck (S1-L1-S2-L2-S3)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQN	Human, residues 2–252, with N-terminal His₈-GGSC(GGS)₄ fusion and C139S and C232A mutations.
His-Nck (S2-L2-S3-L3-SH2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGXEGXFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 106–377, with N-terminal His₈-GGSC(GGS)₄fusion and C139S, C232A, C266S, and C340S mutations.
His-Nck (S3-L3-SH2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 190–377, with N-terminal His₈-GGSC(GGS)₄fusion and C232A, C266S, and C340S mutations.
His-Nck (L3-SH2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLENPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 253–377, with N-terminal His₈-GGSC(GGS)₄fusion and C266S and C340S mutations.
His-Nck (S1)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSGSAAAS	Human, residues 2–61, with N-terminal His₈-GGSC(GGS)₄ fusion.
His-Nck (S2)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDGSAAAS	Human, residues 106–165, with N-terminal His₈-GGSC(GGS)₄fusion and C139S mutation.
His-Nck (S3)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNGSAAAS	Human, residues 190–252, with N-terminal His₈-GGSC(GGS)₄fusion and C232A mutation.
His-Nck_Acidic (FL(L1 KtoE))	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSAREASIVENLEDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 2–377, with C139S, C232A, C266S, and C340S mutations. The basic N-terminal portion of L1 has the following basic to acidic mutations: K64E, K69E, and K72E.
His-Nck_Basic (FL(L1 basic))	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPKSASPADKSFVKPGKRLYKLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human, residues 2–377, with C139S, C232A, C266S, and C340S mutations. The acidic C-terminal portion of L1 has the following acidic to basic mutations: D88K, D95K, D99K, E102K, and D106K.
His-Nck_Neutral (FL(L1 GGSA)₁₀)	GHMHHHHHHHHGGSCGGSGGSGGSGGSLEMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERGGSAGGSAGGSAGGSAGGSAGSGGSAGGSAGGSAGGSAGGSAGSDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKCSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYCIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS	Human full-length Nck with L1 replaced by a GGSA linker, residues 1–58, 109–377, with an N-terminal His₈-GGSC(GGS)₄ fusion and C139S, C232A, C266S, and C340S mutations.
N-WASP	GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED	Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505.
His-N-WASP	MGHHHHHHDYDIPTTENLYFQGSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED	Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505 with N-terminal His₆ fusion.
N-WASP_Basic	GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKGKEKKKGKAKKKRITKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED	Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505. Basic Region (186-200) has been doubled.
His-N-WASP_Basic	MGHHHHHHDYDIPTTENLYFQGSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKGKEKKKGKAKKKRITKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED	Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505 with N-terminal His₆ fusion. Basic Region (186-200) has been doubled.
N-WASP_Neutral	GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYA ADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTGGSGGSGGSGGSGGSADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED	Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505. Basic Region (186-200) has been removed and replaced with (GGS)₅.
His-N-WASP_Neutral	MGHHHHHHDYDIPTTENLYFQGSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTGGSGGSGGSGGSGGSADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED	Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505 with N-terminal His₆ fusion. Basic Region (186-200) has been removed and replaced with (GGS)₅.
N-WASP BPVCA	GSEFKEKKKGKAKKKRAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPHPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVPASSGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVSDGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDDDDEEDFEDDDEWED	Rat, N-WASP fusion of basic, proline-rich, and VCA domains, residues 183–193 and 273–501.
N-WASP BPVCA (R/A R/A)	GSEFKEKKKGKAKKKRAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPHPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVPASSGNKAALLDQIAEGAQLKKVEQNSRPVSCSGRDALLDQIAQGIQLKSVSDGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDDDDEEDFEDDDEWED	Rat, N-WASP fusion of basic, proline-rich, and VCA domains, residues 183–193 and 273–501 with R410A and R438A mutations.
N-WASP BP	GSEFKEKKKGKAKKKRLTKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPPPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVP	Rat, N-WASP fusion of basic, proline-rich, residues 183–239 and 273–396.
SLP-76	GHMDNGGWSSFEEDDYESPNDDQDGEDDGDYESPNEEEEAPVEDDADYEPPPSNDEEALQNSILPAKPFPNSNSMFIDRPPSGKTPQQPPVPPQRPMAALPPPPAGRNHSPLPPPQTNHEEPSRSRNHKTAKLPAPSIDRSTKPPLDRSLAPFDREPFTLGKKPPFSDKPSIPAGRSLGEHLPKIQKPPLPPTTERHERSSPLPGKKPPVPKHGWGPDRRENDEDDVHQRPLPQPALLPMSSNTFPSRSTKPSPMNPLSSHMPGAFSESNSSFPQSASLPPFFSQGPSNRPPIRAEGRNFPLPLPNKPRPPSPAEEENCSLNEGSLEVLFQ	Human, residues 101–420, with a Cys added near the C-terminus for labeling.
Sos1	GPLGSNDTVFIQVTLPHGPRSASVSSISLTKGTDEVPVPPPVPPRRRPESAPAESSPSKIMSKHLDSPPAIPPRQPTSKAYSPRYSISDRTSISDPPESPLLPPREPVRTPDVFSSSPLHLQPPPLGKKSDHGNAFFPNSPSPFTPPPPQTPSPHGTRRHLPSPPLTQEVDLHSIAGPPVPPRQSTSQHIPKLPPKTYKREHTHPSMC	Human, poly-proline rich region, residues 1117–1319 with a Cys added at the C-terminus for labeling.
WASP	GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGRKCLTLATAVVQLYLALPPGAEHWTKEHCGAVCFVKDNPQKSYFIRLYGLQAGRLLWEQELYSQLVYSTPTPFFHTFAGDDCQAGLNFADEDEAQAFRALVQEKIQKRNQRQSGDRRQLPPPPTPANEERRGGLPPLPLHPGGDQGGPPVGPLSLGLATVDIQNPDITSSRYRGLPAPGPSPADKKRSGKKKISKADIGAPSGFKHVSHVGWDPQNGFDVNNLDPDLRSLFSRAGISEAQLTDAETSKLIYDFIEDQGGLEAVRQEMRRQEPLPPPPPPSRGGNQLPRPPIVGGNKGRSGPLPPVPLGIAPPPPTPRGPPPPGRGGPPPPPPPATGRSGPLPPPPPGAGGPPMPPPPPPPPPPPSSGNGPAPPPLPPALVPAGGLAPGGGRGALLDQIRQGIQLNKTPGAPESSALQPPPQSSEGLVGALMHVMQKRSRAIHSSDEGEDQAGDEDEDDEWDD	Human WIP WASP EVH1 binding motif, residues 451–485 fused to human WASP, residues 39–502.

Experiment	His₈-pLAT	His₁₀- Ezrin (ABD)	Grb2	Sos1	pSLP-76	Nck	N-wasp	WASP	Actin	Myosin II	KCl	ATP
pLAT → Sos1 (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125nM	125nM	0nM	0nM	0nM	0nM	0nM	0nM	75mM	1mM
pLAT → Sos1 +Actin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	125 nM	0 nM	0 nM	0 nM	0 nM	250 nM	0 nM	75 mM	1 mM
pLAT → Sos1 +Actin + Myosin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	125 nM	0 nM	0 nM	0 nM	0 nM	250 nM	100 nM	75 mM	1 mM
pLAT → pSLP-76 (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	0 nM	0 nM	0 nM	0 nM	0 nM	75 mM	1 mM
pLAT → pSLP-76 +Actin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	0 nM	0 nM	0 nM	250 nM	0 nM	75 mM	1 mM
pLAT → pSLP-76 +Actin + Myosin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	0 nM	0 nM	0 nM	250 nM	100 nM	75 mM	1 mM
pLAT → Nck (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	0 nM	0 nM	0 nM	0 nM	75 mM	1 mM
pLAT → Nck +Actin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	0 nM	0 nM	250 nM	0 nM	75 mM	1 mM
pLAT → Nck +Actin + Myosin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	0 nM	0 nM	250 nM	100 nM	75 mM	1 mM
pLAT → N-WASP (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	125 nM	0 nM	0 nM	0 nM	75 mM	1 mM
pLAT → N-WASP +Actin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	125 nM	0 nM	250 nM	0 nM	75 mM	1 mM
pLAT → N-WASP +Actin + Myosin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	125 nM	0 nM	250 nM	100 nM	75 mM	1 mM
pLAT → Sos1 +Actin + Myosin (Contraction) For Grb2, Grb2_Basic	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	125 nM	0 nM	0 nM	0 nM	0 nM	250 nM	100 nM	50 mM	0.05 mM
pLAT → N-WASP +Actin + Myosin (Contraction) For N-WASP_WT, N-WASP_Neutral, N-WASP_Basic	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	125 nM	0 nM	250 nM	100 nM	50 mM	0.05 mM
pLAT → WASP (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	0 nM	125 nM	0 nM	0 nM	75 mM	1 mM
pLAT → WASP +Actin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	0 nM	125 nM	250 nM	0 nM	75 mM	1 mM
pLAT → WASP +Actin + Myosin (Steady-State)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	0 nM	125 nM	250 nM	100 nM	75 mM	1 mM
pLAT → WASP +Actin + Myosin (Contraction)	20 nM (sol’n) 500 molecules /μm²	five nM (sol’n) ~100 molecules /μm²	125 nM	62.5 nM	62.5 nM	125 nM	0 nM	125 nM	250 nM	100 nM	50 mM	0.05 mM

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Composition regulates condensate movement with actin networks.

Figure 1—source data 1

Reconstitution of LAT condensate formation and movement using different component mixtures.

Reconstitution of LAT condensate formation and movement using different component mixtures in actin networks.

Reconstitution of LAT condensate formation and movement using different component mixtures in steady-state active actomyosin networks.

pLAT → N-WASP condensates bind to and move with moving actin filaments.

Reconstitution of LAT condensate movement using different component mixtures in contracting actomyosin networks.

Basic regions of Nck and N-WASP mediate interaction of LAT condensates with actin filaments.

Figure 3—source data 1

Figure 3—source data 2

Figure 3—source data 3

Reconstitution of LAT condensate movement using different component mixtures containing N-WASPNeutral or N-WASPBasic in contracting actomyosin networks.

Single particle tracking of LAT condensates in activated Jurkat T cells.

LAT condensates change composition as they move across the IS.

Figure 4—source data 1

Grb2 is maintained as LAT condensates move across the IS in activated Jurkat T cells.

Nck dissipates from LAT condensates as they move across the IS in activated Jurkat T cells.

Grb2 fused to a basic molecular clutch can couple LAT condensates to actin.

Reconstitution of movement in LAT condensates containing Grb2Basic in contracting actomyosin networks.

LAT condensates containing Grb2Basic deviate tend to deviate from a radial trajectory across the IS.

Formin activity is necessary for Nck dissipation from LAT condensates.

Nck dissipates from LAT condensates as they move across the IS in activated Jurkat T cells treated with DMSO.

Nck is maintained in LAT condensates as they move across the IS in activated Jurkat T cells treated with the formin inhibitor SMIFH2.

Sequences of constructs used in the study.

Experimental conditions used in steady state and actomyosin contraction assays.

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Jonathon A Ditlev

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Anthony R Vega

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Darius Vasco Köster

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Xiaolei Su

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Tomomi Tani

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Ashley M Lakoduk

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Ronald D Vale

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Satyajit Mayor

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For correspondence

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Khuloud Jaqaman

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Michael K Rosen

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Reconstitution of LAT condensate movement using different component mixtures containing N-WASP_Neutral or N-WASP_Basic in contracting actomyosin networks.

Reconstitution of movement in LAT condensates containing Grb2_Basic in contracting actomyosin networks.

LAT condensates containing Grb2_Basic deviate tend to deviate from a radial trajectory across the IS.