CCL5 phase separates with heparin in solution.

(A) Confocal images of assembling status of 20 μM CCL5-Cy3 mixed with heparin at different ratios and 5% PEG. Scale bar=10 μm. (B) Turbidity changes with increase of heparin concentration were measured at 620 nm (Figure 1-Source Data 1). (C) Fusion of phase-separated droplets formed at the ratio of CCL5-Cy3: heparin=1:2. The white arrows indicate the dynamic fusion of two adjacent droplets. (D) Representative FRAP results of droplets formed by CCL5-Cy3: heparin=1:2 depicted in Figure 1A, showing the intensity of fluorescence pre- and after photobleaching (Figure 1-Source Data 2). The images of representative droplets in different recovery stages are shown. (E) Confocal images of assembling status of 44AANA47-CCL5 or CCL5 in the presence of heparin. Scale bar=10 μm. (F) Comparison of CCL5 and44AANA47-CCL5 turbidity in the presence of heparin (Figure 1-Source Data 3). Data are mean ± s.d. n=3 (for B, F). Normal distribution was assessed by the Shapiro-Wilk (SW) normality test. P values were determined by unpaired two-tailed t-tests.

Co-phase separation of CCL5 and heparin establishes chemokine gradient

(A) CCL5-EGFP or 44AANA47-CCL5-EGFP were bound to heparin beads or Ni-NTA beads, respectively, and were placed in Matrigel in 96-well plate. After incubation for 12 hr images were taken to quantify the fluorescence intensity. (B) Quantification of the fluorescence signals along the lines with arrows indicated in Figure 2A (Figure 2-Source Data 1). (C) Illustration of in vitro chemotaxis assay. (D) Heparin beads or Ni-NTA beads bound with CCL5 or 44AANA47-CCL5 were placed in the lower chamber. THP-1 cells (3×105 cells) were added to upper chamber. After 4 hr, THP-1 in the lower chamber was collected and counted (Figure 2-Source Data 2). Data are mean ±s.d. n=3. Normal distribution was assessed by the Shapiro-Wilk (SW) normality test. P values were determined by unpaired two-tailed t-tests.

CCL5 phase separates with heparan sulfate on the cell surface

(A) Microscopy images of CCL5-Cy3 on the surface CHO-K1 and CHO-677 as well as CHO-K1 treated with 1 mg/mL heparin. From left to right, the images are fluorescent-field, bright-field and overlay of two illuminations. Scale bar=10 μm. (B) Z-stack scanning of CCL5-Cy3 phase separation on CHO-K1 cell surface. The cell was imaged by confocal microscope with the Z-stack method. Scale bar=10 μm. (C) FRAP of the condensates formed by CCL5-Cy3, showing the intensity of fluorescence pre- and after photobleaching (Figure 3-Source Data 1). The size of the representative droplets in different recovery stages are shown above of the graph. (D) Graphical illustration of the cell-based chemotaxis assay. CHO cells (1×105 cells/well) were plated onto the lower chamber for 24 h (fully attached) and CCL5 or 44AANA47-CCL5 or heparin were added as indicated. THP-1 cells (3×105) were placed on upper chambers. After 4 hr, a small volume of medium in the lower chamber was aspirated to count THP-1 transmigrated through the membrane. (E) Quantification of THP-1collected from the lower chamber (Figure 3-Source Data 2). Data are mean ±s.d. n=3. Normal distribution was assessed by the Shapiro-Wilk (SW) normality test. P values were determined by unpaired two-tailed t-tests.

Formation of chemokine gradient on the cell surface by phase separation

(A) HUVEC cells were seeded on the plates for 24 h and stained with Dil (Cell membrane red fluorescent probe). After washing with PBS, CCL5-EGFP transfected CHO-K1 were added and co-cultured for 24 hr before taking the images (Scale bar=10 μm). (B) Fusion events of droplets formed by CCL5-EGFP on HUVEC cell surface. The white arrows in cropped images indicate the fusion of two droplets with time. (C) FRAP of the condensates formed by CCL5-EGFP in HUVEC cell surface, showing the intensity of fluorescence pre and after photobleaching (Figure 4-Source Data 1). The images of representative droplets in different recovery stages are shown. (D) HUVEC cells were seeded on the plate and firmly adhered before adding the 400 CCL5-EGFP transfected CHO-K1 cells placed in 50% of Matrigel. After 1 hr co-culture, confocal images were taking showing CCL5-EGFP diffusion on the surface of HUVEC. Dotted white circle indicates the source cell of CCL5-EGFP transfected CHO-K1. Scale bar=100 μm. (E) Quantification of the fluorescence intensity shows decreased signals of CCL5-EGFP as the distance from the source cells increased (Figure 4-Source Data 2). (F) The chemotaxis assay (same experimental conditions as described in Figure 1) shows higher activity of CCL5-EGFP transfected CHO-K1 cells than wild type CHO-K1 cells (Figure 4-Source Data 3). Data are mean ±s.d. n=3. Normal distribution was assessed by the Shapiro-Wilk (SW) normality test. P values were determined by unpaired two-tailed t-tests.

Transmigration of human peripheral blood cells

The cells (CHO-K1, CHO-677, or HUVEC) (1×105 cells/well) as indicated were plated onto the lower chamber for 24 hr (fully attached) and CCL5 was added for 1 hr. PBMC isolated from three healthy volunteer donors were placed in the upper chamber (total number of cells: Sample 1: 8.65^105; Sample 2: 2.31^106; Sample 3: 2.23^106). After culturing for 4 hr, the total transmigrated cells in the lower chamber were counted (Figure 5-Source Data 1).

Chemokines phase separation promotes cell recruitment in vivo.

Mice were treated by intraperitoneal injection of CCL5 or the reagents as indicated. After 18h, the animals were sacrificed and peritoneal lavage was collected. Total cell number was counted by automated cell counter (Figure 6-Source Data 1). Data are mean ±s.d. n=4. Normal distribution was assessed by the Shapiro-Wilk (SW) normality test. P values were determined by unpaired two-tailed t-tests.

Schematic model of CCL5 phases separation along with heparan sulfate on the cell surface.

The inflammatory cells secrete chemokines that are immobilized by heparan sulfate in the form of Liquid-Liquid phase separation and subsequently diffused to form a concentration gradient.

Protein expression and purification.

(A)-(B) SDS-PAGE showed the molecular weight and purity of CCL5 (A) and CCL5-EGFP (B).

The effect of salt concentration on phase separation.

(A) Confocal images of assembling status of 20 μM CCL5-Cy3 mixed with heparin with increasing salt concentration. Scale bar=10 μm. (B) Turbidity changes with different increasing salt concentrations. (C) Confocal images of assembling status of A22K-CCL5 or CCL5 in the presence of heparin. Scale bar=10 μm. (D) FRAP of the aggregates formed by A22K-CCL5-Cy3, showing the intensity of fluorescence pre- and after photobleaching.

Comparison of binding of CCL5 and 44AANA47-CCL5 to heparin tetrasaccharide.

(A) Binding mode of heparin tetrasaccharide to 44AANA47-CCL5 or CCL5 shown by molecular docking. On the right are the details framed by the dotted line. (B) Comparison of binding free energy of different interacting forces between CCL5/44AANA47-CCL5 and heparin tetrasaccharide. The mutation led to change in electrostatic interaction forces.

Chemotactic function of CCL5-EGFP.

Labeled CCL5 has similar activity as CCL5, 50 nM CCL5, CCL5-EGFP or CCL5-Cy3 were added to the lower chamber of the Transwell. THP-1 cells were added to upper chambers. Data are mean ± s.d. n=3. P values were determined by unpaired two-tailed t-tests. NS, Not

Diffusion of the CCL5-Cy3 in heparin-beads or Ni-NTA beads.

Beads bound with CCL5-Cy3 placed in Matrigel were imaged and reconstructed by Leica DMi8.

44AANA47-CCL5 didn’t immobilize to the surface of CHO-K1.

CHO-K1 were treated with 500 nM 44AANA47-CCL5. From left to right, the pictures showed bright-field, fluorescent-field and overlay of two illuminations.

Diffusion of CCL5-EGFP on cell surface.

(A) Confocal images of Transfected CHO-K1 and HUVEC cells. Scale bar=100 μm. The yellow boxes are shown in higher magnification on the right. Scale bar=10 μm. CCL5-EGFP was expressed by transfected CHO-K1 and then adhere to the surface of HUVEC and formed phase separation. (B) The range of CCL5-EGFP diffusion on HUVEC cell surface at different time. The black dashed box showed the final image size. (C) Fluorescence intensity along the dotted lines indicated in Figure 4—figure supplement 1B. (D) Confocal images of Transfected CHO-K1 and CHO-K1 or CHO-677. CCL5-EGFP was expressed by transfected CHO-K1 and then adhere to the surface of CHO-K1 and formed phase separation but CHO-677 completely lack the signals. Scale bar=25 μm.