CAMSAP2 is dynamically localized at the Golgi during the reorientation process in directional cell migration.

(A) Immunofluorescence staining of HT1080 located at the foremost edge of the cell wound after 0, 0.5, and 2 h migration. White arrows represent the direction of cell migration. Shown are representative immunofluorescence staining images. Scale bar = 10 μm.

(B) Quantification of the localization of CAMSAP2 on the Golgi after 0, 0.5, and 2 h migration. Each symbol represents an individual cell. The co-localization of representative immunofluorescence staining plots corresponding to each time point are counted. Values = means ± SEM. unpaired two-tailed Student’s t-test, **p < 0.01 (N = 3; 0 h: n = 28; 0.5 h: n = 23; 2 h: n = 20).

(C) Immunofluorescence staining of HT1080 after 2 h of migration in wound healing assay. The cell in the white dashed box shows a different cell state. The cell in the white dashed box on the 1st is the cell closest to the edge of the wound and has already completed the process of directional cell migration: the Golgi is polar and is located at the anterior edge of the nucleus. At this point CAMSAP2 is more localized to the Golgi. The cells in the white dashed box on the 2nd have not yet completed the process of cell directed migration: the Golgi is relatively dispersed and has lost its polarity. At this point the localization of CAMSAP2 on the Golgi is substantially reduced. Golgi was stained with mouse anti-GM130 antibody (red). Nuclei were stained with DAPI (blue). CAMSAP2 was stained with rabbit anti-CAMSAP2 antibody (green). On the right is a magnified image of the white dashed box. Scale bars = 5 μm. White arrows represent migration direction.

(D) Co-localization of CAMSAP2 and GM130 was analyzed using the scatterJ plug-in in ImageJ software. The horizontal and vertical axes of the scatterplot represent the gray values obtained for each pixel point in each channel, and the closer the image is to the diagonal, the higher the degree of co-localization is indicated.

(E) Quantification of the localization of CAMSAP2 on the Golgi in different cell states in the (C). Each symbol represents an individual cell. Values = means ± SEM, unpaired two-tailed Student’s t-test, ***p < 0.001 (N = 3; complete: n = 20; partial: n = 19).

Search for polarity-establishing key proteins that can localize to the Golgi and interact with CAMSAP2.

(A) GO analysis of proteins reported in the literature that may localize on the Golgi (Golgi TAG-IP/Whole-Cell lysate meaningful proteins), and the figure shows the BP (biological process), the protein we focus on that can establish and maintain cell polarity. GO analysis of proteins was plotted by https://www.bioinformatics.com.cn (last accessed on 20 June 2024), an online platform for data analysis and visualization.

(B) Venn diagram. Proteins in (A) that can establish and maintain polarity and proteins that may interact with CAMSAP2 were selected for enrichment, and the enriched proteins are displayed in the diagram. The CAMSAP2 interactants were derived from the study by Wu et al., 2016, which provides the source of these interactants.

(C) Immunoblot showing the results of GFP-IP. Immunoprecipitation showing GFP-CAMSAP2 and Flag-MARK2 interactions. The cells used in the experiment were 293T cells. The interaction between CAMSAP2 and MARK2 is referenced from Zhou et al., 2020.

(D) Immunofluorescence staining of HT1080 and MARK2 KO cells in wound healing assay after 2 h of migration. Golgi was stained with mouse anti-GM130 antibody (red). Nuclei were stained with DAPI (blue). CAMSAP2 was stained with rabbit anti-CAMSAP2 antibody (green). F-actin was stained with phalloidin (white). We fixed the cells after 2 hours of migration with methanol for staining and assessed Golgi reorientation by measuring the angle between the Golgi centroid-nucleus centroid axis and the direction of cell migration. Black arrows point to the direction of migration. Scale bar = 5 μm.

(E) Images demonstrate cells completing Golgi reorientation. If the absolute value of this angle is within 90 degrees, it indicates that the Golgi is positioned at the front edge of the nucleus and oriented in the migration direction, signifying proper Golgi reorientation. Conversely, if the absolute angle falls between 90 and 180 degrees, it suggests that the Golgi is positioned behind the nucleus, indicating incomplete Golgi reorientation. (N = 3; HT1080: n = 35; MARK2 KO: n = 45).

(F) Quantification of the proportion of cells in (E) that can complete Golgi reorientation properly. Values = means ± SEM, unpaired two-tailed Student’s t-test, **p < 0.01 (N = 3; HT1080: n = 35; MARK2 KO: n = 45).

(G) Sequencing result of the MARK2 KO cells near the sgRNA sequence.

(H) Overlay of end-point phase-contrast images with the result of cell tracking after 8 h of migration in a wound healing assay in control HT1080 and MARK2 KO HT1080 cells. Scale bar = 100 μm.

(I) Plots show quantification of wound closure area after 8 h of migration in HT1080 cells and MARK2 KO HT1080 cells. Values = means ± SEM, unpaired two-tailed Student’s t-test, **p < 0.01 (N=3).

MARK2 affects CAMSAP2 localization on the Golgi apparatus.

(A) Immunostaining of control HT1080 and MARK2 KO cells with rabbit anti-CAMSAP2 (green) and mouse anti-GM130 (red). Nuclei were stained with DAPI (blue). Scale bar = 5 μm.

(B) Area of the Golgi defined by the Golgi marker GM130 from (A). Each symbol represents an individual cell. Values = means ± SEM, unpaired two-tailed Student’s t-test, ***p < 0.001 (N = 3; HT1080: n = 104; MARK2 KO: n = 76).

(C) ImageJ software was used to calculate CAMSAP2 co-localization with the Golgi marker GM130. Values = means ± SEM, unpaired two-tailed Student’s t-test, ***p < 0.001 (N = 3; HT1080: n = 20; MARK2 KO: n = 20).

(D) Immunoblot of extracts from control HT1080 cells and MARK2 KO cells with rabbit anti-CAMSAP2, mouse anti-GAPDH, or mouse anti-GM130 antibodies. GAPDH was used as a loading control.

(E) The CAMSAP2/GAPDH ratios from (D) were determined using ImageJ software. Values = means ± SEM, unpaired two-tailed Student’s t-test, p > 0.05, N = 4.

(F) The GM130/GAPDH ratios from (D) were determined using ImageJ software. Values = means ± SEM, unpaired two-tailed Student’s t-test, p > 0.05, N = 3.

(G) Enrichment of Golgi fraction from control RPE1 cells and MARK2 knock down cells by density gradient differential centrifugation.

(H) Immunoblot of homogenate and Golgi-fraction from RPE1 cells and MARK2 knock down cells with rabbit anti-CAMSAP2, rabbit anti-MARK2, or mouse anti-GM130 antibodies. CAMSAP2, GM130 and MARK2 are enriched in the Golgi fraction of RPE1 cells and MARK2 knockdown cells (GM130, Golgi).

(I) The CAMSAP2/GM130 ratios from (H) were determined using ImageJ software. Values = means ± SEM, unpaired two-tailed Student’s t-test, **p < 0.01, N = 3.

MARK2 phosphorylation of serine at position 835 of CAMSAP2 affects the localization of CAMSAP2 on the Golgi and regulates Golgi structure.

(A) Mass spectrometry was used to detect the possible amino acid phosphorylation of CAMSAP2 by MARK2. The phosphorylation sites illustrated in Figure 4A are derived from our analysis of the original mass spectrometry data. These sites were included based on their high confidence scores and data reliability. Importantly, only serine residues met the stringent criteria for inclusion, as no threonine or tyrosine residues had sufficient evidence for phosphorylation.

(B) Proteins are expressed in HEK293 cells, and the band shift is examined using western blotting. The arrowhead labels the shifted bands. Confirm the site specificity of MARK2 phosphorylation.

(C) The site phosphorylated by MARK2 is shown in different CAMSAP2 isoforms. Upper: sequence alignment of CAMSAP2 orthologs across species. Bottom: sequence alignment of CAMSAP2, CAMSAP1 and CAMSAP3 in mouse and human. Arrowheads indicate the CAMSAP2 S835.

(D) In CAMSAP2 KO HT1080 cells, the expression of HA-CAMSAP2 (S835D) compensates for the CAMSAP2 KO effect on Golgi apparatus defects more effectively than HA-CAMSAP2 (S835A). Immunostaining of CAMSAP2 KO HT1080 cells expressing HA-CAMSAP2 (S835D) and HA-CAMSAP2 (S835A). Immunostaining of control cells with rat anti-HA (green) antibodies and mouse anti-GM130 antibodies (red). Nuclei were stained with DAPI (blue). Scale bar = 5 μm.

(E) Area of the Golgi defined by the Golgi marker GM130 from (D). Each symbol represents an individual cell. Values = means ± SEM, unpaired two-tailed Student’s t-test, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 (N = 3; HT1080: n = 39; CAMSAP2 KO: n = 50; CAMSAP2 KO (HA-CAMSAP2): n = 50; CAMSAP2 KO (HA-CAMSAP2-S835A): n = 49; CAMSAP2 KO (HA-CAMSAP2-S835D): n = 35).

(F) ImageJ software was used to calculate Manders’ coefficient M1 values of HA-CAMSAP2 (S835A), HA-CAMSAP2 (S835D) co-localization with the Golgi marker GM130. Values = means ± SEM, unpaired two-tailed Student’s t-test, *p < 0.05 (N = 3; CAMSAP2 KO (HA-CAMSAP2): n = 23; CAMSAP2 KO (HA-CAMSAP2-S835A): n = 40; CAMSAP2 KO (HA-CAMSAP2-S835D): n = 32).

Identification of different interacting proteins with CAMSAP2 S835A/S835D.

(A) Stable expression of CAMSAP2 S835A and CAMSAP2 S835D in MARK2 KO HT1080 was observed for Golgi reorientation at the cell wounding edge after 2 h migration. The arrow points in the direction of the migration. On the right is a split-channel plot of the white dashed box on the left. We fixed the cells after 2 hours of migration with methanol for staining and assessed Golgi reorientation by measuring the angle between the Golgi centroid-nucleus centroid axis and the direction of cell migration. Black arrows point to the direction of migration. Scale Bar = 5 μm.

(B) Images demonstrate cells completing Golgi reorientation. If the absolute value of this angle is within 90 degrees, it indicates that the Golgi is positioned at the front edge of the nucleus and oriented in the migration direction, signifying proper Golgi reorientation. Conversely, if the absolute angle falls between 90 and 180 degrees, it suggests that the Golgi is positioned behind the nucleus, indicating incomplete Golgi reorientation. (N = 3; HT1080: n = 34; MARK2 KO: n = 32; GFP-CAMSAP2: n = 28; GFP-CAMSAP2 S835D: n = 40; and GFP-CAMSAP2 S835A: n = 39).

(C) Quantification of the proportion of cells in (B) that can complete Golgi reorientation properly. Values = means ± SEM, unpaired two-tailed Student’s t-test (N = 3; HT1080: n = 34; MARK2 KO: n = 32; GFP-CAMSAP2: n = 28; GFP-CAMSAP2 S835D: n = 40; and GFP-CAMSAP2 S835A: n = 39).

(D) Immunoblot showing the results of GFP-IP. There was no significant difference in the binding ability of CG-NAP to GFP-CAMSAP2, GFP-CAMSAP2 S835D and GFP-CAMSAP2 S835A. The cells used in the experiment were 293T cells. CG-NAP (AKAP450) is indeed a 450 kDa protein, and the marker at 310 kDa represents the molecular weight marker’s upper limit, above which CG-NAP is observed.

(E) The quantitative results of the immunoblots presented in (D) are shown graphically. The signal intensity of CGNAP was normalized to the intensity of GFP in each mutant. Values = means ± SEM, unpaired two-tailed Student’s t-test, p>0.05, N = 3.

(F) Immunoblot showing the results of GFP-IP. Myc-CLASP2 can bind to GFP-CAMSAP2, GFP-CAMSAP2 (S835D) and GFP-CAMSAP2 (S835A) with the same ability. The cells used in the experiment were 293T cells.

(G) Quantitative results of the immunoblots shown in Figure (F) are shown graphically. The signal intensity of Myc-CLASP2 was normalized to the intensity of GFP in each mutant. Values = means ± SEM. unpaired two-tailed Student’s t-test, p>0.05, N = 3.

Differential interactions of USO1 with CAMSAP2 S835A/D.

(A) Schematic overview of TurboID-mediated neighbor biotinylation assays using His-TurboID-CAMSAP2 (S835A), His-TurboID-CAMSAP2 (S835D), and His-TurboID.

(B) Venn diagram of the three sets, generated using an online tool. Each colored circle represents a different dataset, and overlapping regions indicate common proteins. Proteins were identified that do not interact with His-TurboID but interact differently with FLAG-CAMSAP2 (S835A) and FLAG-CAMSAP2 (S835D).

(C) Immunoblotting showing the results of GFP-IP. Flag-USO1 exhibits a stronger binding affinity with GFP-CAMSAP2 S835D than with FLAG-CAMSAP2 S835A. The cells used in the experiment were 293T cells.

(D) Quantification of immunoblotting is shown in (I). The signal intensity of USO1 was normalized to the intensity of GFP in each mutant. Values = means ± SEM, unpaired two-tailed Student’s t-test, *p < 0.05, N = 3.

(E) Cell extracts were analyzed by immunoblotting using rabbit anti-USO1 and mouse anti-GAPDH to confirm the depletion of USO1. HT1080 cells were transfected with either control or USO1 siRNA for 72 h.

(F) Quantification of the localization of CAMSAP2 on the Golgi in different cells in (E). Each symbol represents an individual cell. Values = means ± SEM, unpaired two-tailed Student’s t-test, *p < 0.05 (N = 3; control: n = 77; USO1 siRNA: n = 64).

(G) Cells were stained with rabbit anti-CAMSAP2 (green) and mouse anti-GM130 (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar = 5 µm.

(H) The left rectangular dashed box represents the five steps of cell migration. The right circular dashed box responds to the regulatory mechanism that governs the second half of Golgi reorientation. The second half of Golgi reorientation: the process by which dispersed unpolarized Golgi stacks to form a completely polarized Golgi.

Information of Primers

Information of Antibodies

CAMSAP2 is essential for Golgi reorientation during directed migration.

(A) Sequencing result of the CAMSAP2 KO cell line near the sgRNA sequence.

(B) Immunoblot of extracts from HT1080 cells prepared from control or CAMSAP2 KO cells with rabbit anti-CAMSAP2, mouse anti-GAPDH, or mouse anti-GM130 antibodies. GAPDH was used as a loading control.

(C) Immunostaining of control HT1080 cells and CAMSAP2 KO cells with rabbit anti-CAMSAP2 (green) and mouse anti-GM130 (red). Nuclei were stained with DAPI (blue). Scale bar = 5μm.

(D) Area of the Golgi defined by the Golgi marker GM130 from (C). Each symbol represents an individual cell. Values = means ± SEM, unpaired two-tailed Student’s t-test, ****p < 0.0001 (N = 3; HT1080: n = 104; CAMSP2 KO: n = 120).

(E) The GM130/GAPDH ratios from (B) were determined using Image J software. Values = means ± SEM, unpaired two-tailed Student’s t-test, p > 0.05 (N=4).

(F) Immunofluorescence staining of HT1080 and CAMSP2 KO cells in wound healing assay after 2 h of migration. Rabbit anti-CAMSAP2 antibody (green), mouse anti-GM130 antibody (red). Nuclei were stained with DAPI (blue). F-actin was stained with phalloidin (white). We fixed the cells after 2 hours of migration with methanol for staining and assessed Golgi reorientation by measuring the angle between the Golgi centroid-nucleus centroid axis and the direction of cell migration. Black arrows point to the direction of migration.

(G) Images demonstrate cells completing Golgi reorientation. If the absolute value of this angle is within 90 degrees, it indicates that the Golgi is positioned at the front edge of the nucleus and oriented in the migration direction, signifying proper Golgi reorientation. Conversely, if the absolute angle falls between 90 and 180 degrees, it suggests that the Golgi is positioned behind the nucleus, indicating incomplete Golgi reorientation. (N = 3; HT1080: n = 53; CAMSAP2 KO: n = 63).

(H) Quantification of the proportion of cells in (G) that can complete Golgi reorientation properly. Values = means ± SEM, unpaired two-tailed Student’s t-test, ***p < 0.001 (N = 3; HT1080: n = 53; CAMSP2 KO: n = 63).

(I) Overlay of end-point phase-contrast images with the result of cell tracking after 8 h of migration in a wound healing assay in control HT1080 and CAMSAP2 KO HT1080 cells. Scale bar = 100 μm.

(J) Plots show quantification of wound closure area after 8 h of migration in HT1080 cells. Values = means ± SEM, unpaired two-tailed Student’s t-test, **p < 0.01 (N=3).

CAMSAP2 is dynamically localized at the Golgi during the reorientation process in directional cell migration.

(A) Live images of HT1080 cells expressing mCherry-galactosyltransferase during Golgi reorientation. Scale bar = 5 μm.

(B) Patterns of different cell states after 2 h of migration in wound healing assay. Black arrows point in the direction of the wound. Green dotted structures represent CAMSAP2. Red striped structures represent the Golgi apparatus. The purple round structure represents the nucleus. Red cells, which have completed Golgi reorientation, have Golgi bodies located at the leading edge of the nucleus towards the side of the cell wound, when the vast majority of CAMSAP2 is localized to the Golgi. Blue cells, which received the signal of cell directional migration, are in the process of Golgi reorientation, the Golgi is located in the center of the cell in a disrupted form and has lost its proper polarity, at this time, very few CAMSAP2 are localized on the Golgi. Green cells, which did not receive a signal for cell directional migration, are in a standard physiological state.

(C) Upper: Immunofluorescence staining plots of HT1080 near the wound after 0 h, 0.5 h, and 2 h migration. Black arrows represent the direction of cell migration. Scale bar = 20 μm. Lower: Pattern plots of cell states located at the foremost edge of the cell wound after 0, 0.5, and 2 h migration. Black arrows represent the direction of cell migration. The dashed line represents the edge of wound healing. Different colors represent different states of cells, as in (A).

MARK2 affects CAMSAP2 localization on the Golgi apparatus.

(A) Diagram of domains for CAMSAP2, CAMSAP2-1149F, CAMSAP2-1222F, CAMSAP2-CKK.

(B) Immunoblot showing the results of GFP-IP. Immunoprecipitation showing GFP-CAMSAP2 1149F and Flag-MARK2 interactions. The cells used in the experiment were 293T cells.

(C) Cells expressing GFP-MARK2 and GFP were stained for chicken anti-GFP (green), mouse anti-GM130 (grey) and rabbit anti-CAMSAP2 (red). Scale bar = 5 μm.

(D) Area of the Golgi defined by the Golgi marker GM130 from (C). Each symbol represents an individual cell. Values = means ± SEM, unpaired two-tailed Student’s t-test, ****p < 0.0001 (N = 3; GFP: n = 33; GFP-MARK2: n = 28).

(E) The Pearson’s correlation coefficient of CAMSAP2 and GM130 in (C). Values = means ± SEM, unpaired two-tailed Student’s t-test, ****p < 0.0001 (N = 3; GFP: n = 17; GFP-MARK2: n = 20).

(F) Immunoblot of extracts from control RPE1 cells and siRNA MARK2 cells. α-tubulin was used as a loading control.

(G) Area of the Golgi defined by the Golgi marker GM130 from (C). Values = means ± SEM, unpaired two-tailed Student’s t-test, ***p < 0.001 (N = 3; RPE1: n =93; siRNA-1 MARK2: n = 56; siRNA-2 MARK2: n = 75).

(H) Immunostaining of control RPE1 cells and siRNA MARK2 cells with rabbit anti-CAMSAP2 (green) and mouse anti-GM130 (red). Nuclei were stained with DAPI (blue). Scale bar = 5 μm.

(I) The Pearson’s correlation coefficient of CAMSAP2 and GM130 from control RPE1 cells and siRNA-2 MARK2 cells. Values = means ± SEM, unpaired two-tailed Student’s t-test, **p < 0.01 (N = 3; RPE1: n = 30; siRNA-2 MARK2: n = 28).

MARK2 phosphorylation of serine at position 835 of CAMSAP2.

(A) CBB staining of a gel with GFP-CAMSAP2, GST and GST-MARK2. GFP-CAMSAP2 expressed in SF9 was purified. GST and GST-MARK2 expressed in E. coli was purified.

(B) Western blot play Kinase assay in vitro. GST or GST-MARK2 was incubated with GFP-CAMSAP2 in kinase buffer (50 mM Tris-HCl pH 7.5, 12.5 mM MgCl2, 1 mM DTT, 400 μM ATP) at 30℃ for 30 min. Samples were boiled with loading buffer to stop the reaction.

(C) Kinase assay samples developed in Phos-tag SDS-PAGE. Phosphorylation was not detected. HEK293 cells were co-transfected with the indicated plasmids, and the band shift of CAMSAP2 mutants were examined by western blotting. Phos-tag was used in SDS-PAGE and the arrowheads indicate the shifted bands cause by phosphorylation.

(D) Detection of phosphorylation at S835 in CAMSAP2 by mass spectrometry. Upper: analysis of phosphorylation results. Below: The mass increase in b4, b5, b6, b7, b8, b10, b11 and b12 indicate the phosphorylation modification occurred on Ser1.

Identification of different interacting proteins with CAMSAP2 S835A/S835D.

(A) Immunoblotting showed the expression of CAMSAP2, CAMSAP2 S835A and CAMSAP2 S835D in the stable cell line.

(B) Overlay of end-point phase-contrast images with the result of cell tracking after 8 hr migration in a wound healing assay. Scale bar = 100 μm.

(C) Plots show quantification of wound closure area after 8 hr of migration. Values = means ± SEM, unpaired two-tailed Student’s t-test, **p < 0.01 (N=3).

(D) Immunoblotting demonstrated biotinylation efficiency. His-TurboID-CAMSAP2 (S835A), His-TurboID-CAMSAP2 (S835D), and His-TurboID were expressed in HT1080 cells, which were then incubated for 10 min by replacing the medium containing 50 mM biotin, and total cell lysis was detected by a streptavidin probe.

(E) Immunoblotting demonstrates proteins that can interact with CAMSAP2. Bands of each construct are marked with black arrow.

(F) Western blotting shows the interaction between CAMSAP2 mutants and USO1.

(G) Schematic diagrams of CAMSAP2 domain mutants used to map the USO1 binding site.

Significant changes in microtubules anchored to the Golgi apparatus during cell migration.

(A) Cells expressing Flag-USO1 were stained for mouse anti-Flag (green) and rabbit anti-CAMSAP2 (red). Nuclei were stained with DAPI (blue). Scale bar = 5 µm.

(B) White lines in (A) used for line scan analyses reflect co-localization of Flag-USO1 and GM130.

(C) Immunofluorescence staining of cells at the edge of the wounding after 0.5 h and 2 h migration. Black arrows point to the direction of migration. USO1(red), and GM130 (green). Scale bar = 5 μm.

(D) White lines in (D) used for line scan analyses in (C) reflect co-localization of USO1 and GM130.

(E) Immunofluorescence staining of cells at the anteriormost edge of the wound after 0.5 h and 2 h migration. GM130 (red), and α-tubulin (gray). Scale bar = 5 μm. Black arrow points to the direction of migration.

(F) Quantification α-tubulin and GM130 distribution at the anteriormost edge of the wound after 0.5 h and 2 h migration. Distance from the center of the Golgi is shown on the horizontal axis and normalized fluorescence intensity on the vertical axis.