Nuclear-capture of endosomes drives depletion of nuclear G-actin to promote SRF/MRTF gene expression and cancer cell invasiveness

doi:10.21203/rs.3.rs-50672/v1

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Nuclear-capture of endosomes drives depletion of nuclear G-actin to promote SRF/MRTF gene expression and cancer cell invasiveness

https://doi.org/10.21203/rs.3.rs-50672/v1

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published 24 Nov, 2021

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Signals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that an RTK, cMET promotes Rab17-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2’s cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

General Cell Biology & Physiology

nuclear G-actin

actin polymerisation

nuclear import

EphA2

cell:cell repulsion

invasiveness

Serum-response factor (SRF)

Myocardin-related transcription factor (MRTF)

pancreatic adenocarcinoma (PDAC)

Heptocyte growth factor (HGF)

Rab17

Author contributions

Experimental work primary data analysis was performed by SM, MM, AP-G, LM, GB and JN. Computational modelling was designed and performed by MN. Bioinformatic analyses were performed by HH and AH. SZ designed and supervised the proteomic approach to characterising the plasma membrane to nuclear traffickome. Experiments were conceived and designed by SM, MN, SZ and JN. SM and JN wrote the manuscript.

Competing interests

The authors declare no competing interests

Acknowledgements

This work was funded by Cancer Research UK.

Many thanks to Drs. Christian Baarlink and Robert Grosse for the generous gift of H1299 cells expressing GFP-MRTF.

Many thanks to Prof. Anne Ridley for the generous gift of GFP-tagged RhoG expression vector.

There is NO Competing Interest.

Movie1.mov
Internalisation and centripetal transport of EphA2/Rab17-positive endosomes. H1299 cells were transfected with EphA2-GFP (green) in combination with Rab17-mCherry (red). Transfected cells were challenged with HGF and monitored using confocal time-lapse fluorescence imaging. Arrows indicate EphA2/Rab17-positive vesicles that move centripetally from the cell’s periphery to the juxta-nuclear region.
Movie2.mov
Interaction of EphA2-positive vesicles with α and β1 importins in the perinuclear region. H1299 cells were transfected with EphA2-GFP (green) in combination with mCherry- importin-α5 (red channel; movie 2) or mCherry-importin-β1 (red channel, movie 3), imaged by fluorescence time-lapse microscopy and movies collected. Arrows indicate where EphA2 and importin-positive structures come into contact for an extended period.
Movie3.mov
Interaction of EphA2-positive vesicles with α and β1 importins in the perinuclear region. H1299 cells were transfected with EphA2-GFP (green) in combination with mCherry- importin-α5 (red channel; movie 2) or mCherry-importin-β1 (red channel, movie 3), imaged by fluorescence time-lapse microscopy and movies collected. Arrows indicate where EphA2 and importin-positive structures come into contact for an extended period.
Movie4.mov
H1299 cells were transfected with wild-type EphA2-GFP (WT; green signal movies 4 & 6) or an NLS mutant of EphA2-GFP (NLS2; green signal movie 5) in combination with mCherry-Lifeact (red signal) in the presence (movie 6) or absence (movies 4 & 5 ) of an siRNA targeting RhoG (siRhoG). Cells were challenged with HGF at the start of the movie and monitored using high resolution time-lapse fluorescence imaging. Arrows indicate events in which actin polymerisation was observed to increase in the vicinity of an EphA2-positive vesicle.
Movie5.mov
H1299 cells were transfected with wild-type EphA2-GFP (WT; green signal movies 4 & 6) or an NLS mutant of EphA2-GFP (NLS2; green signal movie 5) in combination with mCherry-Lifeact (red signal) in the presence (movie 6) or absence (movies 4 & 5 ) of an siRNA targeting RhoG (siRhoG). Cells were challenged with HGF at the start of the movie and monitored using high resolution time-lapse fluorescence imaging. Arrows indicate events in which actin polymerisation was observed to increase in the vicinity of an EphA2-positive vesicle.
Movie6.mov
H1299 cells were transfected with wild-type EphA2-GFP (WT; green signal movies 4 & 6) or an NLS mutant of EphA2-GFP (NLS2; green signal movie 5) in combination with mCherry-Lifeact (red signal) in the presence (movie 6) or absence (movies 4 & 5 ) of an siRNA targeting RhoG (siRhoG). Cells were challenged with HGF at the start of the movie and monitored using high resolution time-lapse fluorescence imaging. Arrows indicate events in which actin polymerisation was observed to increase in the vicinity of an EphA2-positive vesicle.
Movie7.mov
A computational approach was used to model nucleocytoplasmic actin dynamics according to the schematic in Fig. 4a. The movies depict model cells in which the nuclear envelope is clearly shown and the alterations to G-actin levels are represented by the Viridis colour palette. The influence of altering the rates of actin polymerisation (Ecμ1) and cofilin phosphorylation (Ecμ2) on depletion of nuclear G-actin was evaluated. The left-hand movie and graph (black line) depict predicted alterations to G-actin distribution following activation of both juxtanuclear actin polymerisation and cofilin phosphorylation effected by increasing the rates Ecμ1 and Ecμ2 to values greater than zero (Ecμ1>0 and Ecμ2>0). The centre movie and graph (red line) depict alterations to G-actin distribution occurring following activation of actin polymerisation (Ecμ1>0), but not cofilin phosphorylation (Ecμ2=0). The right-hand movie and blue line in the graph depict predicted G-actin dynamics following activation of cofilin phosphorylation (Ecμ2>0), but not actin polymerisation (Ecμ1=0).
Marcoetal.Spreadsheet1.xlsx
Supplementary Dataset 1
Marcoetal.Spreadsheet2.xlsx
Supplementary Dataset 2
FigureS1.jpg
Internalisation, centripetal transport and nuclear capture of endosomes. a, H1299 cells were surface-labelled with NHS-SS-Biotin at 4°C (lower panels) or left unlabelled (lower panels), and then warmed to 37°C in the of HGF for 20 min. Biotin remaining at the cell surface was removed by incubation with the membrane-impermeant reducing agent, MesNa at 4°C, and the nuclei were purified and fixed. Purified nuclei were counterstained with osmium tetroxide, incubated with streptavidin-conjugated gold (10μm), attached to formvar grids and imaged using transmission electron microscopy. The left panels display a portion of the nuclear membrane and the right panels display higher magnification sections featuring nuclear-captured endosomes. The arrows in the lower panels indicate the location of streptavidin-conjugated gold particles. b, H1299 cells were incubated in the presence of HGF for the indicated times and lysed. Nuclei were purified from these lysates using centrifugation, and the presence of the indicated compartment markers in these preparations was determined using Western blotting. Histone H2α was used as loading control. c, H1299 cells were transfected with EphA2-GFP (green) in combination with Rab17-mCherry (red). Transfected cells were challenged with HGF and monitored using confocal time-lapse fluorescence imaging and movies collected (Supplementary movie 1). Bar, 10 μm. Stills (captured at the indicated times following HGF addition) from these movies are presented in the inserts. The arrow indicates an EphA2/Rab17-positive endosome which buds from the plasma membrane and moves centripetally toward the perinuclear region. d, H1299 cells were transfected with constructs encoding GFP, GFP fused to a sequence corresponding to the putative nuclear localisation signal from the juxtamembrane region of EphA2 (NLS-GFP WT) or EphA’s NLS with the alanine substitutions indicated in figure 1h (NLS-GFP [NLS1] and NLS2-GFP [NLS2]). Cell were imaged using fluorescence confocal microscopy. Bar, 20 μm. Arrows indicate nuclear localisation of GFP e, H1299 cells were transfected with EphA2-GFP (green) in combination with mCherry- importin-α5 (upper panels, red) or mCherry-importin-β1 (lower panels, red), imaged by Marco et al. Supplement Nuclear-capture of endosomes drives SRF activation 2 fluorescence time-lapse microscopy and movies collected (Supplementary movies 2 & 3). Still from these movies (selected at 10s intervals) are displayed in the right panels. Bar, 10μm. f, H1299 cells were transfected with either wild-type EphA2 (EphA2WT) or EphA2s with mutations in its nuclear localisation sequence (EphA2NLS1 and EphA2NLS2). Transfected cells were treated with either HGF (left panels) or ephrinA1-Fc soluble ligand (right panels) for the indicated times. Cell were lysed and lysates phosphorylation of EphA2 at Ser897 (left panels) and Tyr588 (right panels) determined using Western blotting. Actin was used as loading control.
FigureS2.jpg
The role of nuclear-capture in HGF-driven expression of MRTF/SRF target genes a, Primary mouse cell lines were derived from pancreatic adenocarcinoma (PDAC) from KPC (Pdx1-Cre, KrasG12D/+, p53R172H/+, EphA2-/-) mice. EphA2 knockout cells stably expressing wild-type GFP-tagged EphA2 (EphA2-/- + WT), or GFP-tagged EphA2s with mutated NLS sequences (EphA2-/- + NLS1 and EphA2-/- + NLS2). Cells were challenged with HGF or were left unchallenged for 20 min and the levels of the indicated mRNAs (FosB; JunB; ATF3; and Zfp36) were determined using qPCR. The HGF-induced fold change in mRNA level is plotted on the y-axis, and the black line is the mean of at least 5 independent experiments ± sem, ***P<0.001, **P<0.01, *P<0.05 paired t-test. Paired data are denoted by dots of the same colour. b - c, H1299 cells were transfected with siRNAs targeting Rab17 (siRab17), EphA2, (siEphA2) or non-targeting control (nt). Cells were challenged with HGF and the HGF-induced change in levels of the indicated mRNAs (FosB; JunB; ATF3; Zfp36; SRF; CFL1, IER5, FOSL2; CYR61; MYL9; VCL; ITGB1 and ACTA2) determined as for (a). The black lines are the means of at least 3 independent experiments ± sem, ***P<0.001, **P<0.01, *P<0.05 paired t-test. Paired data are denoted by dots of the same colour. d, H1299 incubated in the presence or absence of HGF for 20 min. Cells were then lysed and the DNA sheared by sonication. Elk-1 was immunoprecipitated from lysates and the quantity of the indicated genes (FosB & JunB) determined using qPCR. The HGF-induced fold change in immunoprecipitated genes is plotted on the y-axis. Values are mean ± sem, n=6 independent experiments. e, H1299 cells were transfected with siRNAs targeting SRF (siSRF) or a non-targeting control (nt). Cells were plated onto glass surfaces and cell scattering in the presence and absence of HGF determined as for Fig. 2c. ****P<0.0001, one-way analysis of variance (ANOVA) of 3 independent experiments.
FigureS3.jpg
Nuclear G-actin dynamics and MRTF/SRF target gene expression a, H1299 cells were transfected with GFP-RhoG in combination with EphA2-GFP and incubated in the presence or absence of HGF for 20 min. Cells were lysed, immunoprecipitated (IP) using magnetic beads coupled to an antibody recognising RhoG or a non-specific control antibody (IgG). The presence of RhoG and EphA2 in the immunoprecipitates was determined by Western blotting. b-d, H1299 cells were transfected with an expression vector encoding a non-polymerisable mutant of actin (R62D actin) or vector control (empty vector) (b) or an siRNA targeting RhoG (siRhoG) or non-targeting control (nt) (c, d). Cells were challenged with HGF or were left unchallenged for 20 min and the levels of the indicated mRNAs (FosB; JunB; ATF3; and Zfp36) were determined using qPCR. The HGF-induced fold change in mRNA level is plotted on the y-axis, each bar is the mean of at least 4 independent experiments ± sem, ***P<0.001, **P<0.01, *P<0.05 paired t-test. Paired data are denoted by dots of the same colour. In (d), cell scattering in the presence and absence of HGF determined as for Fig. 2c. ****P<0.0001, one-way analysis of variance (ANOVA) of 3 independent experiments. e-g, H1299 cells were transfected with an siRNA targeting exportin-6 (siXPO6) or non-targeting control (nt). Cells were challenged with HGF or were left unchallenged for 20 min and the levels of G-actin in purified nuclei determined using flow cytometry as for figure 3c (e), expression of the indicated MRTF/SRF target genes determined using PCR (f). Each bar is the mean of at least 3 independent experiments ± sem, **P<0.01, *P<0.05 paired t-test. Paired data are denoted by dots of the same colour. Cell scattering in the presence and absence of HGF was measured as for figure 2c (g). ****P<0.0001, one-way analysis of variance (ANOVA) of 3 independent experiments.
FigureS4.jpg
Inhibition of juxtanuclear actin polymerisation does not oppose HGF-driven expression of MRTF/SRF target genes a, A computational approach was used to model nucleocytoplasmic actin dynamics according to the schematic in Fig. 4a. The graphs represent the predicted alteration to cytoplasmic F-actin (left), total G-actin (centre panel) and phosphocofilin (p-cofilin; right panel) following activation of actin polymerisation (Ecμ1) and/or cofilin phosphorylation (Ecμ2). The black lines depict predicted alterations effected by increasing the rates Ecμ1 and Ecμ2 to values greater than zero (Ecμ1>0 and Ecμ2>0). The red lines depict alterations occurring following activation of actin polymerisation (Ecμ1>0), but not cofilin phosphorylation (Ecμ2=0), and the blue lines depict predicted actin dynamics and phosphocofilin levels following activation of cofilin phosphorylation (Ecμ2>0), but not actin polymerisation (Ecμ1=0). b, H1299 cells were transfected with wild-type EphA2-GFP (EphA2; green) in combination with mCherry-Lifeact (Lifeact; red). Transfected cells were incubated in the presence of the arp2/3 inhibitor, CK-666 or vehicle control. Cells were monitored using high resolution time-lapse fluorescence imaging following challenge with HGF and movies collected. Stills (captured 20 min following HGF addition) from these movies are presented. Bar, 10 μm. The juxtanuclear regions is delineated with a dotted line. c, H1299 cells were incubated in the presence or absence (vehicle) of the arp2/3 inhibitor, CK-666 and then challenged with HGF for 20 min. HGF driven expression of the indicated MRTF/SRF target genes was determined using PCR as for figure 2f. Each bar is the mean of 3 independent experiments ± sem, P values as determined using paired t-test.
FigureS5.jpg
Simulation domains for computation modelling of nucleocytoplasmic actin dynamics Schematic representation of the cell as two static circular compartments, Ω𝑐 and Ω𝑛, which represent the cytoplasm and the nucleus, respectively. The external boundaries of the cytoplasm and nucleus are respectively represented by Γ𝑐 and Γ𝑛.

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Nuclear-capture of endosomes drives depletion of nuclear G-actin to promote SRF/MRTF gene expression and cancer cell invasiveness

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