LIMK1 co-localizes with microtubules in endothelial cells
To examine the relative intracellular distribution of LIMK1 in endothelial cells, human umbilical vein endothelial cells (HUVECs), were serum starved, fixed with glutaraldehyde, and costained with rabbit anti-LIMK1 and mouse anti-α-tubulin antibodies. Optical sections (0.5 μm-thick confocal sections) of stained cells revealed a striking co-localization of LIMK1 and MTs (, upper panel), where most of the LIMK1 protein was found along microtubules.
LIMK1 co-localizes with microtubules in HUVECs.
As high levels of actin monomer, tubulin subunits and cytoskeletal binding proteins are present in the cytoplasm, the resolution of cytoskeletal polymers could be reduced, making the detailed analysis of the localization of polymer binding proteins rather difficult. Therefore, to determine whether a certain pool of LIMK1 protein is associated with microtubule cytoskeleton, we have adapted a novel fixation approach that allows the extraction of cytosolic proteins, including monomeric tubulin, from living cells, preserving only polymeric cytoskeletal structures (17
). The extraction of cytosolic LIMK1 and tubulin from living HUVECs was confirmed by Western blotting (). Importantly, we showed that a certain fraction of endogenous LIMK1 could not be extracted from the living cells and was associated with MTs (, lower panel). To quantify the relative amount of the co-localized LIMK1 and tubulin, we used the Zeiss Enhanced Co-localization Tool software. Relative cell surface area was selected for each cell. Co-localization coefficient was calculated as (c1
(%) =100%* pixelsCh1, coloc
/ pixelsCh1, total
) and found to be equal to 85±4.5% suggesting a high extent of colocalization.
To demonstrate that endogenous LIMK1 and tubulin can form a complex in vitro, tubulin was immunoprecipitated from HUVECs lysates and the immunoprecipitates were analyzed by SDS-PAGE followed by probing with anti-LIMK1 antibody (). The data showed that tubulin was specifically immunoprecipitated together with endogenous LIMK1, whereas Protein A/G agarose () and nonimmune sera did not precipitate tubulin or LIMK1 (data not shown), suggesting that the interaction between tubulin and LIMK1 was specific.
The PDZ domain mediates the interaction between LIMK1 and tubulin
LIMK1 interacts with F-actin via its kinase domain, as it was determined using an in vitro
cosedimentation assay (9
). To identify the LIMK1 domain(s) that mediate the interaction with tubulin, we transfected COS-7 cells with Myc-tagged full length LIMK1 or Flag-tagged deletion mutants consisting of the two LIM domains, the PDZ domain, or the kinase domain of LIMK1 (). Tubulin was immunoprecipitated from cell lysates and the presence of the LIMK1 proteins was determined using either anti-Myc or anti-Flag antibodies. Tubulin was immunoprecipitated together with Myc-tagged LIMK1 but not with Myc-tagged zyxin used as a negative control (, left panel). In addition, Myc-tagged LIMK1 was immunoprecipitated together with endogenous tubulin, whereas non-immune serum did not precipitate Myc-tagged LIMK1 or tubulin (, right panel).
LIMK1 interacts with tubulin via its PDZ domain.
Immunoprecipitation of cell lysates with anti-tubulin antibody followed by Western blotting with anti- Flag antibody revealed that the PDZ and not the LIM or the kinase domain interacted with tubulin ().
Modulation of the microtubule cytoskeleton induces changes in LIMK1 localization
We analyzed the intracellular distribution of LIMK1 in HUVECs stimulated with thrombin, a multifunctional enzyme that plays a central role in the regulation of biochemical, transcriptional, and functional responses of endothelial cells (reviewed in (20
)). To test the effect of thrombin on microtubule organization in endothelial cells, we treated HUVECs with 25 nM thrombin for 10 min and found pronounced MTs destabilization resulting in disassembly of the peripheral microtubule network (). Changes in the relative amount of MTs were measured using Zeiss Enhanced Colocalization Tool software. The degree MTs disassembly was expressed as a ratio of the MTs area to the area of the whole cell. The data showed that approximately 44% of the MTs underwent disassembly upon thrombin stimulation (), consistently with previously published results (21
). In addition, the level of acetylated tubulin, representing the stable microtubule pool, was decreased, further confirming the destabilization of MT upon thrombin stimulation ().
Thrombin induced LIMK1 translocation requires MTs destabilization.
Importantly, upon thrombin treatment the cell morphology changed together with the pattern of LIMK1 staining; the apparent filamentous staining of LIMK1 became more homogenous as it translocated to the periphery of the cell (). Colocalization coefficient calculated as (c1 (%)=100%* pixelsCh1, coloc/ pixelsCh1, total) of LIMK1 and tubulin significantly decreased from 85±4.5% to 47±6.2%. To determine if thrombin-induced changes to LIMK1 staining pattern and localization was due to MTs destabilization, we treated endothelial cells with 1 μM nocodazole for 5 min. Treatment with nocodazole resulted in MTs destabilization similar to that seen after incubation with thrombin (). Similar to cells treated with thrombin, addition of nocodazole also resulted in more homogenous LIMK1 staining and its re-distribution to the periphery of the cell (). Colocalization coefficient of LIMK1 and tubulin was significantly from 85±4.5% to 25±7.9%.
To determine if MTs destabilization was required for thrombin-dependent changes of LIMK1 intracellular distribution, microtubule cytoskeleton was stabilized by taxol, an agent that binds microtubules and counteracts the effects of GTP hydrolysis (22
). In cells treated with taxol, LIMK1 was co-localized with MTs (). Stimulation of these cells with thrombin did not affect microtubule structure and did not change the pattern of LIMK1 localization (). Together, these results indicate that modulation of the microtubule cytoskeleton induces changes in LIMK1 localization.
Thrombin enhances the interaction of LIMK1 with F-actin
We have determined that MTs and F-actin are located at different intracellular compartments with greater proportion of MTs found in the apical part of the cell, whereas F-actin is found in the basal part of the cell (). Thrombin that induced MTs destabilization and actin polymerization did not promote the co-localization of MTs and F-actin (). Moreover, co-localization coefficient calculated as (c1 (%)=100%* pixelsCh1, coloc/ pixelsCh1, total) was equal to ~1.61% suggesting the absence of colocalization.
LIMK1 co-localizes with F-actin upon thrombin stimulation.
As LIMK1 was reported to interact with F-actin in in-vitro
) and in-vivo
), we analyzed the relative cellular distribution of endogenous Factin and LIMK1 in HUVECs using confocal microscopy. Surprisingly, in unstimulated cells very little, if any, colocalization of LIMK1 with F-actin was observed (). Co-localization coefficient of LIMK1 and actin was equal 11±6.4. Latrunculin A, an inhibitor of actin polymerization, dramatically decreased F-actin staining but had no effect on the pattern of LIMK1 staining, supporting the notion that LIMK1 did not interact with Factin in these cells. Interestingly, upon thrombin stimulation, we detected pronounced co-localization between LIMK1 and F-actin, especially at the cell periphery (). Colocalization coefficient was significantly increased from 11±6.4 to 43±6.4.
The kinase activity of LIMK1 is required for the interaction with tubulin and actin
In endothelial cells, thrombin was shown to activate Rho and its target Rho kinase (25
). The Rho-associated kinase ROCK activates LIMK1 by phosphorylation at threonine 508 within the kinase activation loop (26
). Inhibition of Rho kinase activity by the pharmacological inhibitor, Y27632, prevented thrombin-induced actin stress fiber formation in endothelial cells (21
). Similarly, inhibition of Rho kinase activity prevented thrombin-induced depolymerization of microtubules (21
). Initially, we determined whether thrombin could induce activation of LIMK1 by ROCK. Endogenous LIMK1 was immunoprecipitated from HUVECs treated with or without 25 nM thrombin and was used in in vitro
kinase assay using cofilin as a substrate. Data showed that stimulation of HUVECs with thrombin for 10 min significantly increased LIMK1- dependent cofilin phosphorylation, as it was detected using specific antiphospho-cofilin antibodies (). Pre-treatment of HUVECs with ROCK inhibitor Y27632 for 10 min completely abolished thrombin-induced cofilin phosphorylation (), which suggest that LIMK1 is activated upon thrombin stimulation by ROCK. Therefore, we have tested the possibility that inhibition of ROCK would also prevent the thrombindependent change of LIMK1 localization in endothelial cells. Pretreatment of HUVECs with Y27632 for 10 min inhibited thrombin-induced actin polymerization and microtubule depolymerization (). Interestingly, Y27632 also abolished LIMK1 translocation in cells challenged with thrombin (), suggesting that ROCK was involved in the thrombin-dependent LIMK1 translocation.
LIMK1 activity is required for its interaction with tubulin and actin.
Initially, we determined the ability of overexpressed wild type LIMK1 or kinase-dead LIMK1 (D446A) to phosphorylate cofilin and to be phosphorylated by the Rho kinase, ROCK2. In vitro kinase assay showed that wild-type LIMK1 phosphorylated cofilin () and that this phosphorylation was significantly increased by ROCK2. As previously demonstrated, LIMK1 (D446A) could not phosphorylate cofilin (). Similarly, antibody specific to phosho-T508 of LIMK1 detected basal phosphorylation of wild-type LIMK1 and kinase-dead LIMK1 (D446A) that was further enhanced by ROCK2 ().
To determine whether ROCK2 could affect the interaction between wild-type LIMK1 and kinase-dead mutant with tubulin and actin, COS-7 were transfected with wild-type LIMK1 or LIMK1 (D446A) in the absence or presence of ROCK2. Forty-eight hours later, the cells were lysed and immunoprecipitated with either anti-α-tubulin or anti-β-actin antibodies. Western blot analysis showed that the amount of wild-type LIMK1 and LIMK1 (D446A) associated with tubulin was greatly decreased in the presence of ROCK2 (). These data suggest that kinase activity is not required for complex formation with tubulin and for the modulation of LIMK1 association with tubulin upon activation.
In contrast, the amount of wildtype LIMK1 associated with actin was greatly increased in the presence of ROCK2 (), supporting the notion that activation of LIMK1 led to increased association with actin. Importantly, while the interaction between LIMK1 (D446A) and actin is similar to that of wild type LIMK1, no changes were observed in the presence of ROCK2 (). These data suggest that LIMK1 activation increases its association with actin.
LIMK1 is required for thrombininduced MTs destabilization and actin polymerization
To determine the ability of LIMK1 to modulate the microtubule and actin cytoskeleton in HUVECs, we studied the effects of overexpressed wild type LIMK1, LIMK1 (D446A), and small interference RNA (siRNA). Overexpression of wild type LIMK1 induced MTs destabilization; with the relative amount of microtubules decreased by ~52 percent (). Similarly, acetylated tubulin was dramatically decreased in cells expressing wild type LIMK1 (). Thrombin that induced MTs destabilization in non-transfected cells did not cause any further changes in microtubule organization in the cells transfected with wild-type LIMK1 (). In contrast, expression of kinase-dead LIMK1 did not induce MTs destabilization in endothelial cells (). Importantly, it attenuated thrombin-induced MTs destabilization and preserved ~80% of acetylated microtubules ().
LIMK1 activity is required for MTs destabilization.
We used small interfering RNA targeted against LIMK1 to determine LIMK1 role in the thrombin-induced modulation of microtubule cytoskeleton. HUVECs were transfected with or without siRNA against LIMK1 or Gα13 (negative control). Twenty-four hours later, cells were lysed and probed with antibodies against LIMK1, LIMK2, and Hsp90 (). Data showed that siRNALIMK1 but not siRNA-Gα13 induced significant down-regulation of the LIMK1 protein. Importantly, LIMK2 expression was not affected under any experimental condition suggesting that LIMK1 siRNA was specific ().
To demonstrate that LIMK1 is required for MTs destabilization induced by thrombin, HUVECs were transfected with GFP in the absence or presence of siRNA-LIMK1 and MTs stability was analyzed in the cells expressing GFP. In control experiment using oligonucleotide conjugated to a fluorescent probe, we determined that the cotransfection efficiency with GFP was ~95%. Data showed that siRNALIMK1 did not affected MTs stability in non-stimulated HUVECs. Importantly, downregulation of LIMK1 inhibited MTs destabilization induced by thrombin ().
Similar to its effect on fibroblasts and epithelial cells, wildtype LIMK1 induced formation of actin stress fibers in endothelial cell (), whereas stimulation of the HUVECs with thrombin did not further increase F-actin staining. LIMK1 (D446A) did not promote stress fiber formation, but it attenuated actin stress fiber formation upon thrombin stimulation (). Importantly, down-regulation of endogenous LIMK1 using siRNA also attenuated stress fiber formation upon thrombin stimulation (). Together, these data indicate that LIMK1 is required for thrombin-induced MTs destabilization and actin polymerization.
LIMK1 activity is required for actin polymerization.