PMC

Immunolocalization of Hsp27 human neutrophils before and after LT treatment: phase and immunofluorescence micrographs of neutrophils exposed to 1 μM FMLP, for 5 min and stained with anti-Hsp27 antibodies that recognized total Hsp27 and p82 Hsp27. LT-treated cells were incubated with 50 ng/ml of LT for 2 h. Arrows in the control images point to the direction of neutrophil polarity. Arrowheads in the LT-treated cells point to the most rounded cells. Note the marked reduction in p82 phospho-Hsp27 content in these cells (bottom right panel). See text. Scale bars=10 μm.

Western blot analysis showing the effects of LT on the phosphorylation of Hsp27 in HeLa cells (A–C) and human neutrophils (D–E). (A) HeLa cell extract subjected to SDS–PAGE followed by western blot analysis using antibodies that recognized total and P82 phosphorylated Hsp27. Cells were treated with 1000 ng/ml LT for 12 h. A total of 50 μg protein extract was loaded in each lane. (B) HeLa cell extract subjected to two-dimensional gel electrophoresis before and after treatment with LT (1000 ng/ml × 12 h) and followed by western blot analysis as described in (A). Letters identify the various phosphorylated isoforms of Hsp27, d being the most phosphorylated form (most acidic isoelectric point) and a being the least phosphorylated form. Note the near complete absence the d isoform in LT-treated cells. A total of 600 μg of protein was loaded for each two-dimensional gel. Arrow points to missing polypeptide seen in in LT-treated cells. (C) Effects of concentration and incubation time on LT inhibition of Hsp27 phosphorylation. Western blots were performed as in (A). For the different concentrations of LT, the incubation time was 12 h. For the time course, cells were exposed to 1000 ng/ml of LT. A total of 50 μg protein extract was loaded per lane. (D) Neutrophil extract subjected to SDS–PAGE. A polyclonal anti-total Hsp27 antibody was used. Anti-p82 Hsp27 failed to crossreact with Hsp27 in human neutrophil extracts subjected to SDS denaturation. (E) Neutrophil extract subjected to two-dimensional gel electrophoresis exactly as described in (B). Note the marked reduction in the most phosphorylated (d) isoform of Hsp27 in LT-treated cells. Arrow points to the main phosphorylation isoform missing in LT-treated cells.

Schematic diagrams showing the basic components of the signal-transduction pathways for actin assembly, and showing the phosphorylation cycle for the shuttling of actin monomers by Hsp27 to regions of new actin assembly. (A) The signal-transduction pathways that allow FMLP receptor binding to activate the uncapping of actin filaments, Arp2/3 nucleation and the release of actin monomers by Hsp27. The first two pathways on the left-hand side have been emphasized. All three pathways are initiated by the heterotrimeric G-protein complex linked to the FMLP receptor. The far left pathway involves phosphoinositide kinases that generate PtdIns (4,5) and PtdIns (3,4,5) that inactivate the barbed end capping proteins gelsolin and CapZ, resulting in free barbed ends. The middle pathway is less well understood, but results in the activation of the GTPase Cdc 42, which in turn activates N-WASP, which binds to and activates Arp2/3 to nucleate the formation of new actin filaments. The far right pathway involves the activation of MAP kinase kinase kinases (MKKKs) to phosphorylate MAP kinase kinases including MAP kinase kinases 3 and 6 (MEK 3/6). These proteins then phosphorylate p38 MAP kinase that phosphorylates and activates MAPKAP kinase 2/3 to phosphorylate Hsp27 causing the release of sequestered actin monomers. LT cleaves the amino-terminus of MEK 3/6, preventing downstream phosphorylation of Hsp27. (B) Model of how Hsp27 phosphorylation and dephosphorylation could serve to shuttle actin monomers to sites of new actin filament assembly. LT blocks MEK 3/6 function and prevents Hsp27 phosphorylation. See text for details.

Effects of RNAi knockdown of Hsp27 and addition of Hsp27 WT and Hsp27AA and Hsp27EE on Listeria actin-based motility. (A) Western blot analysis of Hsp27 in HeLa cell extracts from untreated cells (control), cells treated with RNAi randomer (A) or specific antisense RNA constructs (#1–3), and bar graph comparing the velocities of Listeria in control and RNAi knockdown cells, 3–4 h after initiation of infection (brackets depict the s.e.m. of n=56–125). Methods were identical to and . (B) Western blot analysis of Hsp27 in HeLa cell extracts from untreated, RNAi knockdown cells (RNAi #3, labeled ko), RNAi knockdown cells transfected with EV, WT hamster Hsp27, Hsp27AA (AA) or Hsp27EE (EE). Specific antibodies directed against human Hsp27 and hamster Hsp27 were used to detect knockdown and rescue, respectively. Below is a bar graph quantifying the velocity of Listeria actin-based motility in control and Hsp27 knockdown cells rescued with the various constructs. For WT, AA and EE, two bars are shown. The left-hand bar shows the average velocity of cells rescued by transfection, and the right hand bar shows the average velocity of cells rescued by the microinjection of recombinant proteins (needle concentration 8 μM for all constructs, estimated cytoplasmic concentration 0.8 μM). Brackets represent the s.e.m. of n=20–161. (C) Bar graph quantifying the velocity of Listeria actin-based motility in control and Hsp27 knockdown HeLa cells (RNAi #1) in the presence and absence of 50 ng/ml LT. Note that the addition of LT to Hsp27 knockdown cells did not add to the reduction in Listeria velocity associated with Hsp27 knockdown alone. (D) Effects of addition of increasing concentrations of either recombinant pseudophosphorylated Hsp27EE (open circles) or pseudo-unphosphorylated Hsp27AA (closed circles) to brain extract (10 mg/ml) containing Listeria. Following a 90-min incubation, the lengths of the Listeria actin tails were measured (see ). The 0 point represents the mean lengths of Listeria tails in the absence of added exogenous Hsp27. Brackets represent the s.e.m. of n=40–50 determinations per concentration.

Effects of purified Hsp27 on purified pyrene-labeled actin assembly. (A) Effects of Hsp27 on spectrin-4.1 nucleation. Spectrin 4.1 nuclei (20 μg/ml) were incubated with increasing concentrations of WT Hsp27 (open circles) and then added to a final concentration of 1 μM pyrenyl actin. Minimal impairment of nucleation was observed. However, preincubation of the same concentration of G-actin with WT Hsp27 caused a concentration-dependent inhibition in the rate of actin assembly, after addition of spectrin 4.1 (closed circles). The 0 concentration points represent the rate of actin assembly in the absence of Hsp27 in each of the two experiments (for the actual kinetic curves, see ). (B) Effects of Hsp27 on the pointed end assembly rate of actin (a measure of actin monomer sequestration). The assembly rates of purified pyrenyl actin (2 μM) nucleated by gelsolin–actin seeds (molar ratio 1:20 gelsolin to actin) were determined for each concentration of Hsp27EE (open circles), Hsp27AA (closed circles) as well as WT Hsp27 (closed squares). A concentration-dependent decrease in actin assembly was seen with Hsp27AA and WT Hsp27 with complete inhibition being observed at final concentrations of 8–12 μg/ml. The 0 concentration points represent the rates of actin assembly in the absence of Hsp27 in each of the three experiments (for the actual kinetic curves, see ). (C) Effects of Hsp27 on the actin critical concentration. Left graph: the effects of unphosphorylated WT Hsp27. Increasing concentrations of monomeric pyrenyl actin were incubated with buffer alone (open circles), buffer containing 50 μg/ml (closed circles) and 100 μg/ml (closed squares) of Hsp27 followed by the addition of F-actin nuclei and a final concentration of 0.1 M KCl and 1 mM MgCl₂. The fluorescence intensity at steady state (1.5–2 h) was plotted versus actin concentration. Right graph: the effects of pseudophosphorylated Hsp27EE. The identical conditions described for WT Hsp27 were used. Closed circles represent 50 μg of Hsp27EE and open circles 100 μg/ml. (D) Effects of WT Hsp27 on the disassembly rate of actin filaments. A 2 μM portion of spectrin-4.1 nucleated pyrenyl actin filaments was diluted to 100 nM in buffer containing increasing concentrations of Hsp27 (closed symbols, μg/ml shown for each curve). Note the marked slowing down of actin assembly as compared to F-actin diluted in buffer alone (open circles). Identical curves were seen with pseudo-nonphosphorylated Hsp27AA (data not shown). (E) Effects of MAPKAP-2 phosphorylation of Hsp27 on actin monomer sequestration. Incubation of G-actin with WT Hsp27 (5 μg/ml black solid squares and 13 μg/ml black solid circles) reduced the actin assembly rate as compared to gelsolin nuclei incubated in buffer (open circles). Incubation of the same concentrations of Hsp27 with 2.5 μg/ml of MAPKAP-2 for 30 min at 25°C eliminated this inhibition (gray circles represent 13 μg/ml Hsp27 and gray squares 5 μg/ml Hsp27). The identical conditions used in (B) were employed except that the initial monomeric actin concentration was 1 μM. Inset shows western blots of Hsp27 protein using anti-total Hsp27 and anti-p82 Hsp27 antibodies after incubation with buffer or MAPKAP-2. Densitometry revealed that 45% of the total Hsp27 was phosphorylated by the kinase under the conditions of our experiment.

Effects of LT and the p38 inhibitor SB203580 on in vivo actin assembly. (A) Fluorescent micrographs of left: control HeLa cells infected with Listeria for 4 h stained with Alexa-488 phalloidin (left panel). Note the long actin filament tails. Right: LT-treated HeLa cells were first infected with Listeria for 1 h followed by 2 h incubation with 50 ng/ml LT (right panel). Cells were fixed and stained 4 h after initiation of infection. Note the shorter actin tails. Arrows mark the beginning and end of each tail. Scale bar=10 μm. (B) Left: bar graph comparing the mean velocities of intracellular Listeria in control (white bar), LT-treated (50 ng/ml, dark gray bar) and SB203580-treated (100 μM, light gray bar) HeLa cells. Brackets show the s.e.m. of n=66–322 determinations. To allow comparisons on different days, mean velocities were divided by the mean control velocity for each experiment. The actual mean velocities were 0.066±0.04 μm/s control versus 0.028±0.02 μm/s for LT-treated cells and 0.096±0.011 control versus 0.047±0.002 for SB203580-treated cells, P<0.0001. Cells were treated with LT as describe in (A) or with SB203580 for 15–30 min, and measurements made 4 h after initiation of infection. Middle: bar graph comparing the effects of increasing concentrations of LT on the velocity of Listeria actin-based motility. Same experimental conditions as above. Right: bar graph comparing the effects of increasing concentrations of SB203580 on Listeria actin-based motility. Black bar, velocity 30 min after washing out (WO) SB203580. Experimental conditions as described above. (C) Effects of LT (50 ng/ml) and SB203580 (100 μM) on Shigella actin-based motility. Cells were treated with LT and SB203580 as described in (B). Measurements were made 2 h after initiation of infection. (D) Graph comparing the rate and extent of f-met-leu-phe-stimulated actin assembly in control (open circles) and SB203580-treated (100 μM) (closed circles) neutrophils. Cells were stimulated with 1 μM FMLP at time 0, fixed with formalin at the times depicted, permeabilized with Triton and stained with Alexa 488, followed by FACS analysis of 5000–10 000 cells at each time point as described previously (). (E) LT effects on p38 phosphorylation in HeLa cells. Upper left panel: HeLa cell extract subjected to western blot analysis before and after treatment with LT (1000 ng/ml × 12 h). Anti-total and phospho-p38 kinase antibodies were used. Protein load is 50 μg for all three panels. Lower left panel: time course and concentration dependence of LT-mediated reduction in p38 MAP kinase phosphorylation. For time-course experiments, HeLa cells were treated with 1000 ng/ml of LT. For concentration experiments, HeLa cells were incubated with LT for 12 h. Upper right panel: neutrophil extract subjected to one-dimensional SDS–PAGE and western blot analysis as in HeLa cells.