Masaki Osawa

FtsZ in Bacterial Cytokinesis: Cytoskeleton and Force Generator All in One

SUMMARY FtsZ, a bacterial homolog of tubulin, is well established as forming the cytoskeletal framework for the cytokinetic ring. Recent work has shown that purified FtsZ, in the absence of any other division proteins, can assemble Z rings when incorporated inside tubular liposomes. Moreover, these artificial Z rings can generate a constriction force, demonstrating that FtsZ is its own force generator. Here we review light microscope observations of how Z rings assemble in bacteria. Assembly begins with long-pitch helices that condense into the Z ring. Once formed, the Z ring can transition to short-pitch helices that are suggestive of its structure. FtsZ assembles in vitro into short protofilaments that are ∼30 subunits long. We present models for how these protofilaments might be further assembled into the Z ring. We discuss recent experiments on assembly dynamics of FtsZ in vitro, with particular attention to how two regulatory proteins, SulA and MinC, inhibit assembly. Recent efforts to develop antibacterial drugs that target FtsZ are reviewed. Finally, we discuss evidence of how FtsZ generates a constriction force: by protofilament bending into a curved conformation.

Reconstitution of contractile FtsZ rings in liposomes.

FtsZ is a tubulin homolog and the major cytoskeletal protein in bacterial cell division. It assembles into the Z ring, which contains FtsZ and a dozen other division proteins, and constricts to divide the cell. We have constructed a membrane-targeted FtsZ (FtsZ-mts) by splicing an amphipathic helix to its C terminus. When mixed with lipid vesicles, FtsZ-mts was incorporated into the interior of some tubular vesicles. There it formed multiple Z rings that could move laterally in both directions along the length of the liposome and coalesce into brighter Z rings. Brighter Z rings produced visible constrictions in the liposome, suggesting that FtsZ itself can assemble the Z ring and generate a force. No other proteins were needed for assembly and force generation.

Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction: is it a mechanoresponsive molecule?

Fluid shear stress (FSS) induces many forms of responses, including phosphorylation of extracellular signal–regulated kinase (ERK) in endothelial cells (ECs). We have earlier reported rapid tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in ECs exposed to FSS. Osmotic changes also induced similar PECAM-1 and ERK phosphorylation with nearly identical kinetics. Because both FSS and osmotic changes should mechanically perturb the cell membrane, they might activate the same mechanosignaling cascade. When PECAM-1 is tyrosine phosphorylated by FSS or osmotic changes, SHP-2 binds to it. Here we show that ERK phosphorylation by FSS or osmotic changes depends on PECAM-1 tyrosine phosphorylation, SHP-2 binding to phospho-PECAM-1, and SHP-2 phosphatase activity. In ECs under flow, detectable amounts of SHP-2 and Gab1 translocated from the cytoplasm to the EC junction. When magnetic beads coated with antibodies against the extracellular domain of PECAM-1 were attached to ECs and tugged by magnetic force for 10 min, PECAM-1 associated with the beads was tyrosine phosphorylated. ERK was also phosphorylated in these cells. Binding of the beads by itself or pulling on the cell surface using poly-l–coated beads did not induce phosphorylation of PECAM-1 and ERK. These results suggest that PECAM-1 is a mechanotransduction molecule.

Curved FtsZ protofilaments generate bending forces on liposome membranes

We have created FtsZ‐YFP‐mts where an amphipathic helix on the C‐terminus tethers FtsZ to the membrane. When incorporated inside multi‐lamellar tubular liposomes, FtsZ‐YFP‐mts can assemble Z rings that generate a constriction force. When added to the outside of liposomes, FtsZ‐YFP‐mts bound and produced concave depressions, bending the membrane in the same direction as the Z ring inside liposomes. Prominent membrane tubules were then extruded at the intersections of concave depressions. We tested the effect of moving the membrane‐targeting sequence (mts) from the C‐terminus to the N‐terminus, which is approximately 180 degrees from the C‐terminal tether. When mts‐FtsZ‐YFP was applied to the outside of liposomes, it generated convex bulges, bending the membrane in the direction opposite to the concave depressions. We conclude that FtsZ protofilaments have a fixed direction of curvature, and the direction of membrane bending depends on which side of the bent protofilament the mts is attached to. This supports models in which the FtsZ constriction force is generated by protofilament bending.

The hinge-helix 1 region of peroxisome proliferator-activated receptor γ1 (PPARγ1) mediates interaction with extracellular signal-regulated kinase 5 and PPARγ1 transcriptional activation: Involvement in flow-induced PPARγ activation in endothelial cells

ABSTRACT Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Since both flow and PPARγ have atheroprotective effects and extracellular signal-regulated kinase 5 (ERK5) kinase activity is significantly increased by flow, we investigated whether ERK5 kinase regulates PPARγ activity. We found that activation of ERK5 induced PPARγ1 activation in endothelial cells (ECs). However, we could not detect PPARγ phosphorylation by incubation with activated ERK5 in vitro, in contrast to ERK1/2 and JNK, suggesting a role for ERK5 as a scaffold. Endogenous PPARγ1 was coimmunoprecipitated with endogenous ERK5 in ECs. By mammalian two-hybrid analysis, we found that PPARγ1 associated with ERK5a at the hinge-helix 1 region of PPARγ1. Expressing a hinge-helix 1 region PPARγ1 fragment disrupted the ERK5a-PPARγ1 interaction, suggesting a critical role for hinge-helix 1 region of PPARγ in the ERK5-PPARγ interaction. Flow increased ERK5 and PPARγ1 activation, and the hinge-helix 1 region of the PPARγ1 fragment and dominant negative MEK5β significantly reduced flow-induced PPARγ activation. The dominant negative MEK5β also prevented flow-mediated inhibition of tumor necrosis factor alpha-mediated NF-κB activation and adhesion molecule expression, including vascular cellular adhesion molecule 1 and E-selectin, indicating a physiological role for ERK5 and PPARγ activation in flow-mediated antiinflammatory effects. We also found that ERK5 kinase activation was required, likely by inducing a conformational change in the NH2-terminal region of ERK5 that prevented association of ERK5 and PPARγ1. Furthermore, association of ERK5a and PPARγ1 disrupted the interaction of SMRT and PPARγ1, thereby inducing PPARγ activation. These data suggest that ERK5 mediates flow- and ligand-induced PPARγ activation via the interaction of ERK5 with the hinge-helix 1 region of PPARγ.

Liposome division by a simple bacterial division machinery

We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed patches that produced concave distortions when viewed at the equator of the liposome. When viewed en face at the top of the liposome, many of the patches were seen to be small Z rings, which still maintained the concave depressions. We also succeeded in reconstituting the more natural, two-protein system, with FtsA and FtsZ-YFP (having the FtsA-binding peptide instead of the mts). Unilamellar liposomes incorporating FtsA and FtsZ-YFP showed a variety of distributions, including foci and linear arrays. A small fraction of liposomes had obvious Z rings. These Z rings could constrict the liposomes and in some cases appeared to complete the division, leaving a clear septum between the two daughter liposomes. Because complete liposome divisions were not seen with FtsZ-mts, FtsA may be critical for the final membrane scission event. We demonstrate that reconstituted cell division machinery apparently divides the liposome in vitro.

Platelet endothelial cell adhesion molecule-1 is a major SH-PTP2 binding protein in vascular endothelial cells

Platelet endothelial cell adhesion molecule‐1 (PECAM‐1, CD31) is rapidly tyrosine phosphorylated in mechanically stimulated vascular endothelial cells (ECs). A 65‐kDa protein from ECs specifically bound to the c‐Src phosphorylated PECAM‐1 cytoplasmic domain and was identified as a protein tyrosine phosphatase SH‐PTP2 (SHP2, Syp). PECAM‐1 was coimmunoprecipitated by anti‐SH‐PTP2 from EC extracts as a major binding protein, and the level of association increased when PECAM‐1 was tyrosine phosphorylated. This association was mediated by SH2 domains of SH‐PTP2. A rapid translocation of SH‐PTP2 into cell‐cell adhesion sites, where PECAM‐1 was localized, occurred in mechanically stimulated cells. Our results suggest that PECAM‐1 is a component of a mechanosensing machinery acting upstream of SH‐PTP2.

Inside-out Z rings - constriction with and without GTP hydrolysis

The bacterial tubulin homologue FtsZ forms a ring‐like structure called the Z ring that drives cytokinesis. We showed previously that FtsZ–YFP–mts, which has a short amphipathic helix (mts) on its C terminus that inserts into the membrane, can assemble contractile Z rings in tubular liposomes without any other protein. Here we study mts–FtsZ–YFP, where the membrane tether is switched to the opposite side of the protofilament. This assembled ‘inside‐out’ Z rings that wrapped around the outside surface of tubular liposomes. The inside‐out Z rings were highly dynamic, and generated a constriction force that squeezed the tubular liposomes from outside. This is consistent with models where the constriction force is generated by curved protofilaments bending the membrane. We used this system to test how GTP hydrolysis by FtsZ is involved in Z‐ring constriction. Without GTP hydrolysis, Z rings could still assemble and generate an initial constriction. However, the constriction quickly stopped, suggesting that Z rings became rigidly stabilized in the absence of GTP hydrolysis. We propose that remodelling of the Z ring, mediated by GTP hydrolysis and exchange of subunits, is necessary for the continuous constriction.

GIT1 Mediates Thrombin Signaling in Endothelial Cells. Role in Turnover of RhoA-Type Focal Adhesions

Abstract— Thrombin mediates changes in endothelial barrier function and increases endothelial permeability. A feature of thrombin-enhanced endothelial hyperpermeability is contraction of endothelial cells (ECs), accompanied by formation of focal adhesions (FAs). Recently, a G protein–coupled receptor kinase-interacting protein, GIT1, was shown to regulate FA disassembly. We hypothesized that GIT1 modulates thrombin-induced changes in FAs. In human umbilical vein ECs (HUVECs), thrombin recruited GIT1 to FAs, where GIT1 colocalized with FAK and vinculin. Recruitment of GIT1 to FAs was dependent on activation of the small GTPase RhoA, and Rho kinase, as demonstrated by adenoviral transfection of dominant-negative RhoA and treatment with Y-27632. Thrombin stimulated GIT1 tyrosine phosphorylation with a time course similar to FAK phosphorylation in a Rho kinase– and Src-dependent manner. Depletion of GIT1 with antisense GIT1 oligonucleotides had no effect on basal cell morphology, but increased cell rounding and contraction of HUVECs, increased FA formation, and increased FAK tyrosine phosphorylation in response to thrombin, concomitant with increased endothelial hyperpermeability. These data identify GIT1 as a novel mediator in agonist-dependent signaling in ECs, demonstrate that GIT1 is involved in cell shape changes, and suggest a role for GIT1 as a negative feedback regulator that augments recovery of cell contraction.

FtsZ from Divergent Foreign Bacteria Can Function for Cell Division in Escherichia coli

FtsZs from Mycoplasma pulmonis (MpuFtsZ) and Bacillus subtilis (BsFtsZ) are only 46% and 53% identical in amino acid sequence to FtsZ from Escherichia coli (EcFtsZ). In the present study we show that MpuFtsZ and BsFtsZ can function for cell division in E. coli provided we make two modifications. First, we replaced their C-terminal tails with that from E. coli, giving the foreign FtsZ the binding site for E. coli FtsA and ZipA. Second, we selected for mutations in the E. coli genome that facilitated division by the foreign FtsZs. These suppressor strains arose at a relatively high frequency of 10(-3) to 10(-5), suggesting that they involve loss-of-function mutations in multigene pathways. These pathways may be negative regulators of FtsZ or structural pathways that facilitate division by slightly defective FtsZ. Related suppressor strains were obtained for EcFtsZ containing certain point mutations or insertions of yellow fluorescent protein. The ability of highly divergent FtsZs to function for division in E. coli is consistent with a two-part mechanism. FtsZ assembles the Z ring, and perhaps generates the constriction force, through self interactions; the downstream division proteins remodel the peptidoglycan wall by interacting with each other and the wall. The C-terminal peptide of FtsZ, which binds FtsA, provides the link between FtsZ assembly and peptidoglycan remodeling.