Intact flagellar motor of Borrelia burgdorferi revealed by cryo-electron tomography: evidence for stator ring curvature and rotor/C-ring assembly flexion.

The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of approximately 1,280 flagellar motors, a approximately 3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

REGULATION OF HGF EXPRESSION BY ΔEGFR-MEDIATED C-MET ACTIVATION IN GLIOBLASTOMA CELLS

Overexpression of the hepatocyte growth factor receptor (c-Met) and its ligand, the hepatocyte growth factor (HGF), and a constitutively active mutant of the epidermal growth factor receptor (∆EGFR/EGFRvIII), occur frequently in glioblastoma. c-Met is activated in a ligand-dependent manner by HGF or in a ligand-independent manner by ∆EGFR. Dysregulated c-Met signaling contributes to the aggressive phenotype of glioblastoma, yet the mechanisms underlying the production of HGF in glioblastoma are poorly understood. We found a positive correlation between HGF and c-Met expression in glioblastoma, suggesting that they are coregulated. This is supported by the finding that in a c-Met/HGF axis-dependent glioblastoma cell line, shRNA-mediated silencing of c-Met, or treatment with the c-Met inhibitor SU11274, attenuated HGF expression. Biologically, c-Met knockdown decreased anchorage-independent colony formation and the tumorigenicity of intracranial xenografts. Building on prior findings that ∆EGFR enhanced c-Met activation, we found that ∆EGFR also led to increased HGF expression, which was reversed upon ∆EGFR inhibition with AG1478. ∆EGFR required c-Met to maintain elevated HGF expression, colony formation of glioblastoma cells, and the tumorigenicity of orthotopic xenografts. An unbiased mass spectrometry-based approach identified phosphotyrosine-related signaling changes that occurred with c-Met knockdown in a glioblastoma cell line expressing ΔEGFR and in parental cells. Notably, phosphorylation of STAT3, a master regulator of the mesenchymal GBM subtype and a known target of ∆EGFR, also decreased when c-Met was silenced in these cells, suggesting that the signals from these receptors converge on STAT3. Using a STAT3 inhibitor, WP1193, we showed that STAT3 inhibition decreased HGF mRNA expression in ΔEGFR-expressing glioblastoma cells. Consistent with these findings, constitutively active STAT3 partially restored HGF expression and anchorage-independent growth of c-Met knockdown glioblastoma cells that overexpressed ΔEGFR. We found that higher levels of HGF and c-Met expression associated with the mesenchymal GBM subtype. Taken together, these results suggest that the activity of c-Met regulates the expression of HGF in glioblastoma cells, that ∆EGFR feeds positively into this autocrine loop, that signaling of the two receptors together modulate HGF expression via STAT3, and that the HGF/c-Met axis may therefore be a good additional target for therapy of mesenchymal GBM tumors.