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Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.
Nauli, S.M. et al. (2008) Circulation 117: 1161–1171.

How do vascular endothelial cells sense shear stress? Significant evidence exists to suggest that physiologic fluid shear stress offers beneficial effects in vivo. For example, vascular regions with steady laminar flow are generally protected from atherosclerosis, and shear stress has been shown to upregulate the expression of vascular endothelial growth factors in osteoblasts. Vascular endothelia are known to release nitric oxide—a potent vasodilator—in response to fluid shear, but the specific mechanosensory apparatus that mediates this response is unknown. In their present study, Nauli and colleagues investigated the role of primary cilia in the activation of shear stress–related physiological response in vascular endothelia. Immunofluorescence staining identified the proteins polycystin-1 and polaris—known components of primary cilia—in aorta of embryonic mice. Cells were observed to respond to applied shear stress by transiently releasing intracellular calcium and nitric oxide; mutant cells defective in cilia structure/function showed no response to shear stress. Prolonged shear stress led to polycystin-1 cleavage in wild-type cells, accompanied by the loss of the shear stress response. In an effort to further elucidate the roles of polycystin-1 and polaris in the shear stress response, cells were subjected to other physical/chemical stimuli, namely apical membrane distortion and exposure to acetylcholine. Both wild-type and mutant cell types showed similar responses to these stimuli despite their different shear stress responses, indicating a role for native polycystin-1 and polaris proteins that is specific to the shear stress signaling mechanism.

Validation of novel tools that detect the elusive O-GlcNAc

The O-GlcNAc (O-linked β-N-acetylglucoasamine) modification is an abundant, highly dynamic, intracellular regulatory modification found in all eukaryotic cells; like phosphorylation, O-GlcNAc modification plays a key role in signaling pathways. Until recently, the O-GlcNAc modification presented several formidable challenges for detection. First, O-GlcNAc is very small, uncharged molecule, so O-GlcNAc-modified proteins do not migrate any differently from unmodified ones. Additionally, being present at very low levels, it is often beyond the detection limits of antibody- and lectin-based detection methods. Finally, O-GlcNAc is very labile; even with mass spectrometry, the molecule simply goes undetected. Clark et al. recently reported on an advanced chemoenzymatic labeling strategy based upon click chemistry that overcomes the limitations of these existing techniques and enables detection of the dynamic O-GlcNAc at unprecedented levels (Clark PM et al., (2008) Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. JACS (web release date August 7, 2008)). The authors initially tested the technique on α-crystallin (a known O-GlcNAc modified protein with a low extent (~10%) of glycoslyation) and detected femtomole levels with the new chemoselective approach (compared to conventional antibody and lectin techniques, by which only microgram levels were detectable). The new method was then applied to nuclear and cytosolic protein fractions from rat forebrain, and a total of 213 proteins, representing 67 known and 146 novel O-GlcNAc proteins, were identified. Besides detecting O-GlcNAc proteins directly in gels (which streamlines proteomic analyses), the authors also demonstrated the how this near-quantitative, simple, and robust two-step chemoenzymatic method can be applied to image O-GlcNAc proteins in cells and tissues.