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Remarkably, we observed the rapid disappearance of some microtubules (red, Figure?2M; Movie S11) in the most distal extracellular region of the axoneme. Given that microtubules are known to confer stiffness to the structures they shape (Hoey et?al., 2012), we reasoned that this unusual arrangement could give endothelial cilia unique stiffness profiles. To test this hypothesis, we modeled the cilium as Oxalosuccinic acid a beam and computed the hydrodynamic load by the local slender-body theory. The bending stiffness is defined as Eb?= M/k, with k being the curvature and M the moment at a cross-section of the cilium. Using our in?vivo measurements, we estimated the bending stiffness (Eb) of endothelial cilia to be in the range of 0.5?MI-773 molecular weight the conventional 9+2 microtubule doublets in the axoneme ( Figures 2N and 2O; Movie S12), demonstrating that the sample preparation did not affect the native cilia ultrastructure. We also tested whether blood flow is involved in?controlling the endothelial cilia microtubule content by characterizing the endothelial cilia ultrastructure in controls at 24 hpf, when flow forces are extremely low, and in the silent heart mutants (sih), which lack a functional tnnt2a gene ( Sehnert et?al., 2002; Figure?S2E). In the absence of flow, the observed cilia ultrastructure was similar to that of controls, suggesting that flow does not affect the microtubule content of endothelial cilia. Together, these results suggest that endothelial cilia have a unique ultrastructure, which by dictating rigidity and bending properties may render the cilia deformable in response to small flow variations. To examine this, we tested the sensitivity of cilia deflection at the earliest stages of flow generation and characterized the relationship between blood flow and cilia bending. We segmented cilia based on their GFP signal and plotted the most acute value of the angle Ponatinib ic50 between the cilia and the endothelial surface over time (Figures 3A�C3C; Movie S13), while simultaneously imaging flow (Figure?3C; Movie S13). At 24 hpf, we found the average cilia angle in the DA lumen to be close to 84�� (Figures 3D, 3F, and S3; Movie S14). This angle progressively decreased with time, reaching 41.5�� �� 2�� at 28 hpf (Figures 3D, 3F, and S3A; Movie S14), when cilia are highly deflected by flow. We also found that the frequency of cilia bending resembled that of the blood flow in response to heart contraction (2.41?Hz and 2.63 �� 0.