The guidance receptor plexin D1 is a mechanosensor in endothelial cells
Authors: Yun Fang
Adam Keen
Vedanta Mehta
Kar-Lai Pang
John S Reader
Armando Del Rio Hernandez
Katrin Nather
Daniel Rozbesky
Michael Ameismeier
E Yvonne Jones
Dimple Karia
Ellie Tzima
Dariusz Lachowski
Youxin Kong
Jianhua Huang
Journal: Nature (2020)
Discipline: Multidisciplinary
Keywords: Shear stress; arteries; blood flow; vascular development; homeostasis
Affiliations: Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK., Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK., Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, UK., Department of Medicine, Duke University, Durham, NC, USA., Department of Medicine, University of Chicago, Chicago, IL, USA., Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. .

Abstract:  Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis1. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses2. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures3-5. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature. 

Retracted: No

PubPeer comments:
 Supplemental Figure 1 contains the uncropped blots that were used in Figures 2-4. As pointed out to me by a reader, not all uncropped blots appear to match the blots shown in the main article. For example, here is Figure 3B, with the PI3K p85 blot marked in red on the left, and the uncropped blot on the right. ![file] Similarly, I have a hard time matching the panels shown in Figure 3D (top) with the corresponding uncropped blots (bottom). I have marked them below with boxes of the same color, but they appear to show different blots. ![file] In Figure 4H (top), the p-Akt, p-ERK1 2, and p-VEGFR2 panels do not appear to match the uncropped blots (bottom).Boxes of the same color highlight blots that should correspond to each other, but they appear to show different blots. Also, the uncropped pERK1 2 and pVEGFR2 blots appear to show two different exposures of the same blot - was it incubated with two antibodies at the same time? ![file] , 
 The uncropped VE-Cadherin and PI3K P85 blots for Extended Figure 7d appear to be showing the same blot (pink box). Can the authors please clarify if this blot was incubated with multiple antibodies at the same time? ![file] The uncropped tVinculin blot for Extended Figure 11 - which does match the published blot - appears to look very similar to the uncropped tVinculin blot for Figure 3D - which does not match the published blot. Could the authors please check? ![file] , 
 Some additional comments from the reader: In Figure 4h, the uncropped pERK1 2 and pVEGFR2 blots appear to be different exposures of the same blot (as pointed out above as well). In addition, the tERK1 2 and tVEGFR2 blots also appear to be different exposures of the same blot. Normally, one would use the same membrane to immunoblot a phosphorylated and a total protein (in other words, the membranes should have been identical u2018verticallyu2019 instead of u2018horizontallyu2019), because the bands of the phosphorylated one should match one or more of the bands of the total protein. It is unexpected that the same blot is used to show two different proteins, but that different blots were created to show phosphorylated and total versions of the same proteins. Could the authors please clarify? ![file] , 
 Some more mismatches between published and uncropped blots. Extended Figure 1. * Colored boxes highlight panels that do NOT appear to match the provided uncropped blots. * In Ext Figure 1F, the G alphaq 11 and GAPDH blots appear to be the different incubations of the same blot, even though the proteins are very similar in size. ![file] Extended Figure 3 * Here, the top panels (phosphorylated proteins) were all run on the same blot, but none match the provided uncropped blots. * The bottom panels (total proteins) do match. ![file] Extended Figure 8 * Colored boxes highlight panels that do NOT appear to match the provided uncropped blots. ![file] , 
 ![file] The p-VEGFR2 band of Figure 4h does not correspond to the region surrounded by the dashed frame in the uncropped image. , 
 The authors publishes an erratum (doi: 10.1038 s41586-020-1979-4) in which they replaced most of the uncropped immunoblots (IBs) and reconciled most of the disparities highlighted in the previous comments. They have not explained yet why the same IB is presented as both VE-cadherin and PI3K P85 in Fig. 7D, as shown above. The erratum provides for the first time IB replicates. IB densitometry has a major role in substantiating the conclusions of the paper. The band pattern of some of them are inconsistent. Further, some of them do not appear to display a quantifiable band. This is in addition to numerous bands of poor quality that are not addressed here. Densitometry of Figure 2A: ![file] VE-cadherin bands of Fig. 3B: ![file] P-VEGFR2 bands in Fig. 4H ![file] Piezo1 replicates of extended data Fig. 1E: were they immunoblotted with the same antibody?: ![file] PECAM-1 and PI3K p85 replicates in extended data Fig. 7D: ![file] Can the authors explain how they quantified the above bands? ,