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. 2004 Mar 15;164(6):811-7.
doi: 10.1083/jcb.200312133.

Primary Cilia of Human Endothelial Cells Disassemble Under Laminar Shear Stress

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Free PMC article

Primary Cilia of Human Endothelial Cells Disassemble Under Laminar Shear Stress

Carlo Iomini et al. J Cell Biol. .
Free PMC article

Abstract

We identified primary cilia and centrosomes in cultured human umbilical vein endothelial cells (HUVEC) by antibodies to acetyl-alpha-tubulin and capillary morphogenesis gene-1 product (CMG-1), a human homologue of the intraflagellar transport (IFT) protein IFT-71 in Chlamydomonas. CMG-1 was present in particles along primary cilia of HUVEC at interphase and around the oldest basal body/centriole at interphase and mitosis. To study the response of primary cilia and centrosomes to mechanical stimuli, we exposed cultured HUVEC to laminar shear stress (LSS). Under LSS, all primary cilia disassembled, and centrosomes were deprived of CMG-1. We conclude that the exposure to LSS ends the IFT in cultured endothelial cells.

Figures

Figure 1.
Figure 1.
Chlamydomonas IFT-71 has amino acid sequence similarity with predicted proteins in other organisms. (A) IFT-71 (GenBank/EMBL/DDBJ accession no. AY505143) was aligned with other predicted proteins using ClustalX. Dark shading indicates amino acid sequences identical to those of IFT-71 and lighter shading indicates conserved amino acid residues. Line boxes indicate the seven peptides sequenced by mass spectrometry and used for the identification of IFT-71. Thick underlines denote long homology region-1 (LHR-1) and LHR-2. Accession nos. are AAK77221.1 for Hs-CMG1, BAC35365.1 and NP_080595.1 for Mm-CMG1, and C18H9.8 for Ce-IFT-71. (B) IFT-71 has 10 exons, thick lines, and nine introns, thin lines, covering a total of 4,602 nt of genomic DNA.
Figure 2.
Figure 2.
IFT-71 is a subunit of a 17S protein complex found around the basal bodies and along Chlamydomonas flagella. (A) Autoradiogram of a partial two-dimensional polyacrylamide map resolving a subset of 35S-labeled polypeptides of IFT complex A and complex B. (A') Western blot of the map shown in A developed by the IFT-71ab. An arrow indicates the prominent spot of IFT-71 that was analyzed by mass spectrometry. (B and B') IFT complex A and complex B in fractions 8 and 9 were resolved from the remaining proteins of a flagellar extract. (B) Autoradiogram of 35S-labeled polypeptides contained in sucrose gradient fractions 1–20 and resolved by PAGE. Migration of molecular weight standards is indicated on the left side. Direction of sedimentation is from right to left. (B') Western blot of the electrophoretogram shown in B developed by hisIFT-71Ab. (C–F) Phase contrast micrographs. (C'–F') Immunofluorescence signals from hisIFT-71Ab applied to cells grown at 21°C. (C') Wild type. (D') fla10-1, temperature-sensitive mutant of anterograde IFT. (E' and F') Temperature-sensitive mutants of retrograde IFT. (E') fla15. (F') dhc1b. Bar, 5 μm.
Figure 3.
Figure 3.
CMG-1 is concentrated around centrioles of HUVEC at interphase and mitosis and in particles located along primary cilia at interphase. (A–F) Immunofluorescence signals from AcTubab following the application to HUVEC. (A'–F') Immunofluorescence signals from hisCMG-1Ab. (A”–F”) Combined immunofluorescence signals from AcTubab and hisCMG-1Ab. Bars, 5 μm.
Figure 4.
Figure 4.
PKD-1 is concentrated around centrioles and in particles along primary cilia of HUVEC at the interphase. (A, B, and inset in A) Immunofluorescence signals from AcTubab. (A', B', and inset in A') Immunofluorescence signals from an antibody specific for the COOH terminus of PKD-1. (B”) Phase contrast micrograph corresponding to B and B'. An arrow indicates the primary cilium, and N marks the nucleus. Bars, 5 μm.
Figure 5.
Figure 5.
Under LSS, primary cilia disassemble, CMG-1 become undetectable in the centrosomal region, and HUVEC orient toward the direction of LSS. (A–C) Immunofluorescence signals from AcTubab. (A'–C') Immunofluorescence signals from hisCMG-1Ab. (A”–C”) Combined immunofluorescence signals from AcTubab and hisCMG-1Ab. (A and B) HUVEC after 1 h of LSS. (C) HUVEC after 2 h of LSS. Bars, 5 μm.

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