Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium. J Cell Biol 1987 Dec;105(6 Pt 2):2973-87
Date
12/01/1987Pubmed ID
3693403Pubmed Central ID
PMC2114726DOI
10.1083/jcb.105.6.2973Scopus ID
2-s2.0-0023553166 (requires institutional sign-in at Scopus site) 57 CitationsAbstract
The ciliary base is marked by a transition zone in which Y-shaped cross-linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y-shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA-binding component seen in cytochemical experiments.
Author List
Horst CJ, Forestner DM, Besharse JCAuthor
Joseph C. Besharse PhD, MA Emeritus Professor in the Cell Biology Neurobiology and Anatomy department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsAnimals, Newborn
Cell Membrane
Cilia
Glycoconjugates
Microscopy, Electron
Microtubules
Molecular Weight
Photoreceptor Cells
Polyethylene Glycols
Proteins
Proteoglycans
Rats
Wheat Germ Agglutinins
