Changes in Adhesion Complexes Define Stages in the Differentiation
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Changes in Adhesion Complexes Define Stages in the Differentiation of Lens Fiber Cells David C. Beebe, Oleg Vasiliev, Jianli Guo, Ying-Bo Shui, and Steven Bassnett PURPOSE. During their differentiation, lens fiber cells elongate, he lens is composed of epithelial cells at various stages of detach from the lens capsule, associate at the sutures, and Tdifferentiation (Fig. 1A, 1B). The surface of the lens nearest degrade all cytoplasmic membrane-bound organelles. Changes the cornea is covered by a simple cuboidal epithelium. In the in the expression or organization of cell adhesion and cyto- avian lens, the epithelium thickens at the lens periphery, form- skeleton-associated proteins were correlated with these events ing the annular pad. Mitosis at the border of the epithelium and during fiber cell differentiation in chicken embryos. the annular pad produces cells that will eventually differentiate into the fiber cells that comprise the bulk of the lens. During METHODS. Fixed or living lenses were sliced with a tissue slicer, their differentiation, fiber cells become progressively longer permeabilized or extracted with detergents, stained with anti- until their tips reach the sutures at the anterior and posterior bodies or fluorescent-labeled phalloidin, and viewed with a poles of the lens. At the sutures, they contact fiber cells from confocal microscope. The distribution of N-cadherin in elon- the opposite side of the lens (Fig. 1B). Shortly after reaching gating and mature fiber cells was determined by Western blot the sutures, fiber cells detach from the lens capsule and are analysis. Reverse transcription–polymerase chain reaction (RT- covered by the next cohort of differentiating cells.1 Fiber cells PCR) was used to determine the distribution of vinculin and deeper in the lens abruptly degrade their nuclei, endoplasmic paxillin transcripts. reticulum, and mitochondria.2–4 There is a sharp boundary RESULTS. Staining for N-cadherin and band 4.1 protein de- only one or two cells wide between the more superficial fiber creased soon after fiber cells detached from the capsule. De- cells that contain these organelles and cells deeper within the tergent extraction of lens sections and Western blots of dis- lens that have no organelles. This boundary defines a stage sected lens regions showed that much of this decrease in through which all fibers pass as they mature. Once it is formed staining was due to epitope masking. Vinculin immunoreactiv- early in lens development, the organelle-free zone increases in ity was barely detectable on the lateral membranes of elongat- size at approximately the same rate as the lens. In addition, just ing fiber cells but increased markedly once these cells reached before chicken embryo lens fiber cells degrade their or- their maximum length and formed the sutures. Staining for ganelles, the plasma membranes of adjacent fiber cells partially paxillin was also low in elongating fiber cells but increased late fuse.5 This process links the cytoplasm of fiber cells that con- in fiber cell differentiation, just before the cells destroyed their tain organelles with the cytoplasm of the fiber cells in the membrane-bound organelles. Spectrin and ankyrin immunore- organelle-free lens core. Elongating fiber cells also have many activity did not change when fiber cells reached the sutures. fewer membrane interdigitations than mature fiber cells 4,6,7 Staining for F-actin increased transiently in cells that had just deeper in the lens. reached the sutures. Messenger RNAs for vinculin and paxillin In equatorial slices, radial cell columns run from deep in the were more abundant in maturing than in elongating fiber lens to the surface of the fiber mass (Fig. 1A). An elongating cells. fiber cell in one of these columns becomes flattened between the cells that differentiated just before and just after it. Thus, in CONCLUSIONS. The adhesion complexes of lens fiber cells cross section, a lens fiber cell has two longer sides, where it change in organization and composition soon after these cells adheres to the cells in its radial column, and four shorter sides, finish elongating and detach from the capsule. Increased stain- where it makes contact with cells in the adjacent columns. ing for vinculin and paxillin defines distinct stages of fiber cell These cell–cell associations cause the cross sections of fibers differentiation that are intermediate between the completion to approximate flattened hexagons. The precise packing of the of cell elongation and the time when lens fiber cells degrade fiber cells may be important for lens transparency, because this their membrane-bound organelles. Remodeling adhesion com- order is disrupted in cortical cataracts. plexes during fiber cell maturation may assure the stability of Cell–cell adhesions may stabilize the packing arrangement fiber–fiber associations, once these cells are no longer tran- of lens fibers. They may also be important for holding cells scriptionally active. (Invest Ophthalmol Vis Sci. 2001;42: together during accommodation, when substantial bending 727–734) and shearing forces must be generated between the cells. Several investigators have suggested that the cytoskeleton plays an active or passive role in accommodation.8–10 To func- tion in this manner, cytoskeletal components must be con- From the Cataract Research Center, Washington University School of Medicine, St. Louis, Missouri. nected, directly or indirectly, to the junctions that hold fiber Supported by National Institutes of Health Grants EY04853, cells together. EY09179 (DCB), and EY09852 (SB), and a National Eye Institute Core Previous studies have identified a number of proteins in lens Grant to the DOVS. SB is the recipient of a Career Development Award fibers that function as cellϪcell adhesion molecules or that and DCB a Jules and Doris Stein Professorship from Research to serve as links between cytoskeletal elements and the compo- Prevent Blindness. nents of cell junctions. One of these is N-cadherin, a transmem- Submitted for publication June 13, 2000; revised November 1, brane protein that forms calcium-dependent homophilic asso- 2000; accepted November 30, 2000. ciations between neighboring cells.11–13 Also present in lens Commercial relationships policy: N. 14 15 Corresponding author: David C. Beebe, Cataract Research Center, fibers are proteins, such as vinculin and plakoglobin, that Department Ophthalmology and Visual Sciences, Washington Univer- are likely to connect adhesive junctions to the cytoskeleton. sity School of Medicine, Campus Box 8096, 660 South Euclid Avenue, These molecules are most abundant along the shorter sides of St. Louis, MO 63110. [email protected] elongating fiber cells.14,15 Several proteins that were first dis- Investigative Ophthalmology & Visual Science, March 2001, Vol. 42, No. 3 Copyright © Association for Research in Vision and Ophthalmology 727 Downloaded from iovs.arvojournals.org on 10/02/2021 728 Beebe et al. IOVS, March 2001, Vol. 42, No. 3 FIGURE 1. Chicken embryo lens sec- tions along the equatorial (A) and midsagittal axes (B). covered in the submembrane cytoskeleton of red blood cells for at least 1 hour with PBS, exposed to secondary antibody in the same have also been identified in the lens. These include spec- buffer used for the primary antibody, washed three times in PBS, and trin16–18 (also called fodrin), ankyrin,19 band 4.1 protein,16 examined with a scanning confocal microscope (model 410; Carl Zeiss, band 4.9 protein,20 and band 3 protein.19 Based on their Thornwood, NY). functions in erythrocytes, these molecules may be important in In some cases, unfixed lens slices (400 or 500 m) underwent stabilizing lens fiber cell plasma membranes or in maintaining detergent extraction for 3 hours at 4°C in a buffer modified from that cell shape. Finally, proteins involved in modulating the assem- described by FitzGerald23 (0.5% Nonidet P [NP]-40, 100 mM KCl, 5 mM bly, function, and stability of the actin cytoskeleton, including MgCl2, 1 mM disodium EDTA, protease inhibitor cocktail [Roche Mo- tropomodulin, tropomyosin, caldesmon, and myosin, have re- lecular Biochemicals, Indianapolis, IN] and 2 mM 2-mercaptoethanol). cently been described in the lens.18,21,22 After extraction, slices were stained as described. Actin filaments are known to interact, directly or indirectly, Primary antibodies and the dilutions used were mouse monoclonal with most of the proteins we have mentioned. Direct associa- anti-N-cadherin (anti-A-CAM, clone GC-4; Sigma), 1:200; rat monoclo- tion of lens actin with spectrin has been demonstrated.17 nal anti-N-cadherin (hybridoma supernatant NC-2), 1:200; mouse Proteins that link the plasma membrane–adhesion complex monoclonal anti-vinculin (clone hVIN-1; Sigma) 1:200; rabbit anti- with other cytoskeletal elements, such as microtubules and ankyrin (chicken erythrocyte; Calbiochem–Novabiochem, La Jolla, intermediate filaments, have not been identified in the lens, CA), 1:200; mouse monoclonal anti-paxillin (clone Z035; Zymed, South although such links may be important in lens fiber cell forma- San Francisco, CA), 1:200; rabbit anti-␣-actinin (Sigma), 1:100; rabbit tion or stability. anti-spectrin (chicken erythrocyte; Sigma), 1:200; mouse monoclonal The present study was designed to determine whether anti-protein 4.1 (chicken erythrocyte; East-Acres Biologicals, South- proteins that contribute to structure and function of the cy- bridge, MA), 1:200; and mouse monoclonal anti-talin (Sigma), 1:200. toskeleton–cell adhesion complex change during fiber cell Secondary antibodies