In Situlocalization of Cytoskeletal Elements in the Human Trabecular
Total Page：16
File Type：pdf, Size：1020Kb
Investigative Ophthalmology & Visual Science. Vol. 31. No. 9. September 1990 Cops right £• Association lor Research in Vision and Ophthalmology In Situ Localization of Cytoskeletal Elements in the Human Trabecular Meshwork and Cornea Robert N. Weinreb* and Mark I. Ryderf The authors compared cytoskeletal elements of the in situ human trabccular-mcshwork cell with in situ human corneal cells using indirect immunofluorcsccncc staining for tubulin and intermediate filaments (vimentin, cytokeratin, and desmin) and NBD-phallacidin staining for f-actin using both fixed frozen and unfixed frozen sections from postmortem eyes. Both f-actin and tubulin were found throughout the cell body of trabecular-meshwork cells, keratocytes, corneal endothelium, and corneal epithelium. The f-actin staining pattern was concentrated at the cell periphery of these four cell types. Vimentin stain was intensely localized in focal areas of the trabecular-meshwork cell, keratocytes, and throughout the corneal cndothelium. A general anticytokeratin antibody was intensely localized in corneal epithelium and endothelium. However, PKK-1 anticytokeratin antibody was seen only in superficial layers of corneal epithelium and not in corneal endothelium. The 4.62 anticytokeratin antibody was not observed in either corneal epithelium or endothelium. None of these three cytokera- tin antibodies were seen in trabccular-mcshwork cells or keratocytes. Desmin stain was not noted in any of these cell types. In general, cytoskeletal staining of unfixed frozen sections showed a similar staining pattern for f-actin and tubulin but a more uniform and intense staining pattern for vimentin and cytokcratin compared with fixed frozen material. The authors conclude that these cytoskclctal stains can differentiate human Irabeciilar-meshwork cells from cells of the cornea in situ. Invest Ophthalmol Vis Sci 31:1839-1847, 1990 Human trabecular-meshwork cells perform many myosin subfragment used to label f-actin)-labeled, of the activities which have been hypothesized to con- critical-point dried cells. Similar techniques have tribute to the normal function of the trabecular been used to localize the major cytoskeletal elements meshwork.1"3 They maintain a flat appearance, re- in cultured bovine trabecular-meshwork cells.15 main attached to trabecular-meshwork beams, and However, the overall cell shape and cytoskeletal orga- possess the ability to spread to cover beams that be- nization of any cell is dependent on its external envi- come denuded. In addition, these cells have phago- ronment. Thus, the nature and organization of cyto- cytic abilities and can regulate the deposition and skeletal elements of cells propagated in serial culture degradation of the extracellular matrix in the trabecu- may be different than the same cells in situ. lar meshwork.2-4"" As in other cells, these structural In the current study, we used fixed frozen and un- and motile functions arc effected through the three- fixed frozen tissue to obtain a comprehensive over- dimensional network of actin filaments (microfila- view of the in situ localization of actin filaments, ments). microtubules, and intermediate filaments tubulin in microtubules. and three different interme- known collectively as the cytoskeleton. diate filament proteins (vimentin. cytokeratin. and Recently, we examined the in vitro organization of desmin) in human trabecular-meshwork cells using actin microfilaments. microtubules. and vimentin fil- fluorescent antibody or NBD-phallacidin labeling (a aments in cynomolgus monkey and human trabecu- fluoresccin-like stain specific for f-actin) of these ele- lar-meshwork cells12"14 with both fluorescent labeling ments. In addition, we compared the in situ localiza- of these cytoskeletal elements and with transmission tion of cytoskeletal elements of trabecular-meshwork electron-microscopic observations of extracted. S-1 (a cells with other cell types from the adjacent cornea. These studies show striking differences in the in situ cytoskeletal labeling patterns among these cell types. From the "Department of Ophthalmology. University of Califor- nia, San Diego, and the ["Department of Oral Biology. University of Materials and Methods California. San Francisco. California. Supported in part by NIH grant EY05990 (R.N.W.). Reprint requests: Robert N. Weinrcb. MD. University of Califor- Postmortem eyes were obtained by enucleation nia. San Diego. Department of Ophthalmology (T-014), La Jolla, within 2 hr after death. A corneoscleral button was CA 92093. excised 2-mm posterior to the limbus to include the 1839 Downloaded from iovs.arvojournals.org on 10/02/2021 1840 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Seprember 1990 Vol. 31 trabecular meshwork. In preliminary experiments, antialpha tubulin (Amersham, Arlington Heights, we found that direct fixation of the corneoscleral IL), (2) mouse monoclonal antivimentin (Amer- buttons resulted in a marked autofluorescence of the sham). (3) mouse monoclonal antidesmin (Amer- sectioned tissue. This autofluorescence was elimi- sham). (4) a general mouse monoclonal anticytoker- nated by storing the buttons in McCarey-Kaufman atin (Amersham) with unknown specific reactivity to media for 6-24 hr before fixation and freezing or particular cytokeratin species. (5) mouse monoclonal before freezing alone. Nine nonglaucomatous eyes PKK-1 anticytokeratin antibody (Labsystems. Hel- from nine patients (aged 23-78 yr) were processed for sinki. Finland) which crossreacts with the 44-kilodal- cytoskeletal labeling. ton (kD). 46-kD, 52-kD, and 54-kD cytokeratins of In the fixation and freezing procedure, the corneo- HcLa cells, and (6) mouse monoclonal anticytokera- scleral tissue from eight eyes were fixed in 2.0% para- tin 4.62 (Miles Laboratories. Naperville, IL) which formaldehyde in 0.1 M phosphate-buffered saline reacts with the 40-kD (no. 19) cytokeratin seen in 16 (PBS) with 10 mM of sodium azide at 4°C for 4-6 hr. differentiated simple epithelium. Each monoclonal washed three times with PBS. and stored in PBS with antibody was diluted in PBS with 0.1% Triton X-100. sodium azide for 3-14 days. A segment of tissue con- 1.0% bovine scrum albumin, and 10 mM sodium taining cornea, sclera, and trabecular meshwork was azide at the following concentrations: antialpha tu- excised from each corneoscleral button, mounted in bulin, 1:50: antivimentin, general anticytokeratin, OCT compound (Miles Laboratories, Naperville. IL) and antidesmin. 1:7; and PKK-1 and 4.62 anticyto- and frozen with dry ice and liquid nitrogen. In the keratins. 1:20. freezing-alone procedure, the corneosclcral tissues All primary incubations were done for 60 min at from five eyes were directly mounted in OCT com- 20°C. The slides were then washed three times with pound and frozen without prior fixation. The frozen PBS with sodium azidc and incubated for 40 min at blocks of tissue were then transferred to a Slec HR 20°C with 50 ml of a 1:50 dilution of rhodamine- cryostat (London, England). For each piece of tissue. conjugated goat anti-mouse antibody (Cappel, Mal- 5-nm thick sections were cut and then collected on vern. PA) in the same buffer as used for the primary Chrome-Alum-treated slides (Becton-Dickinson. monoclonal incubation. Sunnyvale, CA). They were allowed to air dry for 1-2 Control incubations included incubating several hr. One slide from each tissue block was stained with sections with the primary monoclonal antibodies Mayer's hematoxylin and eosin (H & E) (Roboz Sur- alone and the secondary rhodamine-conjugated anti- gical, Washington, DC) to identify historically the body alone. After three more washes in PBS with different tissues. The remaining slides from each sodium azide, the sections were incubated with 50 JUL block were immersed in acetone for 20 min at of 1.5 mg/ml NBD-phallacidin (Molecular Probes, -20°C, air dried, and labeled for tubulin. vimentin. Junction City. OR) in PBS with sodium azide for 30 cytokeratin, ordesmin, using an indirect rhodamine- min at room temperature, washed in PBS with so- 12 conjugated antibody technique. Some sections were dium azidc, and mounted in a 1:1 mixture of PBS 12 then labeled for f-actin with NBD-phallacidin. and glycerol. In this labeling technique, the sections were first The H & E- and fluorescent-stained slides were incubated with one of the following antibodies to se- examined and photographed with an Olympus BH lected cytoskeletal elements: (1) mouse monoclonal microscope (Tokyo, Japan) using a 490-nm excita- Figs. 1-8. Fig. I. Low power view of a fixed frozen section of the trabecular meshwork (TM) stained with Mayer's hemotoxylin and eosin (H & E). Corneal cndothclium (CN) and a portion of the ciliary body (CB) are shown, also. X 100. Fig. 2. Higher power H & E fixed frozen section of the trabecular meshwork region. Numerous dark staining nuclei of trabecular meshwork cells can be distinguished (arrows) covering the meshwork of collagen beams. X1000. Fig. 3. High power fixed frozen section of the trabccular meshwork fluoresccntly stained for filamentous act in using NBD phallacidin. The actin localizes both around the nucleus (N) of the trabecular meshwork cell in and within the fine cell processes (CP) which cover the collagen beams of the meshwork. X900. Fig. 4. The same area of trabecular meshwork as in Figure 3 stained for tubulin using indirect rhodaminc conjugated antibody technique. The distribution of tubulin staining is similar to that of actin: it is found around the nucleus of the trabecular meshwork cell and within the fine processes (CP) which cover the collagen beams of the meshwork. X900. Fig. 5. High power fixed frozen section of the trabccular meshwork fluoresccntly stained for vimentin using an indirect rhodaminc conjugated antibody technique. The vimentin stain localizes to a few focal areas of the trabecular meshwork cells (arrows). A slight background fluorescence is noted in the collagen beams of the meshwork. x9()0. Fig. 6. High power unfixed frozen section of the trabccular meshwork stained for vimentin. Note a more uniform stain of vimentin (arrow) throughout the trabccular meshwork cells when compared to the fixed frozen section in Figure 5.