Localization of ZO-1 in the Nucleolus of Corneal Fibroblasts

Abstract

purpose. Within the multidomain structure of ZO-1 are motifs responsible for ZO-1’s localization to intercellular junctions and its newly demonstrated localization to the leading edge of lamellipodia in corneal fibroblasts. Since ZO-1 also has two nuclear localization signals, this study was undertaken to determine whether stimuli associated with wounding would induce nuclear translocation of ZO-1

methods. Immunocytochemistry and immunoblot analysis were used to localize endogenous and exogenous ZO-1 in nuclear and cytoplasmic sites in corneal fibroblasts and 293T fibroblasts, with and without myc-ZO-1 transfection. Cells were serum starved by growth for 48 hours in DMEM/F12 with 0.2% FBS and subsequently were either scrape wounded or treated with 10% FBS, PDGF, or FGF-2 for 6 hours. For immunoblot analysis, after lysis, the nuclear and cytosolic fractions were separated and analyzed by SDS-PAGE. Cells on companion coverslips were fixed with 3% p-formaldehyde and permeabilized with 1% Triton before immunocytochemical detection of ZO-1 and nuclear proteins.

results. ZO-1 was rarely detected in the nucleus of serum-starved corneal fibroblasts. In contrast, it colocalized with nucleolin in the nucleoli of corneal fibroblasts after serum-starved cells were treated with 10% FBS, PDGF, or FGF-2. Immunoblot analysis confirmed the immunocytochemical results: Little ZO-1 was detected in the nuclear fraction of lysates of serum-starved cells, but ZO-1 was found in the nuclear fractions of rabbit corneal and 293T fibroblasts treated with 10% FBS, PDGF, or FGF-2. Furthermore in scrape-wounded corneal fibroblasts, ZO-1 was localized to nucleoli of both serum-starved and serum-treated cells.

conclusions. Localization of ZO-1 to nucleoli of corneal and 293T fibroblasts under proliferative and promigratory conditions suggests a physiologically significant interaction of ZO-1 with proteins in nucleoli during the healing process.

Wounding of the cornea activates quiescent fibroblasts (keratocytes) in the stroma to proliferate and migrate into the wound. ^{1 2 3} We recently reported the detection of ZO-1 in the leading edge of corneal fibroblasts migrating into a scrape wound where ZO-1 colocalized with integrins and polymerizing actin. ⁴ The multiple PDZ protein–protein interaction domains allow ZO-1 to function as a scaffold protein linking the actin cytoskeleton with proteins in the leading edge of the lamellipodia or in cell–cell junctions. ^{5 6}  

Within ZO-1’s domain structure there are also two nuclear localization signals (NLSs)—one in its first PDZ domain and one in its GUK domain—compared with ZO-2, which has only one nuclear localization domain in it first PDZ domain. ^{5 7} Recently ZO-1 has been identified as a member of the NACos family of proteins that localize to the nucleus and adhesion complexes, ⁵ presumably using the NLSs. In epithelial cells, other NACos proteins, including β-catenin, paxillin, symplekin, ZONAB, ZO-2, and ZO-3, have been found in the nucleus, where they may interact with specific transcription factors. ⁵ For example, β-catenin associates with LEF/TCF transcription factors in the nucleus, and ZO-2 interacts with Jun and Fos (AP-1), C/EBP transcription factors ⁸ and the DNA-binding protein scaffold attachment factorB (SAF-B). ⁹ Based on morphology and function, NACos proteins have been localized in nuclear subdomains including nucleoli, Cajal bodies, splicing speckles, and promyelocytic leukemia-PML sites associated with transcription as (co)activators or repressors, with mRNA processing, and with RNA export, respectively. ^{10 11}  

ZO-1 has been immunodetected in the nucleus in subconfluent cultures of epithelial cells (MDCK, LLC-PK1, and CV-1) during the remodeling of cell–cell contacts during wound healing. ^{5 12} However, herein we report that not only does ZO-1 translocate to the nucleus of corneal fibroblasts under conditions that mimic wounding, but it localizes quite specifically to the nucleolus. Nucleolar localization suggests that ZO-1 may participate in sequestration of proteins that control cell-cycle checkpoints and proliferation or regulate rRNA synthesis. ^{10 13 14}  

Methods

Cell Culture

Quiescent corneal fibroblasts were released from rabbit corneas (Pel Freeze, Rogers, AR) by collagenase as described in Masur et al. ¹⁵ The fibroblasts were grown in DMEM/F12 supplemented with 10% fetal bovine serum (10% FBS/DMEM/F12), antibiotics and antimycotics (penicillin 100 U/mL, streptomycin 10 μg/mL, amphotericin B 0.25 μg/mL [Invitrogen, Carlsbad, CA], and gentamicin 0.05 mg/mL [Sigma, St. Louis, MO]). These activated corneal fibroblasts were plated in 100-mm dishes and on 13-mm glass coverslips in 10% FBS/DMEM/F12. After 24 hours, they were made quiescent by serum starvation for 48 hours: DMEM/F12 containing 0.2% FBS (0.2% FBS/DMEM/F12). Finally, they were left untreated or treated with 10 ng/mL platelet-derived growth factor (PDGF) or 10 ng/mL fibroblast growth factor and 5 μg/mL heparin (FGF-2), or 10% FBS for 6 hours before fixation for immunocytochemistry or lysis and fractionation for SDS-PAGE analysis. We also used human 293T fibroblasts (Stuart A. Aaronson, Mount Sinai School of Medicine, New York, NY), which transfect readily to study the subcellular localization of an exogenously expressed myc-tagged ZO-1. 293T cells were cultured in 10% FBS/DMEM/F12. To make the 293T cells quiescent, they were plated on type I collagen (10 μg/mL; Angiotech BioMaterials, Palo Alto, CA) in supplemented serum-free medium (SSFM) consisting of DMEM/F12 supplemented with l-glutamine, glutathione, RPMI vitamin mix, sodium pyruvate (Invitrogen), antibiotic–antimycotic mix, gentamicin (Sigma), and insulin-transferrin-selenium supplement (Invitrogen). ¹⁶  

Transfection

To verify that ZO-1 was translocated to the nucleus, we transfected 293T cells with myc-ZO-1. After 24 hours of growth in SSFM in 100-mm collagen-coated dishes, the 293T cells were transfected with 10 μg of DNA/100-mm plate of full-length myc-ZO-1, ¹⁷ (generously supplied by Alan S. Fanning, Carolina Cardiovascular Biology Center, University North Carolina, Durham, NC) with a lipophilic transfection reagent (Lipofectamine 2000; Invitrogen), according to the manufacturer’s protocol. Seventy-two hours after transfection, 10% FBS or PDGF or FGF-2 was added for 6 hours. These were compared to nontransfected 293T cells grown for 96 hours in SSFM. Lysis and fractionation were performed and analyzed by SDS-PAGE and immunoblot. ^{18 19}  

In Vitro Scrape-Wound Model

Confluent cell cultures grown for 48 hours in 10% FBS/DMEM/F12, or in 0.2% FBS/DMEM/F12 were wounded by a single scratch with a P200 pipette tip across the diameter of the coverslip. After wounding, media were replaced with fresh media of the same composition for 4 to 6 hours and then fixed for immunocytochemistry. 

Immunocytochemistry

Cells were fixed with 3% p-formaldehyde (PFA; Fisher Scientific, Pittsburgh, PA) in PBS (pH 7.4; 20 minutes, 25°C), and permeabilized with 1% Triton X-100 in PBS (10 minutes, 25°C). In preliminary experiments, we found that this concentration of and duration of exposure to Triton was necessary for antibodies to gain consistent access to the nucleus. After blocking nonspecific binding with 3% normal goat serum (20 minutes, 25°C), the cells were incubated with primary antibodies, which were diluted according to the supplier’s instructions in PBS with 3% normal goat serum, 0.1% Triton, 1% BSA, and 0.02% sodium azide. The following antibodies were used for immunocytochemistry (and Western blot analysis, except where noted): rabbit anti-ZO-1 (61-7300, lot 41191369; Zymed, San Francisco, CA), mouse anti-nucleolin (7G2; Serafin Pinol-Roma, Sophie Davis School of CCNY, New York, NY), rabbit anti-Nopp140 (Thomas Meier, Albert Einstein College of Medicine, New York, NY), mouse anti-SC-35 (BD Biosciences, San Jose, CA), mouse anti-PML (5E10; Ineke van der Kraan, Amsterdam, The Netherlands), rabbit anti-Connexin-43 (Cx43; Elliot Hertzberg, Albert Einstein College of Medicine, Bronx, NY), and rabbit anti-FAK (Upstate Biotechnology, Lake Placid, NY). The primary antibodies were visualized with anti-IgG raised against the appropriate animal species conjugated to Alexa Dyes 488 or 568 (Invitrogen-Molecular Probes, Eugene, OR). Microscopy was then performed with systems (Axioskop or Axiovert 200 microscope; Carl Zeiss Meditec, Inc., Dublin, CA, or Laser Scanning Confocal Microscope; Leica, Deerfield, IL) equipped with CCD cameras, and images were collected (PhotoShop; Adobe Systems, San Jose, CA). Experiments were performed at least three times with similar results and representative images are shown. 

Cell Lysis, SDS-PAGE, and Immunoblot Analysis

Cells grown and treated as just described were rinsed twice in ice-cold PBS (pH 7.4) and gently resuspended in lysis buffer composed of 0.5% Nonidet P-40, 1 mM dithiothreitol, 100 mM NaF, 3 mM sodium vanadate, 10 mM sodium pyrophosphate, and protease inhibitors (Complete; Roche, Indianapolis, IN). Subcellular fractionation was performed as published. ¹⁸ The lysates were centrifuged at 1000g for 5 minutes at 4°C, and the supernatant was saved as the cytosolic fraction (CF). The pellet was resuspended in lysis buffer: 10 mM Tris (pH 8.4), 140 mM NaCl, 1.5 mM MgCl₂, (identified as lysis buffer B ¹⁸ ), and a one-tenth volume of the detergent (3.3% [wt/vol] sodium deoxycholate and 6.6% [vol/vol] Tween 40) was added while vortexing at a low speed. The suspension was incubated on ice for 5 minutes, and the nuclear fraction (NF) was pelleted by centrifugation at 1000g for 3 minutes at 4°C, and the supernatant was added to the CF. The nuclear pellet was rinsed once in lysis buffer and lysed in RIPA buffer (150 mM NaCl, 2 mM EDTA, 1% DOC, 0.1% SDS, 1% Triton X-100, 10% glycerol, 50 mM HEPES [pH 7.5], 100 mM NaF, 3 mM sodium vanadate, and 10 mM sodium pyrophosphate protease inhibitor [Complete; Roche]) on ice. ¹⁸ A 20- to 100-μg protein aliquot of each fraction was analyzed by SDS-PAGE. This protocol successfully separates nuclei and cytosol as indicated by reprobing the blots for SC-35 or RNA polymerase II as a nuclear marker, (immunodetected by mouse anti-SC-35 [BD Bioscience] or rabbit anti-RNA Pol II [Santa Cruz Biotechnology, Santa Cruz, CA]), and for α-tubulin, as a cytoplasmic marker (immunodetected by mouse anti-α-tubulin; Sigma). We immunodetected ZO-1 using rabbit anti-ZO-1 (61-7300, Lot: 41191369, Zymed) and myc, using mouse anti-myc (9E10; Santa Cruz Biotechnology). 

Results

ZO-1 in the Nucleus of Corneal Rabbit Fibroblasts

ZO-1 was immunodetected in the cell–cell contacts as previously shown (Fig. 1A , double arrows). Unexpectedly, when we optically sectioned through the nucleus using confocal microscopy we immunodetected ZO-1 in discrete structures within the nuclei of rabbit corneal fibroblasts, which were grown in 10% FBS (Fig. 1B , single arrow). This result was consistent with previous sequence analysis that indicated that ZO-1 has two nuclear localization sequences. These sequences have been proposed as the basis for localization of ZO-1 in the nucleus of subconfluent epithelial cells cultured in a wound area. ^{5 12 20}  

To understand the potential role that nuclear ZO-1 may play, we first asked whether ZO-1 was in the nucleus of rabbit corneal fibroblasts when the cells were growth arrested at the G₀/G₁ under conditions of serum starvation (0.2% FBS, 48 hours). We did not detect ZO-1 in the nuclei of serum-starved fibroblasts (Fig. 2A ; N). However, ZO-1 was detected in the nucleus if serum-starved cultures were treated with 10% FBS for 6 hours (Fig. 2B , single arrow). Because PDGF and FGF-2 have been shown to promote migration and proliferation in models of wound-healing, ²¹ we tested whether these growth factors could promote nuclear translocation of ZO-1 in serum-deprived corneal fibroblasts. The cells were then incubated (6 hours, 37°C), with or without 10% FBS, PDGF, or FGF-2. In contrast to serum-starved cells where ZO-1 was found only in the cell–cell contacts (Fig. 2A , double arrows) and not in the nucleus in fibroblasts treated with 10% FBS, PDGF or FGF-2, ZO-1 was found in discrete sites resembling nucleoli (Figs. 2B 2C 2D , single arrows) as well as cell–cell contacts (double arrows). ZO-1 was detected in nuclei in 45%, 32%, and 30% of fibroblasts treated with 10% FBS, PDGF, and FGF-2, respectively, but was not found in nuclei of serum-starved fibroblasts (Fig. 2E) . 

To control for the possibility that our immunocytochemical protocol allowed for nonspecific localization by rabbit antibodies we substituted rabbit antibodies to other cytoplasmic proteins: connexin-43 (Cx43), focal adhesion kinase (FAK), or preimmune IgG. We did not detect nuclear localization of either Cx43 or FAK or preimmune IgG in either serum-starved or treated fibroblasts (data not shown). Nuclear localization of ZO-1 seems dependent on protein synthesis since treatment with 10 μg/mL cycloheximide 1 hour before the addition of 10% FBS inhibited the translocation of ZO-1 into the nucleus. In contrast, ZO-1 was still localized in the cell–cell contacts after inhibition of protein synthesis (data not shown). 

Immunoblot Detection of ZO-1 in the Nuclear Fraction

To confirm the immunocytochemical data, we evaluated the distribution between the nucleus and cytosol of endogenous ZO-1 in rabbit corneal fibroblasts or exogenous ZO-1 in 293T cells after serum treatment. We used an established protocol to separate the NF and CF. ¹⁹ and used SC-35 or RNA Pol II as a marker for the NF and α-tubulin as a marker for the CF. Significant amounts of ZO-1 were immunodetected in both the NF and CF of rabbit corneal fibroblasts grown in serum (Fig. 1C)and also in 293T cells transfected with myc-ZO-1 and grown in serum (Fig. 1D) . Similar nuclear and cytosolic localization was detected with an antibody to ZO-2 (data not shown). 

To confirm that serum induces the nuclear translocation of endogenous ZO-1, we grew 293T cells in serum-free medium. We compared these with the 293T cells grown in serum-free medium and treated with 10% FBS for 6 hours before lysis. ZO-1 was detected in the NF only after treatment with FBS (Fig. 3A , lane 1). Because 293T cells are easily transfected (compared to primary corneal fibroblasts), we could evaluate the distribution of exogenously expressed myc-ZO-1 in 293T cells grown in SSFM or SSFM treated with 10% FBS or growth factor. Seventy-two hours after transfection, the cells were treated with 10% FBS, PDGF, or FGF-2 or not treated for 6 hours and then lysed. The lysates were fractionated into NF and CF, separated by SDS-PAGE, and immunoblotted with anti-myc to detect myc-ZO-1, with anti-SC-35 to verify the NF and with anti-α-tubulin to verify the CF. myc-ZO-1 was immunodetected in the CFs of all the cells whether serum-free or treated with 10% FBS or growth factors. In contrast, myc-ZO-1 was not immunodetected in the NF of serum-free 293T cells (Figs. 3B 3C ; lanes 2 and 3, respectively) but was immunodetected in the NF of cells treated with 10% FBS (Fig. 3B ; lane 1) and cells treated with FGF-2 or PDGF (Fig. 3C ; lanes 1, 2). These data demonstrate that myc-ZO-1 was expressed in 293T cells but was only translocated into the nucleus of cell treated with 10% FBS, PDGF, or FGF-2. This extends our finding on FBS-, PDGF-, and FGF-2-induced nuclear translocation of endogenous ZO-1 in rabbit corneal fibroblasts and 293T cells. These data also confirm that ZO-1 nuclear translocation is in response to treatment of cells with 10% FBS, or growth factors, and that ZO-1 nuclear translocation does not occur simply because of increased ZO-1 protein expression. 

Defining the Subnuclear Localization of ZO-1

The nucleus contains several morphologic and functional subdomains including nucleoli, splicing speckles, and PML bodies. These nuclear bodies are involved in rRNA synthesis, mRNA processing, transcriptional regulation of DNA and cell-cycle regulation respectively. ^{10 11} Because of their size, shape, and number, the ZO-1 containing nuclear structures resembled nucleoli. To confirm that they were nucleoli, we performed dual immunocytochemical localization with antibodies to nucleoli, splicing speckles, and PML bodies. We used rabbit corneal fibroblasts grown under standard conditions of serum-starvation followed by treatment with or without 10% FBS (6 hours, 37°C). Nucleoli, splicing speckles, and the PML nuclear bodies were identified with antibodies to nucleolin, SC-35, and PML, respectively. ^{22 23 24} All were detected in their characteristic morphology (Fig. 4) . Furthermore unlike ZO-1, the structures identified by these antibodies were restricted to the nucleus and were in the same location, both in serum-deprived rabbit corneal fibroblasts (Figs. 4A 4C 4E)and in fibroblasts treated with 10% FBS (Figs. 4B 4D 4F) . The nucleolar morphology was also confirmed with antibody to the nucleolar marker Nopp140 (data not shown). Using sequential confocal microscopy, we demonstrated that ZO-1 colocalized with nucleolin in rabbit corneal fibroblasts (Figs. 5A 5B 5C , single arrows) but not with either SC-35 or PML (data not shown). We concluded that nuclear ZO-1 was concentrated in the nucleoli of rabbit corneal fibroblasts. 

ZO-1 Localization in Fibroblasts Migrating into a Scrape Wound

We previously demonstrated in migrating fibroblasts that ZO-1 was strongly expressed at the leading edge of lamellipodia, where it colocalized with G-actin. ⁴ Since in the present study we found that ZO-1 was detected in nucleoli of cells treated with 10% FBS, PDGF, or FGF-2, conditions that induce cell proliferation, we wondered whether ZO-1 would also translocate to nucleoli after wounding. We scrape wounded confluent cultures of rabbit corneal fibroblasts grown with or without 10% FBS for 6 hours after serum starvation. We found ZO-1 in nucleoli of the migrating fibroblasts, even in cultures that were serum-deprived (Figs. 6A 6B , single arrow). In contrast to nonwounded, serum-starved cultures where ZO-1 was not detected in nucleoli (Fig. 2A) , cells distal to the wound also had nucleolar ZO-1 (Figs. 6C 6D)presumably as a result of signals transmitted from the cells adjacent to the wound through gap junctions. ²⁵ We also confirmed that ZO-1 was immunodetectable in the leading edge of lamellipodia (Figs. 6A 6Barrowhead) and in cell–cell contacts (Figs. 6A 6B , double arrows). Because ZO-1 was found in nucleoli in the cultures of fibroblasts migrating into the wound under serum-starvation, we concluded that wounding provides signals that are sufficient to cause the nuclear translocation of ZO-1. Thus, wounding as well as treatment of cells with 10% FBS, PDGF, or FGF-2 induced ZO-1 to translocate to the nucleolus. 

Discussion

We recently demonstrated that ZO-1 localizes to the leading edge of lamellipodia in activated corneal fibroblasts migrating into a wound. ⁴ We now report that ZO-1 is detected in nucleoli of corneal fibroblasts that are activated by scrape wounding or by treatment of 10% FBS, PDGF, or FGF-2. Since quiescent corneal fibroblasts (keratocytes) are activated in situ by endogenous growth factors including PDGF (synthesized by the epithelium and stored in the corneal basement membrane) and FGF-2 (secreted by both epithelial and fibroblast cells), ²¹ this finding suggests a dynamic role for nucleolar ZO-1. ZO-1 in the nucleolus is in a position to interact with resident proteins and other proteins translocated there after stimulation. ¹⁰ ZO-1’s domain structure provides several interaction sites. Previous research has focused on the domains that bind to the actin cytoskeleton and that bind to transmembrane proteins of cell–cell junctions directly (occludins and connexins) or indirectly (via β-catenin to cadherins). Other interacting domains include three PDZs domains in the N-terminal, a guanylate kinase site (GuK), a Src homology domain (SH3) and the C-terminal actin-binding domain and of special interest, two putative nuclear localization signals. ⁷  

This is the first report of detection of ZO-1 in nucleoli. Earlier studies in epithelial cells have identified conditions in which ZO-1 was detected in the nucleus, but it was not detected in nucleoli. Specifically ZO-1 was immunodetected in the nucleus when epithelial cell cultures were subconfluent but localized to the cytoplasm at epithelial cell–cell junctions as cell–cell contacts matured or when cell cultures were more confluent and less proliferative. ¹² Similarly, ZO-2 was detected in the nucleus in sparse cultures of epithelial cells where it partially colocalized with splicing factor SC-35. ²⁶ Gottardi et al., ¹² found that the translocation of ZO-1 into the nucleus of MDCK cells was dependent on new protein synthesis, which we found was also true for the localization of ZO-1 to nucleoli. In contrast to ZO-1 and ZO-2, symplekin was mainly cytoplasmic in subconfluent cultures and found in the nucleoplasm in confluent epithelial cultures. ²⁷  

Why has ZO-1 not been reported previously in nucleoli? In addition to identifying conditions that stimulated the translocation of ZO-1 into the nucleus, we attempted to increase the sensitivity of methods to detect this molecule immunocytochemically by altering the postfixation permeabilization. In consideration of previous reports of ZO-1 or ZO-2 in the nuclei of epithelial cells, ^{12 26 27} we tested various combinations of times and concentrations of incubation with Triton X-100 after 3% PFA fixation, and found that permeabilization by 1% Triton X-100 for 10 minutes produced consistent detection of ZO-1 in nucleoli of corneal fibroblasts treated with 10% FBS. (We also included 0.1% Triton X-100 in all steps in the immunodetection.) With the use of this protocol, ZO-1 was consistently detectable by wide-field fluorescence microscopy as a bright, specific signal in nucleoli of cells treated with 10% FBS or growth factors for 6 hours (Figs. 2 6) . Furthermore, ZO-1 was consistently absent from the nuclei of starved cells. The validity of the protocol was supported by two additional immunocytochemical controls: the previously described patterns of other nuclear proteins (SC-35, PML and nucleolin) were correctly immunodetected (Figs. 4 5) , and no nucleolar signal was generated with preimmune rabbit IgG (data not shown). We hypothesize that incubation with increased concentration of Triton X-100 and the longer permeabilization time enhanced penetration of the nuclear membrane by the antibodies and/or made the ZO-1 in the nucleolus more antibody accessible. It is possible that a similar permeabilization protocol would reveal nucleolar ZO-1 in epithelial cells in a scrape-wound model or after appropriate growth factor stimulation. ^{12 28}  

The nucleolar localization of ZO-1 is intriguing, since the nucleolus is the site of ribosomal rRNA transcription, processing, and subsequent assembly of processed rRNA with ribosomal proteins to form ribosomal subunits. ²⁹ In addition, the nucleolus can act as a “molecular basin” to retain proteins and thus prevent them from interacting with their potential downstream partners until a specific cell-cycle or metabolic stage. These include several proteins that control cell-cycle checkpoints, such as MDM2, Cdc14, Pch2, and pRB1. ^{10 13 30} For instance, p19^ARF protein in the nucleolus sequesters MDM2 protein from the nucleoplasm and prevents MDM2 from inhibiting p53. ^{10 31} If ZO-1 in the nucleolus interacted with the p19^ARF protein through its SH3 domain, p19^ARF would not be available to interact with MDM2, which would remain in the cytoplasm, inhibiting p53 and cell-cycle progression, resulting in cell proliferation. 

The fact that ZO-1 was not detected in the nucleoli of serum-starved fibroblasts but was detected in nucleoli of cells treated with either FBS, PDGF, or FGF-2 treatment, suggests that the nucleolar translocation is related to the G₁/S transition. Similarly, fibroblasts’ activation as they migrate into the scrape wound was accompanied by ZO-1 in the nucleolus even in the absence of addition of serum or growth factors. The multiple sites of ZO-1 detection suggest that ZO-1 performs multiple functions related to adhesion and cell density signals at cell–cell junctions, actin assembly at the leading edge, and cell-cycle regulation in the nucleolus. ZO-1 is now a candidate for carrying signals by its translocation to nucleoli. ³²  

Our findings suggest future experiments aimed at investigating the how and why of ZO-1 translocation to the nucleolus and its function there. Such studies could add significantly to our understanding of the contribution of ZO-1 in the transition of cells from the G₁ to S phase and ZO-1’s role in cell-cycle regulation and cell migration and proliferation during corneal wound healing. 

 Supported by a grant from the National Eye Institute R01-EY09414 (SKM), an NEI Core Grant P30 EY001867, a Research Fellowship from Fight for Sight, Prevent Blindness America (MB), and Research To Prevent Blindness (Department of Ophthalmology Mount Sinai School of Medicine). The Microscopy Shared Research Facility is supported in part with funding from National Institutes of Health-National Cancer Institute shared resources Grant 1 R24 CA095823.

 Submitted for publication July 4, 2006; revised November 15 and December 27, 2006; accepted February 28, 2007.

 Disclosure: M. Benezra, None; R. S. Greenberg, None; S. K. Masur, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

ZO-1 was detected in cell–cell junctions and discrete bodies in the nuclei of corneal fibroblasts. Corneal fibroblasts were grown in DMEM/F12 with 10% FBS for 48 hours processed for immunocytochemical and imaged via confocal microscopy. (A, B) Images of the same X-Y field, imaged at two different Z-levels, 0.81 μm apart. In (A), imaged toward the base of the cells, ZO-1 is detected only in cell–cell junctions (double arrows) and not in the nucleus (N). In (B), where the image was through the middle of the cells, ZO-1 was detected in small bodies within the nuclei (single arrow) in addition to cell–cell contact regions (double arrows). Scale bar, 10 μm. (C) Analysis was performed on the CF and the NF lysates of the corneal fibroblasts grown in the presence of 10% FBS for 72 hours. Antibody to ZO-1 detected bands in both the NF and CF at ∼220 kDa. (D) Immunoblot analysis was also performed on the lysates of 293T cells transfected with myc-ZO-1 24 hours after seeding, and grown in media with 10% FBS for 72 hours before lysis. Myc-ZO-1 was successfully expressed in the 293T cells and was detectable in the NF and CF. After lysis and separation fractionation, the CF was identified by antibody to α-tubulin and the NF was identified by SC-35 or RNA pol II, indicating successful fractionation.

Figure 2.

 

ZO-1 was absent from the nuclei of serum-starved fibroblasts but was immunodetected in nucleolar structures of fibroblasts treated with 10% FBS, PDGF, or FGF-2. After 48 hours of growth in DMEM/F12 with 0.2% FBS (serum starvation), ZO-1 was absent from nuclei (N) and was detected only in cell–cell contacts (double arrows) (A). ZO-1 was detected in nucleoli (single arrow) of fibroblasts that were treated with 10% FBS, (B), 10 ng/mL PDGF (C), or 10 ng/mL FGF-2 with 5 μg/mL heparin (FGF-2) (D) for 6 hours after serum-starvation. In treated cells, ZO-1 was also detected in the cell–cell contacts (double arrows). (E) The percentage of cells with immunodetected ZO-1 in nuclei: serum-starved cells (0.2% FBS) or serum-starved cells that were treated with 10% FBS, PDGF, or FGF-2 for 6 hour. In each condition, a total of 100 cells were counted from different areas of the coverslips and the data indicate the percentage of cells that had ZO-1 in the nucleus.

Figure 3.

 

In 293T cells, myc-tagged and endogenous ZO-1 was detected in the NF only after 10% FBS, PDGF, and FGF-2 treatment. (A) Endogenous ZO-1 was detected in the NF of 293Tcells treated with 10% FBS. Immunoblot analysis was performed on CFs and NFs of 293T cells grown serum-free for 96 hours and left untreated or treated with serum for 6 hours. The immunoblot was probed for the presence of endogenous ZO-1. Detection of SC-35 and α-tubulin in the NF and CF, respectively, demonstrated the successful separation of fractions. As in the rabbit corneal fibroblasts (Fig. 1C) , endogenous ZO-1 was detected in the NF of 293T cells after FBS treatment. (B, C) 293T cells were transfected 24 hours after seeding with myc-ZO-1. 72 hours after transfection, cells were treated with 10% FBS (B), 10 ng/mL PDGF or 10 ng/mL FGF-2 (C) for 6 hours. After lysis and separation of the NF, identified by immunodetection of SC-35 and the CF, identified by immunodetection of α-tubulin, immunoblot analysis with anti-myc antibody to detect myc-ZO-1 was performed. myc-ZO-1 was successfully expressed in the 293T cells and was detectable in the CF of 293T cells under all conditions. myc-ZO-1 was detected in the NF only in fibroblasts cells treated with 10% FBS (B) or a growth factor (+) (C) and was absent from the NF of serum-starved cells (−).

Figure 4.

 

Antibodies to nuclear proteins reveal the nuclear organization in rabbit corneal fibroblasts. Three subnuclear domains were immunodetected in cells grown under serum-starvation (A, C, E) or subsequent to treatment with 10% FBS for 6 hours (B, D, F). Antinucleolin was used to detect the nucleolus (A, B), anti-SC-35 was used to detect splicing speckles (C, D), and anti-PML was used for PML bodies (E, F). These antibodies did not bind to any sites outside the nucleus. In contrast to our finding that ZO-1 was detected in the nucleus only after FBS (Fig. 2B) , these proteins were detectable in the nuclei of cells grown in serum-depleted medium (0.2% FBS) as well as after 10% FBS. Furthermore, the immunodetected patterns that we observed for nucleolin, SC-35, and PML were unaltered in cells treated with 10% FBS. The number and pattern of nucleolin containing bodies (A) was strikingly similar to those detected by anti-ZO-1 (Figs. 1 2) . Scale bar, 10 μm.

Figure 5.

 

ZO-1 was colocalized with nucleolin in the nucleolus of fibroblasts. Immunocytochemistry with anti-ZO-1 and anti-nucleolin was performed on rabbit corneal fibroblasts treated with 10% FBS for 6 hours after 48 hours of serum starvation. Sequential confocal micrographs identified nucleolin, red channel (A) and ZO-1, green channel (B). Superimposition of the two channels revealed colocalization of ZO-1 with nucleolin in the same nucleoli, orange (C). Arrows: nucleoli that have nucleolin (A, red) and ZO-1 (B, green) and therefore appear orange in (C). The field was scanned independently for each channel to eliminate cross-talk between the two channels. Scale bar, 10 μm.

Figure 6.

 

ZO-1 translocates to nucleoli of wounded corneal fibroblasts. Confluent cultures of rabbit corneal fibroblasts were scrape wounded after 48 hours of serum starvation and grown in 0.2% (A, C) or 10% FBS (B, D) and fixed 6 hours after wounding. As we previously reported, ZO-1 was found in the leading edge of lamellipodia of cells at the wound margin (arrowhead), (A, B) as well as in the cell–cell contacts (double arrows) (A, B, D). ZO-1 was found in the nucleoli (single arrow) of cells migrating into the wound in serum-starved (A) and 10% FBS-treated (B) cultures. ZO-1 was also detected in cells distal from the wound edge in cells that were not (C) or were (D) treated with 10% FBS. Scale bar, 10 μm.

The authors thank Victor Friedrich, Robert Hennigan, and Rumana Huq for expert advice in microscopy in the Microscopy Shared Research Facility of Mount Sinai School of Medicine.